Genetics and Genetic Testing in Pancreatic Cancer

Genetics and Genetic Testing in Pancreatic Cancer

Accepted Manuscript Genetics and Genetic Testing in Pancreatic Cancer David C. Whitcomb, MD PhD, Celeste Shelton, MS, Randall E. Brand, MD PII: DOI: ...

1MB Sizes 3 Downloads 265 Views

Accepted Manuscript Genetics and Genetic Testing in Pancreatic Cancer David C. Whitcomb, MD PhD, Celeste Shelton, MS, Randall E. Brand, MD

PII: DOI: Reference:

S0016-5085(15)01089-6 10.1053/j.gastro.2015.07.057 YGAST 59947

To appear in: Gastroenterology Accepted Date: 31 July 2015 Please cite this article as: Whitcomb DC, Shelton C, Brand RE, Genetics and Genetic Testing in Pancreatic Cancer, Gastroenterology (2015), doi: 10.1053/j.gastro.2015.07.057. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.

ACCEPTED MANUSCRIPT

Invited Review: Gastroenterology, 2015 Special issue: Genetics, Genetic Testing and Biomarkers of Digestive Diseases.

RI PT

Genetics and Genetic Testing in Pancreatic Cancer

David C Whitcomb MD PhD1-4*, Celeste Shelton MS1, and Randall E Brand MD1,4 1

SC

Department of Medicine Division of Gastroenterology, Hepatology and Nutrition, Department of Human Genetics, 3 Department of Cell Biology & Molecular Physiology. 4 Pittsburgh Cancer Institute University of Pittsburgh and UPMC, Pittsburgh, PA.

*Corresponding Author

EP

TE D

David C Whitcomb MD PhD Medical Arts Building, Room 401.4 3708 Fifth Ave, Pittsburgh PA 15213 412 578 9515 Fax 412 578-9547

M AN U

2

AC C

No conflict of interest.

1

ACCEPTED MANUSCRIPT

Abstract

RI PT

Genetic testing of germline DNA is used in patients suspected of being at risk of pancreatic ductal adenocarcinoma (PDAC) to better define the individual’s risk and to determine the

mechanism of risk. A high genetic risk increases the pretest probability that a biomarker of

SC

early cancer is a true positive and warrants further investigation. The highest PDAC risk is

generally associated with a hereditary predisposition. However, the majority of PDAC results

M AN U

from complex, progressive gene-environment interactions that currently falls outside the traditional risk models. Over many years the combination of inflammation, exposure to DNAdamaging toxins and failed DNA repair promote the accumulation of somatic mutations in pancreatic cells –PDAC risk being increased by already present oncogenic germline mutations.

TE D

Predictive models and new technologies are needed to classify patients into more accurate and mechanistic PDAC risk categories that can be linked to improved surveillance and preventative

EP

strategies.

AC C

Keywords: Familial pancreatic cancer Hereditary Breast and Ovarian Cancer syndrome Hereditary pancreatitis Genetic testing

2

ACCEPTED MANUSCRIPT

Introduction The pancreas is a simple organ with two functions: produce digestive enzymes and signal the

RI PT

cells of the body that food is being digested and absorbed. The organ has three general cell types: acinar cells that synthesize digestive enzymes as inactive zymogens, duct cells that

produce bicarbonate-rich juice to flush the zymogens into the intestine, and islet of Langerhans

SC

cells that produce hormones such as insulin, glucagon, pancreatic polypeptide and

somatostatin. The pancreas is generally protected from the environment, although it is

M AN U

sensitive to smoking, alcohol consumption and other uncommon factors. As a simple organ the pancreas provides a great model to understand the relationship between genetics, biomarkers and utility of genetic testing.

TE D

Nearly 50,000 individuals in the US are expected to be diagnosed with pancreatic cancer in 2015 (1). There are no strong environmental factors that predispose an individual to pancreatic

EP

cancer – even alcohol and smoking confer relatively low risk with a relative risk of ~2 (2, 3). A strong family history of pancreatic cancer is also uncommon. It has been estimated from case-

AC C

control and cohort studies that up to 10% of patients with pancreatic cancer will have a first or second-degree relative with pancreatic cancer (4, 5). However, germline mutations responsible for the increased risk for developing pancreatic cancer are identified as contributing risk factors in only about 20% of these cancer-prone families. Thus, the majority of pancreatic cancers arise in patients with no strong environmental risk exposure or hereditary predisposition for this problem.

3

ACCEPTED MANUSCRIPT

The primary form of pancreatic cancer discussed here is pancreatic ductal adenocarcinoma

RI PT

(PDAC). PDAC has several characteristic features. The term “ductal” refers to the appearance of features of ductal epithelium such as tubular elements and mucin production rather than the cell of origin. PDAC also generates a dense, fibrotic stroma or desmoplastic reaction. Tumors

SC

contain cancer cells with high rates of mitosis, an irregular, heterogeneous and infiltrative

growth pattern, inflammatory cell infiltration and areas of necrosis. PDAC demonstrates a high

M AN U

affinity for perineural invasion, early metastasis and the generation of a catabolic state in patients resulting in rapid weight loss, cachexia and early demise. Greater than 70% of patients will die within the first year of diagnosis. Major highlights of current knowledge on PDAC is provided in Table 1.

TE D

This review will specifically focus on genetic and environmental risk for PDAC, risk evaluation and counseling, and biomarker interpretation in very high-risk patients such as members of pancreatic cancer-prone kindreds. Recommendations for any type of surveillance program is

EP

currently limited to these families since the pre-test probability that an early biomarker of

AC C

pancreatic cancer is a true positive is high enough to warrant further, sometimes radical, interventions in such individuals.

PDAC as a Disorder of Complex, Progressive Gene-Environment Interactions Fundamentally, PDAC is an acquired complex genetic disorder. There are many important factors driving the development of PDAC, but these causative or disease maintenance factors alone are neither necessary nor sufficient to cause the disease. The development of PDAC 4

ACCEPTED MANUSCRIPT

requires several sequential complex steps, with each step involving the convergence of multiple

RI PT

risk factors and random pathologic events.

PDAC is a disease of the elderly.

In the United States the median age of onset of PDAC for all races and both sexes is 71 years

SC

(SEER) (Figure 1). The fact that pancreatic cancer typically occurs later in life is in line with general hypotheses and computer models predicting that multiple sequential steps are

M AN U

required, each resulting in accumulation of multiple stochastic (random) “hits” to pancreatic cell DNA from specific types of environmental factors (6, 7). However, the sequence and rate of progression between steps may vary among individuals, and there are currently no biomarkers to accurately track progression. When PDAC develops earlier in life than expected, it generally

9).

TE D

suggests that some of the required events were not acquired randomly, but were inherited (8,

EP

PDAC Incidence Varies by Country

Epidemiologic data also suggests that the incidence of PDAC varies around the world (Figure 2).

AC C

The highest rates of pancreatic cancer for men and women are in the Czech Republic, with an age-standardized rate (ASR) of 11.9/100,000 for men and 7.9/100,00 for women (10). Other Eastern Europe countries also suffer from a high ASR for PDAC. High rates of PDAC for both men and women are found in industrialized European countries, the United States (ASR 7.5), Israel (ASR 7.6), and Japan (ASR 8.5) (10). Lower rates are seen in South America with the

5

ACCEPTED MANUSCRIPT

exception of French Guyana (ASR 8.1), Uruguay (7.7) and Argentina (ASR 7.2) (10). The lowest rates are in Africa and Southern Asia.

RI PT

PDAC is Associated with Toxins

Multiple epidemiology studies indicate that some toxins generate an increased risk for PDAC. The most important is cigarette smoking, estimated to increase the odds of developing PDAC by

SC

about a factor of 2 (11). Heavy alcohol use also increases the risk of PDAC, but only by a factor

M AN U

of about 1.5 (12). Alcohol and smoking are also important because the increase risk of chronic pancreatitis (below). Exposure to chlorinated hydrocarbons or polycyclic aromatic hydrocarbons also increase risk of PDAC, by a factor of about 1.4 to 4.4 (13). Finally, dietary factors can increase risk, especially N-nitroso-containing foods (14). The mechanisms of action

TE D

of these toxins vary, but it is believed that key features are that they may cause both DNA damage and injury-inflammation. Thus, multiple environmental and occupational exposures affect PDAC risk, but the independent effect sizes are too small to explain PDAC in individual

EP

cases.

AC C

PDAC is Associated with Inflammation One of the highest risks for PDAC is hereditary pancreatitis (HP), an autosomal dominant genetic disorder caused by gain-of-function mutations in the PRSS1 gene coding for cationic trypsinogen (15, 16). Pancreatic inflammation in HP begins at a median age of 10 years, but the marked increase in incidence of PDAC does not occur until the 6th decade of life (17-19). The risk of PDAC in patients with HP is nearly 70 times the general population (17-21). However,

6

ACCEPTED MANUSCRIPT

the PRSS1 mutations are not enriched in PDAC tumors from non-HP cases (22), suggesting that the generation of inflammation, rather than the mutation in PRSS1 itself, is the driving factor.

RI PT

Thus, it is believed that the high risk of PDAC is due to the effects of chronic inflammation, and that risk is markedly increased in the context of toxic environmental factors that could directly lead to DNA damage, such as smoking (17, 20, 23). But even in HP, only a minority of patients

SC

develop PDAC, the incidence does not appear to be randomly distributed within all families, and the risk of PDAC does not correlate with severity of inflammation and fibrosis (24). These

M AN U

observations suggest that high risk of PDAC in the general population could also represent a combination of existing pathogenic cancer gene variants, plus environmental factors and pancreatic inflammation.

TE D

While HP serves as more homogenous model of chronic pancreatitis, it appears that chronic pancreatitis from any cause increases the risk of PDAC (2, 17, 21, 25-29). The risk of PDAC in patients with acute or unspecified pancreatitis is estimated to increased about 5-6 fold, while

EP

the risk of PDAC from chronic pancreatitis appears to be higher, noting that the type of study (case-control versus cohort) and timing between diagnosis of pancreatitis and PDAC (problem

AC C

of reverse causality) can affect calculated risk (2, 21, 29, 30). It is not known if the critical event to activate oncogenic processes is only a short term injury and inflammatory response to trigger the oncogenic process (31, 32), or if chronic pancreatitis, defined by recurrent or persistent inflammation with fibrosis causing morphologic changes (33), is required. Certainly an argument can be made that a chronic inflammatory disorder affecting the pancreas can increase the risk of PDAC (2, 23, 25, 28, 34-36).

7

ACCEPTED MANUSCRIPT

PDAC Requires Complex Gene x Environment Interactions

RI PT

Globally, the ASR of pancreatic cancer is not uniform among populations and geographical locations (Figure 2). This observation suggests that environmental and/or genetic factors are important. Further analysis suggests that genetic variants or environment factors alone are not

SC

sufficient, but must interact in complex ways to increase the risk of PDAC (Table 2). For

example, India, especially Southern India, has high rates of chronic pancreatitis, yet lower rates

M AN U

of pancreatic cancer than seen in the United States or Europe (26, 37). This fact suggests lower rates of underlying pancreatic cancer risk gene variants in this population resulting in less PDAC development within the context of a strong inflammatory milieu. Furthermore, while some of the lowest rates of PDAC are in Africa, the highest age-standardized rates of PDAC in the USA

TE D

are among African Americans (38), suggesting that environment is a critical risk modifier. These data support a disease model with three risk components:, (A) germline and acquired genetic variants resulting in failure to repair DNA damage (e.g. familial pancreatic cancer syndromes),

EP

(B) exposure to toxic factors that cause DNA damage (e.g. smoking); and (C) inflammation to

AC C

accelerate the overall process of PDAC development (e.g. hereditary or chronic pancreatitis).

If PDAC is indeed a progressive complex disorder as we describe, then it follows that improvements in risk assessment, early detection, diagnosis and management requires a more holistic and integrative approach. A new approach requires both the appreciation of the complex genetic and environmental interactions, a link to accurate predictive models of the

8

ACCEPTED MANUSCRIPT

natural history, the effects of targeted preventative or managing treatments, and better

RI PT

biomarkers to monitor the process.

Molecular Oncogenesis Models

While the genesis of PDAC is only partially understood, the overall process results in the

SC

accumulation of specific classes of mutations that, in turn, result in a malignant neoplasm. Two lines of research have been especially productive in improving our understanding of PDAC

(Supplementary Materials).

Molecular pathology

M AN U

carcinogenesis: molecular pathology and genetically engineered mice (GEM) models

TE D

In 2001, a consensus histopathology-genetic model, defined as the pancreatic intraepithelial neoplasia (PanIN) sequence was published that proposed the progression of normal pancreatic duct cells to PDAC manifested by progressive changes in these cells (39). This model integrates

EP

a series of proposed somatic mutations resulting in the morphological changes in the pancreatic duct that are classified by pathologists as normal, PanIN-1A, PanIN-1B, PanIN-2,

AC C

PanIN-3, carcinoma in situ, and PDAC. The process of acquiring somatic gene mutations appears to begin with the activating KRASG12D mutation followed by a series of loss of function mutations in the tumor suppressor CDKN2A (p16), then TP53 (p53), SMAD4, BRCA2 and others (39, 40). The model has been extremely useful for coordinating and harmonizing communications among physicians and scientists, although the process does not necessarily begin in the normal duct, but may actually begin in the acinar cell, especially in the context of

9

ACCEPTED MANUSCRIPT

inflammation (41). The use of next generation sequencing techniques has strengthened and

RI PT

enhanced these early concepts (42-46).

Integrated Disease model.

Disease models are important, not because they are perfectly accurate, but rather because they

SC

are useful in organizing complex and interacting data elements, in understanding fundamental principles and in developing new hypotheses for testing. A simple model for complex acquired

M AN U

diseases, such as pancreatic cancer, can be divided into three phases: disease risk without symptoms, disease activity with progression, and disease endpoint.

The overall process of PDAC oncogenesis is complex, but appears to begin with several key early events. Germline mutations are present in all cells of the body from birth. Since each cell

TE D

has two copies of each gene, they typically function normally even if one copy is non-functional. In carriers, the risk of the loss of key processes, such as growth regulation and DNA repair, is

EP

high since only one random mutation in the functional gene is needed to completely knock out

AC C

its protective function.

The likelihood that a critical mutation will occur in a pancreatic cell is increased by toxins, such as components of cigarette smoke and inflammation – especially chronic pancreatitis (2, 25, 28, 35, 36). Inflammation is likely important because of several downstream effects, including generation of a toxic milieu, tissue injury with acinar cell de-differentiation to a more duct-like morphology where there is more rapid cell turnover, and increased susceptibility to DNA

10

ACCEPTED MANUSCRIPT

damage (47). Inflammation also activates KRAS in acinar and duct cells, activates sensory nerves, attracts inflammatory cells, activates pancreatic stellate cells and leads to expression of

RI PT

growth factors during regeneration. Recurrent pancreatitis and/or chronic pancreatitis differs from smoking as a risk factor because it specifically targets the pancreas and can initiate or drive the oncogenic process (31, 48-50). Smoking can also be viewed as a secondary risk factor

SC

because it modifies this process and may contribute to DNA damage (20), but by itself it is not

M AN U

sufficient to cause PDAC.

The disease endpoint is the development of a self-replicating cell that has acquired all of the features of an invasive, malignant cancer with unregulated growth, invasion of surrounding

TE D

tissues and metastasis to distant sites (Figure 3).

Risk assessment currently focuses on germline mutations (discussed below), environmental risk factors and the presence of chronic pancreatitis. The process of early detection and early

EP

diagnosis focuses on identifying this cell and its daughters before they spread outside of the pancreas, remains the greatest clinical challenge. Once PDAC is established, then cancer

AC C

treatment become the goal (not discussed here)

Approach to Patients with Inherited Pancreatic Cancer Susceptibility Syndromes

Between 5% and 10% of PDACs are caused by an identifiable pathogenic sequence variant (51). However, this is likely an underestimate given the complexity of gene-gene and gene-

11

ACCEPTED MANUSCRIPT

environment interactions that are beginning to be uncovered (Table 2). Features within a family that are suspicious for a hereditary cancer predisposition include early onset cancer

RI PT

(before the age of 50 years), rare tumors (e.g. male breast cancer, ovarian cancer), multiple primaries (synchronous and/or metachronous), multiple affected close relatives, and particular ethnicities with a higher carrier frequency (e.g. Ashkenazi Jewish) (8). Family history is critical in

SC

determining the appropriate test for an individual. Once a responsible mutation is identified in a family, single-site testing is appropriate for other at-risk family members within testing

M AN U

guidelines. However, if a responsible gene mutation is not identified, family members are still at risk based on their personal and family history, particularly in the presence of a family history of pancreatic cancer. Individuals are considered to be at risk for PDAC if they have (i) an identified genetic syndrome associated with pancreatic cancer (including hereditary

TE D

pancreatitis); (ii) two or more relatives with PDAC with one being a first degree relative (parent, sibling, child); (iii) three or more relatives with PDAC (52). The primary genetic syndromes are

EP

discussed below, followed by some issues to consider before ordering genetic tests.

AC C

Hereditary Breast and Ovarian Cancer syndrome

Hereditary breast and ovarian cancer (HBOC) syndrome is an autosomal dominant condition most commonly caused by mutations in the BRCA1 and BRCA2 genes. HBOC is associated with increased risks for breast cancer (47 – 55% by age 70 years) and ovarian cancer (17 – 39%), as well as prostate cancer, male breast cancer, PDAC, and melanoma (53). Carrier frequency in the general population is 1 in 500, and 1 in 40 in the Eastern European Ashkenazi Jewish population. The BRCA1 and BRCA2 genes both encode tumor suppressors.

12

ACCEPTED MANUSCRIPT

The BRCA1 gene is located at 17q21, and encodes a protein complex, which functions as an E3

RI PT

ubiquitin ligase and tumor suppressor involved in DNA damage signaling, DNA repair (homologous recombination), chromatin remodeling, and transcriptional regulation (54).

Increased cancer risks associated with mutations in BRCA1 are primarily breast and ovarian

SC

cancer. Relative risk for PDAC is estimated to be as high as 2.55 – 2.8 as compared to the general population (55-57). However, the general consensus is that risk of PDAC is still

M AN U

relatively small, and a mutation in BRCA1 alone does not warrant PDAC surveillance.

The BRCA2 gene is located at 13q13.1 and encodes a tumor suppressor involved in homologous recombination to repair double-strand DNA breaks. Pathogenic mutations in BRCA2 are

TE D

associated with somewhat lower risks for breast and ovarian cancer than mutations in BRCA1, but higher risks for melanoma, pancreatic and PDAC. Relative risk for PDAC is estimated between 3- to 9-fold (58). Pancreatic surveillance is recommended to be limited to BRCA1/2

AC C

EP

mutation carriers with a first or second-degree relative with PDAC (52).

Familial Atypical Multiple Mole Melanoma Syndrome

Familial atypical multiple mole melanoma (FAMMM) syndrome is an autosomal dominant condition primarily caused by mutations in CDKN2A. FAMMM is characterized by increased risks for dysplastic nevus and early-onset melanoma. The CDKN2A gene encodes the p16 protein, which functions as a cell cycle regulator. Mutations in CDKN2A are also associated with

13

ACCEPTED MANUSCRIPT

increased risks for PDAC and have been identified in kindreds without melanoma. Risk for PDAC in CDKN2A mutation carriers is estimated to be 58% by 80 years with a 13- to -39-fold risk

RI PT

above the general population (52). PDAC screening is recommended for patients with a known mutation associated with FAMMM (52)

Familial Adenomatous Polyposis

SC

Familial adenomatous polyposis (FAP) syndrome is an autosomal dominant condition

M AN U

characterized by hundreds to thousands of colorectal adenomas that typically develop during adolescence at an average age of 16 years. FAP is primarily caused by mutations in the APC gene, which encodes a multi-domain protein that functions as a tumor suppressor and regulates the Wnt pathway. Colorectal cancer risk is nearly 100% without intervention, and adenomas primarily develop in the left colon. Extracolonic cancers include thyroid (2 – 3%),

TE D

duodenal (4 – 12%), and hepatic (59). One study reported a nearly 5-fold relative risk for PDAC (59). Other reports estimate this risk may be elevated to 2% above the general population risk

AC C

EP

of 1.5% (60).

Lynch Syndrome

Lynch syndrome (Hereditary nonpolyposis colorectal cancer; HNPCC) is an autosomal dominant condition and the most common hereditary colorectal cancer syndrome. It accounts for 1 – 3% of colorectal cancers and 1 – 4% of endometrial cancers in the United States. It is characterized by predominantly right-sided early-onset colorectal adenomas and carcinomas, with an average

14

ACCEPTED MANUSCRIPT

age of cancer onset of about 45 years. Lifetime risks for colorectal cancer range from 22 – 74% in MLH1 and MSH2 mutation carriers (52). Extra colorectal cancer risks include endometrial

RI PT

cancer, ovarian cancer, gastric, and PDAC. The relative risk for PDAC is 9- to 11-fold (61, 62). Pancreatic surveillance is recommended to be limited to Lynch syndrome-associated mutation

SC

carriers with a first or second-degree relative with PDAC (52).

Lynch syndrome is caused by germline mutations in mismatch repair genes (MLH1, MSH2,

M AN U

MSH6, PMS2) or from deletions in EPCAM which result in downstream promoter hypermethylation and silencing of MSH2. Pathogenic mutations impair protein expression of the MMR genes, resulting in defective DNA repair and microsatellite instability. Tumor analysis, particularly, microsatellite instability testing and immunohistochemistry may suggest or rule

TE D

out a diagnosis of Lynch syndrome. Promoter hypermethylation/BRAF analysis is appropriate for tumors that demonstrate a loss of MLH1 (52).

EP

Peutz Jeghers Syndrome

AC C

Peutz Jeghers syndrome (PJS) is an autosomal dominant condition caused by mutations in the STK11 gene. This condition is characterized by hamartomatous gastrointestinal polyps, mucocutaneous hyperpigmentation and elevated risks for gastrointestinal, breast, and ovarian cancer. Relative risks for PDAC in patients with PJS have been estimated to be as high as 132 (63). PDAC screening is recommended for patients with known mutations associated with PJS (52).

15

ACCEPTED MANUSCRIPT

Li-Fraumeni Syndrome

Li-Fraumeni syndrome is an autosomal dominant cancer predisposition syndrome that is most

RI PT

frequently caused by germline mutations in the TP53 gene. Li-Fraumeni syndrome is characterized by early onset cancer, particularly breast, osteosarcoma, soft tissue sarcoma, leukemia, brain tumors, and adrenocortical carcinoma. The relative risk for developing a PDAC

SC

is about 7-fold (64, 65).

M AN U

PALB2

Biallelic mutations in the PALB2 gene are associated with Fanconi anemia, an autosomal recessive condition characterized by chromosome instability, low blood-cell count, and increased risks for acute myeloid leukemia and squamous cell carcinoma. PALB2 interacts with

TE D

BRCA2, and monoallelic truncating mutations have been associated with familial breast cancer (66, 67). The risk for PDAC in PALB2 mutation carriers is not known. PDAC surveillance is recommended by some experts to be limited to PALB2 mutation carriers with a first or second-

AC C

ATM

EP

degree relative with pancreatic cancer (52).

Biallelic mutations in the ATM gene are associated with ataxia telangiectasia (AT), a rare autosomal recessive condition. Primary features of AT include early-onset progressive cerebellar ataxia, oculocutaneous telangiectasia, sensitivity to ionizing radiation, immunodeficiency, and a 35% risk for cancer. The ATM gene encodes a serine-protein kinases involved in the DNA damage response.

16

ACCEPTED MANUSCRIPT

Monallelic mutations in the ATM gene are associated with increased risks for breast cancer and 3-fold relative risk for PDAC (68). A recent study found that ATM mutations account for about

RI PT

2.4% of familial pancreatic cancers (69). PDAC surveillance is recommended to be limited to ATM mutation carriers with a first or second-degree relative with pancreatic cancer (52).

PALLD

SC

A risk variant in the PALLD gene was found to be associated with familial pancreatic cancer in a

M AN U

large kindred (70). PALLD encodes Palladin, a protein component of actin-containing microfilaments that may be involved in cytoskeletal organization, embryonic development, cell motility, scar formation, and neuron development (71). However, the evidence for the association of PALLD with familial pancreatic cancer is controversial and has not been identified

Hereditary Pancreatitis

TE D

in other studies (72, 73).

EP

Hereditary pancreatitis (HP) is most often caused by gain of function mutations in the PRSS1 gene and characterized by an onset of acute pancreatitis, typically in childhood. Acute

AC C

pancreatitis usually progresses to chronic pancreatitis by early adulthood. Symptoms include intense pain, steatorrhea, weight loss, gastrointestinal distress, and nutritional deficiencies. Damage to the pancreas often results in exocrine and endocrine insufficiency. HP has been found to be associated with a greater than fifty-fold increased risk of PDAC in multiple populations (17-20).

17

ACCEPTED MANUSCRIPT

PRSS1-related hereditary pancreatitis is an autosomal dominant condition. The PRSS1 gene codes for cationic trypsinogen, which is normally activated in the duodenum by enterokinase or

RI PT

previously activated trypsin, to become trypsin. Pathogenic mutations in PRSS1 result in the gain-of-function features in the trypsinogen molecule to either promote activation and/or retard degradation (16, 74). Since trypsin is the key activation molecule for most of the other

SC

zymogens, premature and sustained activation of trypsin leads to activation of other digestive enzymes, pancreatic injury from autodigestion, intense inflammation, fibrosis and atrophy.

M AN U

Further research is needed to determine why some HP families have high incidences of PDAC in the absence of obvious environmental factors (e.g. tobacco; alcohol), whereas other HP families are cancer-free. Such observations suggest the presence of risk and/or protective variants that influence the development of PDAC in families with hereditary pancreatitis. Total

TE D

pancreatectomy (75) with or without islet autotransplantation (TPIAT) (76) has been suggested a last-resort option for HP patients at high risk for PDAC . However, caution should be exercised before TPIAT because the surgery can be risky, it is irreversible, it is considered years

EP

before the likely onset of cancer, TPIAT patients often have significant motility disorders, and it

AC C

commits patients to life-long pancreatic enzyme replacement therapy (76). PDAC screening is recommended for patients with known mutations associated with HP (52).

Cystic Fibrosis

Cystic fibrosis is an autosomal recessive condition caused by mutations in the CFTR gene. CFTR encodes the cystic fibrosis transmembrane conductance protein, an epithelial anion channel. This channel conducts chloride and bicarbonate anions in the respiratory system, the sweat

18

ACCEPTED MANUSCRIPT

glands, the intestines and biliary system, but acts primarily as bicarbonate in the pancreas, male reproductive system and nasal sinuses (77-79). Given this dual role of the CFTR channel, cystic

RI PT

fibrosis can be separated into two distinct diseases depending on whether chloride conductance is impaired or preserved. The traditional form of cystic fibrosis is caused by severe biallelic CFTR (CFTRSEV) mutations, which impair chloride and bicarbonate conductance through the CFTR channel (80). A second form of cystic fibrosis is caused by mutations in CFTR that

SC

prevent the transformation of this channel into a bicarbonate-specific channel (CFTRBD) (78).

M AN U

Failure of CFTR to secret bicarbonate arises from combinations of CFTRSEV, CFTR-mild-variable mutations (CFTRM-V) or CFTRBD mutations that impair the ability of the pancreas to flush zymogens out of the pancreatic ducts and predisposes to premature trypsinogen activation, eventually leading to chronic pancreatitis.

TE D

Any combination of CFTR variants that causes chronic pancreatitis increases the risk of PDAC through the chronic inflammation mechanism. Cystic fibrosis has been associated with an increased risk for PDAC (81). However, the risk for PDAC based on CFTR genetics is still small

EP

and screening has not been recommended. With the increasing lifespan for patients with cystic

AC C

fibrosis, it is likely that greater incidence of PDAC will be identified as this population ages.

Other pancreatitis predisposing germline mutations

A growing list of genetic variants that increase susceptibility to pancreatitis or progression to the various features of chronic pancreatitis exists (82). In addition to PRSS1 and CFTR (above) significant associations exist between pancreatitis and variants in SPINK1, CTRC (80, 82-84). CASR (85-87), UBR1 (88), SBDS (89), CEL (90), CTSB, CLDN2 (91), CPA1 (92), GGT1 (93), MMP1,

19

ACCEPTED MANUSCRIPT

and MTHFR (94). Pathogenic variants in GGT1 are of particular interest since they are also significantly associated with risk of PDAC (95).

RI PT

Familial pancreatic cancer

Familial pancreatic cancer (FPC) is defined in a family by two or more first-degree relatives with pancreatic cancer in the absence of an identifiable syndrome (e.g parent-child or siblings) (51).

SC

Risks for pancreatic cancer for an individual increase with each additional affected first-degree

M AN U

relative, and PDAC screening is recommended by some experts for members of familial pancreatic cancer kindreds with a first degree relative affected with PDAC (52). One or two first degree relatives with PDAC are associated with a 4 – 7% risk for developing pancreatic cancer (96, 97). Risk for developing PDAC with three or more first degree relatives is 17 -32% (96, 97). It is important that relatives in a family with familial pancreatic cancer avoid exacerbating

TE D

environmental factors, such as smoking which increases cancer risk and decreases age of onset (98). A responsible genetic mutation has only been identified in about 20% of FPC cases.

EP

Genetic Risk in Non-familial PDAC

AC C

Unlike many other cancers, PDAC generally appears sporadically. This observation is consistent with the current concept that PDAC is a complex disorder with multiple gene-environmental interactions necessary for oncogenesis. Strong genetic risk variants are uncommon and not sufficient to cause PDAC without additional factors. In the United States about 10% of pancreatic cancer cases have at least one relative with PDAC (99), but families with multiple cases of PDAC are uncommon. In most cases, no family history has been identified. However,

20

ACCEPTED MANUSCRIPT

since no strong individual environmental factor has been identified either, it is believed that there must be some hidden genetic or epigenetic risk factors linked to cancer in apparently

RI PT

sporadic cases.

Search for genetic risk using a candidate gene approach

SC

After the discovery that mutations in PRSS1 cause HP, rapid advances in understanding genetic risk for chronic pancreatitis came by using a candidate gene approach. This has not been as

M AN U

successful in attempts to identify PDAC risk genes. Examples include exploration of the vitamin D metabolizing genes (100), Type 2 diabetes mellitus risk genes (101) and melanoma risk genes (102). This approach may be successful in the future as better insights into PDAC susceptibility

TE D

pathways emerge.

GWAS Projects to Discover Additional Genetics Risk for PDAC Several major consortiums completed GWAS projects aimed at identifying the key genetic risk

EP

factors for pancreatic adenocarcinoma. The first major finding was the confirmation that the ABO blood type was a risk factor for PDAC, with A and B being associated with risk, and O being

AC C

protective (103-107). One possible explanation is an association with inflammation linked to H pylori infections of the stomach – and an epidemiology study confirmed an association between PDAC risk and CagA-negative H pylori seropositivity in individuals with non-O blood types (OR 2.78) (108). Another possibility is that blood type ABO-B is associated with elevated serum lipase levels and inflammation associated with chronic pancreatitis (109). However, blood type only accounts for a modest change the risk of developing PDAC.

21

ACCEPTED MANUSCRIPT

The most recent publications on the GWAS approach to discovering the genetic risk factors for

RI PT

PDAC are given in Figure 4). This study combined multiple previous studies with new studies to compare 9,925 pancreatic cancer cases with 11,569 controls (110). As seen from the

Manhattan plot, about a dozen loci have been identified, including the ABO locus (chromosome

SC

9q34.2) with the greatest association. Although statistically significant, the risk of any one of these alleles is relatively small, with odds ratios ranging from 0.88 (slightly protective) to 1.26

M AN U

(slight risk). None of these sites have yet provided the critical insights into pancreatic cancer risk.

Missing Heritability

The risk of environmental toxins and pancreatitis is fairly well defined and cannot explain the

TE D

incidence of PDAC in most populations. Strong evidence of the importance of germline genetic factors is illustrated by familial cancer syndromes, as discussed above, but this only accounts for

EP

a minority of cases. GWAS studies likewise failed to provide clear evidence of the origin of the majority of risk for PDAC (Supplementary Materials). If indeed germline mutations are of

AC C

central importance, where are they hiding? As with GWA studies directed at other complex disorders, most variants identified so far in the PDAC GWAS projects have relatively small increments in risk (111). These limited successes with GWAS in complex disorders propel the question of how the 'missing' heritability can be explained. A number of explanations have been proposed, as summarized in the authoritative review by Manolio, et. Al. (111), but these have not proven to be the answer.

22

ACCEPTED MANUSCRIPT

While the answer is still missing, some insights from the genetics of chronic pancreatitis may be applicable. Chronic pancreatitis is also a complex disorder, and leading research groups

RI PT

recognize that disease in an individual patient arises from the combination of dozens and dozens of common and rare mutations (78, 80, 112, 113). These advances have not come from traditional clinical sign and symptom-defined hypothesis-null hypothesis significance testing

SC

research, dubbed the “scientific method”. Historically, the scientific method, used in the

context of Western medical research, stems from the Germ Theory of Disease, where a single

M AN U

pathogenic factor, such as a microorganism, causes a specific type of illness (114). This paradigm has been the basis of medical education, by law, since the Flexner Report of 1910 (115-117), and has strongly influenced medical taxonomy, diagnostic criteria, biomedical research approaches (based on Koch’s postulates) and thinking. The inadequacy of the Germ

TE D

Theory for cancers became increasingly recognized in the 1980s as the “two hit” hypothesis of cancer development began to emerge – in contrast to the “one hit” of the germ theory. The importance of multiple interacting “hit” models is that with additional factors, global statistical

EP

approaches fail, since no single factor has sufficient independent effect size and frequency differences between cases and controls needed to be detected using chi square or exact tests.

AC C

The fact that most non-infectious disorders are complex (more than one etiologic factor), and that intricate and elaborate gene-environment scenarios exist to mediate these disorders demands the replacement of the germ theory with a new paradigm, such as personalized or precision medicine (114). The challenges with the new paradigm of Precision Medicine are that the fundamental components of disease modeling and simulation of disease progression with

23

ACCEPTED MANUSCRIPT

or without intervention have not been taught to most physicians. Furthermore, the tools needed to apply these principles in every day cases are not typically available.

RI PT

The missing hereditability is illustrated in Figure 5. The traditional statistical approaches fall between the dotted lines, such as genetic linkage studies for families and GWAS for

populations. In chronic pancreatitis, and presumably in PDAC, a wide variety of genetic variants

SC

with highly variable minor allele frequencies underlie risk of disease and disease progression when combined with other variants. Modifier genes are an important example, as they must

M AN U

be fairly common or they would not be found in combination with susceptibility genes. Risk of progressing from recurrent acute pancreatitis to chronic pancreatitis, for example, is increased by CTRC G60G (113) and the high-risk CLDN2 haplotype (91), both with frequencies in the

TE D

general population of over 10%.

Newer approaches are emerging using next generation sequencing (NGS) which provides detailed genetic sequence for thousands of genes in one test. The information cannot be

EP

understood using statistical approaches, but insights can be gained from evaluating known genes in known pathways. Tang et al (118) recently used data from existing GWAS data sets in

AC C

a pathway analysis. Discovery that axonal guidance signaling pathways and α-adrenergic signaling genes were significantly overrepresented in pancreatic cancer provides some new risk mechanism for future research.

24

ACCEPTED MANUSCRIPT

Genetic testing for PDAC risk in Pancreatic Cancer Families Genetic testing can be considered for individuals with PDAC whose personal and/or family

RI PT

histories of cancer are concerning for a hereditary cancer predisposition. Some features that suggest the possibility of an inherited predisposition include the presence of multiple family members with the same or genetically-related cancers, early-onset cancer diagnoses,

SC

individuals with multiple primary malignancies, and individuals with rare tumors. In some

circumstances, ethnic background can increase the likelihood of a hereditary predisposition; for

M AN U

example, individuals of Ashkenazi Jewish descent have an increased likelihood of hereditary breast and ovarian cancer as the result of founder mutations in the BRCA1/2 genes.

There are several benefits of genetic testing for the patient, particularly within familial

TE D

pancreatic cancer families. Some of these families have well-described PDAC predisposing syndromes such as Hereditary Breast Ovarian Cancer, Familial Atypical Multiple Mole Melanoma (FAMMM), Lynch syndrome, Peutz-Jeghers syndrome, Familial Adenomatous

EP

Polyposis (FAP), Li-Fraumeni syndrome and Hereditary Pancreatitis as described above. Although all of these syndromes are associated with an increased risk for developing pancreatic

AC C

cancer, they are more commonly associated with other cancers, with the exception of hereditary pancreatitis (Table 3). Individuals who have been diagnosed with a hereditary cancer syndrome are provided with information about their risks to develop another cancer in the future, which can provide the opportunity for implementation of risk reductive and preventative options. Unfortunately, this benefit of genetic testing is not commonly applicable to individuals with PDAC given the current poor survival rates. Individuals with PDAC may still

25

ACCEPTED MANUSCRIPT

personally benefit from genetic testing through the potential of personalizing their treatment regimen. There is great interest in emerging therapies designed to directly target specific

RI PT

genetic pathways, and these therapies may be more effective in individuals with specific germline gene mutations. For example, a class of therapy called PARP inhibitors is being

SC

investigated in BRCA1/2 mutation carriers.

Genetic testing can also provide important information for unaffected family members.

M AN U

Genetic testing is always best initiated in an individual who has features of the syndrome in question. For those families in which a pathogenic mutation has been identified, mutationspecific (single-site) genetic testing can be offered to other family members. Single-site testing is both more affordable and easier to interpret than full gene sequencing. Family members

TE D

who are found to carry the mutation have defined cancer risks and can undergo increased surveillance and consider preventative options, whereas family members who do not carry the mutation identified in the family are not at a significantly increased risk and can follow the

EP

general population cancer screening guidelines. Enriching the population through genetic testing improves the performance of surveillance interventions by better utilizing health-care

AC C

resources for preventive and surveillance interventions on the appropriate individuals. In addition, the performance of biomarkers improves by a decrease in false positive results as a result of higher disease prevalence in gene mutation carriers.

Health care providers have the opportunity to identify appropriate candidates for genetic risk assessment. Referral to a genetic counselor can be valuable since he or she can elicit a detailed

26

ACCEPTED MANUSCRIPT

family history, provide risk assessment, coordinate testing when appropriate, and interpret test results in the context of family history. As part of the genetic assessment, the certified genetic

RI PT

counselor will address the many nuances involved with genetic testing, which include: 1) selecting the appropriate genetic test; 2) making certain the most informative family member is tested; 3) assisting the patient to ensure proper insurance coverage and clarification of out of

SC

pocket costs; 4) interpreting the results (positive or negative for a mutation) in the context of the family history; 5) Aiding the patient about how to inform family members of the test

M AN U

results; and 6) educating patients found to carry a mutation with proper cancer surveillance recommendations.

Until recently, genetic testing for PDAC has not been widely employed unless an individual met criteria for one of the previously described hereditary cancer syndromes associated with PDAC.

TE D

If genetic testing was performed, the most likely gene was evaluated, followed by step-wise analysis of other genes that could be considered based on personal and family history. This process was both time-consuming and expensive and often failed to identify a cause for the

EP

cancers in the family. The Supreme court ruling in 2013 (Association for Molecular Pathology v.

AC C

Myriad Genetics, Inc., 569 U. S. 12-398), along with technologic advances such as nextgeneration sequencing (NGS), however, have changed the landscape for genetic testing of cancer patients. It is now possible to affordably analyze multiple genes simultaneously through NGS and the dissolution of the patent on BRCA1/2 testing allows for the inclusion of these genes in multiple laboratories’ testing options. As a result, there has been an expansion in commercial laboratories offering NGS panels consisting of cancer susceptibility genes for different cancers, including PDAC.

27

ACCEPTED MANUSCRIPT

Commercially available panels for PDAC typically include sequencing and deletion/duplication

RI PT

studies of APC, ATM, BRCA1, BRCA2, CDKN2A, EPCAM, MLH1, MSH2, MSH6, PALB2, PMS2, STK11, and TP53, as well as deletion/duplication studies of EPCAM. However, caveats of gene panels include the high likelihood of identifying a variant of uncertain significance (VUS) or

SC

identifying a mutation in a gene without established guidelines or clear cancer risks. The

finding of a VUS in the absence of a pathogenic mutation (an inconclusive result) may create

M AN U

confusion and be distressing to patients.

The Role of Biomarkers in surveillance and early diagnosis of PDAC

A biomarker has been defined as a characteristic that is objectively measured and evaluated as

TE D

an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention (119). In the case of PDAC, the pathogen is “self” gone wrong.

EP

Biomarkers of cancer are therefore defined as an abnormal function or level of a “normal” biological process or biochemical. This reality poses a major problem in identifying factors that

AC C

could serve as ideal biomarkers, since biomarkers originating from normal processes cannot serve as indicators of early PDAC with high sensitivity and specificity.

The problem of choosing biomarkers is compounded in a complex disorder because multiple biomarkers may be needed. Since multiple processes must be monitored, each process will require a different biomarker as an indicator of each disease state, functional activity or

28

ACCEPTED MANUSCRIPT

surrogate endpoint. Taken together, controlling pancreatic cancer in a population faces the challenge of classifying patients according to risk and then identifying early biomarkers with

RI PT

characteristics that signal an intervention that saves lives and money.

SC

Future Directions

M AN U

Pancreatic cancer remains one of the most challenging neoplasms to anticipate, to detect, to diagnose and to effectively treat. One of the challenges is the complex nature of the disorder, as no single risk factor or early marker is strong enough to warrant clinical action. Advances will likely require the analysis of multiple risk factors and biomarkers within a single individual

TE D

tracked over time. In addition, better methods are needed to combine imaging features with biological activities, or novel approaches that are yet to be defined and developed.

As a complex disorder with poor prognosis, improvements in disease prediction will require

EP

modeling of multiple environmental, genetic, and physiologic factors. In addition to testing for

AC C

Mendelian PDAC-susceptibility syndromes, genetic testing will involve testing of multiple common risk variants that influence disease. Current panels test do not test for these common low-risk variants that influence disease course. As disease prediction modes become more complex, genetic counseling issues will continue to be central to the selection and interpretation of complex genetic scenarios in the clinical setting. This paradigm-shift will require genetics-professionals informed on current knowledge to aid physicians in the clinical applications of genetic test results.

29

ACCEPTED MANUSCRIPT

Figure 1. Percentage of new cases of PDAC by age group. (Modified from (38)) Figure 2. Pancreatic cancer – Mortality, ASR (age-standardized Rate), both sexes Source: Simple maps - (http://globocan.iarc.fr/Pages/Map.aspx#) (IARC, 150 Cours Albert Thomas, 69372 Lyon CEDEX 08, France - Tel: +33 (0)4 72 73 84 85 - Fax: +33 (0)4 72 73 85 75) -:

M AN U

SC

RI PT

Figure 3. PDAC Carcinogenesis Model. Initial risk factors include inherited genes (green box), DNA-damaging toxins and inflammation (yellow cloud). Inflammation itself can cause acinarductal reprograming. The KRASG12D mutation (yellow box) is a key early event. Continued inflammation activates KRAS and leads to loss or silencing of CDKN2A (orange box) and the reprograming becomes irreversible metaplasia. The process of oncogenesis becomes independent of external risk as a vicious cycle of progressive loss of tumor suppressor genes (purple box) and ability to repair DNA or trigger apoptosis is lost (Blue box). Eventually, PDAC “Stem Cells” emerge, with the capacity of self-replication and production of daughter clones (Red circle). Subclones acquire the capacity of invasion and metastasis (purple circle), leading to recognized features of PDAC. The dashed lines represent germline mutations that accelerate the rate of oncogenesis at various stages and pathways. CIS, carcinoma in situ. Figure 4. Manhattan plot of GWAS data from PDAC association studies. Loci previously associated with pancreatic cancer in individuals of European ancestry are shown in black, 2p13.3 is shown in blue and new loci are shown in red. The red line indicates the cutoff for statistical significance (5 × 10–8). The blue line indicates the cutoff for suggestive evidence of association (1 × 10−6) From (110).

AC C

EP

TE D

Figure 5. Relationship between of the minor allele frequency and effect size (odds ratio) in studies of complex genetic disorders. Family studies focus on highly penetrant rare alleles with large effect (blue) while GWAS focus on common alleles that tend to have small effects (pink). Thus, most statistical approaches rely on identifying associations with characteristics shown within diagonal dotted lines, but do not explain the majority of genetic risk. Here we argue that the missing hereditary for complex disorders is in the combinations of rare, low frequency and common variants of individual low-effect may interact to exert a high combined effect (arrows A and B) that cannot be easily detected by global statistics (green box). Arrow A: various combinations of low-effect alleles generate high-effect sizes through epistatic mechanisms. Arrow B: state-dependent risk alleles are down-stream of susceptibility factors and accelerate disease progression. Some common alleles may have strong effects in specific contexts, such as modifier variants. (Figure modified from (111))

30

ACCEPTED MANUSCRIPT

References: Yeo TP. Demographics, Epidemiology, and Inheritance of Pancreatic Ductal Adenocarcinoma. Seminars in oncology. 2015;42(1):8-18.

2.

Andersen DK, Andren-Sandberg A, Duell EJ, Goggins M, Korc M, Petersen GM, et al. Pancreatitis-diabetes-pancreatic cancer: summary of an NIDDK-NCI workshop. Pancreas. 2013;42(8):1227-37.

3.

Solomon S, Das S, Brand R, Whitcomb DC. Inherited pancreatic cancer syndromes. Cancer journal. 2012;18(6):485-91.

4.

Brand RE, Lynch HT. Hereditary pancreatic adenocarcinoma. A clinical perspective. The Medical clinics of North America. 2000;84(3):665-75.

5.

Permuth-Wey J, Egan KM. Family history is a significant risk factor for pancreatic cancer: results from a systematic review and meta-analysis. Fam Cancer. 2009;8(2):109-17.

6.

Yachida S, Jones S, Bozic I, Antal T, Leary R, Fu B, et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature. 2010;467(7319):1114-7.

7.

Yachida S, Iacobuzio-Donahue CA. Evolution and dynamics of pancreatic cancer progression. Oncogene. 2013;32(45):5253-60.

8.

Lindor NM, Greene MH. The concise handbook of family cancer syndromes. Mayo Familial Cancer Program. Journal of the National Cancer Institute. 1998;90(14):1039-71.

9.

Couch FJ, Johnson MR, Rabe KG, Brune K, de Andrade M, Goggins M, et al. The prevalence of BRCA2 mutations in familial pancreatic cancer. Cancer Epidemiol Biomarkers Prev. 2007;16(2):342-6.

10.

Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al. Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 Lyon, France: International Agency for Research on Cancer (IARC); 2014 [cited 2015 June 12]. Available from: http://globocan.iarc.fr.

SC

M AN U

TE D

EP

AC C

11.

RI PT

1.

Iodice S, Gandini S, Maisonneuve P, Lowenfels AB. Tobacco and the risk of pancreatic cancer: a review and meta-analysis. Langenbeck's archives of surgery / Deutsche Gesellschaft fur Chirurgie. 2008;393(4):535-45.

12.

Lucenteforte E, La Vecchia C, Silverman D, Petersen GM, Bracci PM, Ji BT, et al. Alcohol consumption and pancreatic cancer: a pooled analysis in the International Pancreatic Cancer Case-Control Consortium (PanC4). Ann Oncol. 2012;23(2):374-82.

13.

Andreotti G, Silverman DT. Occupational risk factors and pancreatic cancer: a review of recent findings. Mol Carcinog. 2012;51(1):98-108.

31

ACCEPTED MANUSCRIPT

Risch HA. Pancreatic cancer: Helicobacter pylori colonization, N-nitrosamine exposures, and ABO blood group. Mol Carcinog. 2012;51(1):109-18.

15.

Whitcomb DC, Gorry MC, Preston RA, Furey W, Sossenheimer MJ, Ulrich CD, et al. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nature Genetics. 1996;14(2):141-5.

16.

Gorry MC, Gabbaizedeh D, Furey W, Gates LK, Jr., Preston RA, Aston CE, et al. Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis. Gastroenterology. 1997;113(4):1063-8.

17.

Lowenfels A, Maisonneuve P, DiMagno E, Elitsur Y, Gates L, Perrault J, et al. Hereditary pancreatitis and the risk of pancreatic cancer. Journal of the National Cancer Institute. 1997;89(6):442-6.

18.

Howes N, Lerch MM, Greenhalf W, Stocken DD, Ellis I, Simon P, et al. Clinical and genetic characteristics of hereditary pancreatitis in Europe. Clin Gastroenterol Hepatol. 2004;2(3):252-61.

19.

Rebours V, Boutron-Ruault MC, Jooste V, Bouvier AM, Hammel P, Ruszniewski P, et al. Mortality rate and risk factors in patients with hereditary pancreatitis: uni- and multidimensional analyses. Am J Gastroenterol. 2009;104(9):2312-7.

20.

Lowenfels AB, Maisonneuve P, Whitcomb DC, Lerch MM, DiMagno EP. Cigarette smoking as a risk factor for pancreatic cancer in patients with hereditary pancreatitis. Journal of the American Medical Association. 2001;286(2):169-70.

21.

Raimondi S, Lowenfels AB, Morselli-Labate AM, Maisonneuve P, Pezzilli R. Pancreatic cancer in chronic pancreatitis; aetiology, incidence, and early detection. Best Pract Res Clin Gastroenterol 2010;24(3):349-58.

22.

Hengstler JG, Bauer A, Wolf HK, Bulitta CJ, Tanner B, Oesch F, et al. Mutation analysis of the cationic trypsinogen gene in patients with pancreatic cancer. Anticancer Res. 2000;20(5A):2967-74.

24.

25.

SC

M AN U

TE D

EP

AC C

23.

RI PT

14.

Greer JB, Whitcomb DC. Inflammation and pancreatic cancer: an evidence-based review. Curr Opin Pharmacol. 2009;9(4):411-8. Rebours V, Levy P, Mosnier JF, Scoazec JY, Soubeyrand MS, Flejou JF, et al. Pathology Analysis Reveals That Dysplastic Pancreatic Ductal Lesions Are Frequent in Patients With Hereditary Pancreatitis. Clin Gastroenterol Hepatol. 2009. Lowenfels AB, Maisonneuve P, Cavallini G, Ammann RW, Lankisch PG, Andersen JR, et al. Pancreatitis and the risk of pancreatic cancer. International Pancreatitis Study Group. New England Journal of Medicine. 1993;328(20):1433-7.

32

ACCEPTED MANUSCRIPT

Chari ST, Mohan V, Pitchumoni CS, Viswanathan M, Madanagopalan N, Lowenfels AB. Risk of pancratic carcinoma in tropical calcifying pancreatitits: an epidemicologic study. Pancreas. 1994;9:62-6.

27.

Neglia JP, FitzSimmons SC, Maisonneuve P, Schoni MH, Schoni-Affolter F, Corey M, et al. The risk of cancer among patients with cystic fibrosis. Cystic Fibrosis and Cancer Study Group. N Engl J Med. 1995;332(8):494-9.

28.

Yadav D, Lowenfels AB. The epidemiology of pancreatitis and pancreatic cancer. Gastroenterology. 2013;144(6):1252-61.

29.

Duell EJ, Lucenteforte E, Olson SH, Bracci PM, Li D, Risch HA, et al. Pancreatitis and pancreatic cancer risk: a pooled analysis in the International Pancreatic Cancer CaseControl Consortium (PanC4). Ann Oncol. 2012;23(11):2964-70.

30.

Tong GX, Geng QQ, Chai J, Cheng J, Chen PL, Liang H, et al. Association between pancreatitis and subsequent risk of pancreatic cancer: a systematic review of epidemiological studies. Asian Pacific journal of cancer prevention : APJCP. 2014;15(12):5029-34.

31.

Daniluk J, Liu Y, Deng D, Chu J, Huang H, Gaiser S, et al. An NF-kappaB pathwaymediated positive feedback loop amplifies Ras activity to pathological levels in mice. The Journal of clinical investigation. 2012;122(4):1519-28.

32.

di Magliano MP, Logsdon CD. Roles for KRAS in pancreatic tumor development and progression. Gastroenterology. 2013;144(6):1220-9.

33.

Sarner M, Cotton PB. Classification of pancreatitis. Gut. 1984;25(7):756-9.

34.

Philip B, Roland CL, Daniluk J, Liu Y, Chatterjee D, Gomez SB, et al. A high-fat diet activates oncogenic Kras and COX2 to induce development of pancreatic ductal adenocarcinoma in mice. Gastroenterology. 2013;145(6):1449-58.

35.

Bansal P, Sonnenberg A. Pancreatitis is a risk factor for pancreatic cancer. Gastroenterology. 1995;109:247-51.

37.

38.

SC

M AN U

TE D

EP

AC C

36.

RI PT

26.

Fernandez E, La Vecchia C, Porta M, Negri E, D'Avanzo B, Boyle P. Pancreatitis and the risk of pancreatic cancer. Pancreas. 1995;11:185-9. Balakrishnan V, Unnikrishnan AG, Thomas V, Choudhuri G, Veeraraju P, Singh SP, et al. Chronic pancreatitis. A prospective nationwide study of 1,086 subjects from India. JOP. 2008;9(5):593-600.

Anonymous. SEER Stat Fact Sheets: Pancreas Cancer: National Cancer Institute; 2015 [cited 2015 June 28, 2015]. Available from: http://seer.cancer.gov/statfacts/html/pancreas.html

33

ACCEPTED MANUSCRIPT

Hruban RH, Adsay NV, Albores-Saavedra J, Compton C, Garrett ES, Goodman SN, et al. Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions. The American journal of surgical pathology. 2001;25(5):579-86.

40.

Maitra A, Hruban RH. Pancreatic cancer. Annual review of pathology. 2008;3:157-88.

41.

Habbe N, Shi G, Meguid RA, Fendrich V, Esni F, Chen H, et al. Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice. Proc Natl Acad Sci U S A. 2008;105(48):18913-8.

42.

Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Angenendt P, et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 2008;321(5897):1801-6.

43.

Waddell N, Pajic M, Patch AM, Chang DK, Kassahn KS, Bailey P, et al. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature. 2015;518(7540):495501.

44.

Grant RC, Selander I, Connor AA, Selvarajah S, Borgida A, Briollais L, et al. Prevalence of germline mutations in cancer predisposition genes in patients with pancreatic cancer. Gastroenterology. 2015;148(3):556-64.

45.

Humphris J, Chang DK, Biankin AV. Inherited susceptibility to pancreatic cancer in the era of next-generation sequencing. Gastroenterology. 2015;148(3):496-8.

46.

Mafficini A, Amato E, Fassan M, Simbolo M, Antonello D, Vicentini C, et al. Reporting tumor molecular heterogeneity in histopathological diagnosis. PLoS One. 2014;9(8):e104979.

47.

Kiraly O, Gong G, Olipitz W, Muthupalani S, Engelward BP. Inflammation-induced cell proliferation potentiates DNA damage-induced mutations in vivo. PLoS Genet. 2015;11(2):e1004901.

48.

Rhim AD, Mirek ET, Aiello NM, Maitra A, Bailey JM, McAllister F, et al. EMT and dissemination precede pancreatic tumor formation. Cell. 2012;148(1-2):349-61.

50.

SC

M AN U

TE D

EP

AC C

49.

RI PT

39.

Ling J, Kang Y, Zhao R, Xia Q, Lee DF, Chang Z, et al. KrasG12D-induced IKK2/beta/NFkappaB activation by IL-1alpha and p62 feedforward loops is required for development of pancreatic ductal adenocarcinoma. Cancer Cell. 2012;21(1):105-20. Huang H, Liu Y, Daniluk J, Gaiser S, Chu J, Wang H, et al. Activation of nuclear factorkappaB in acinar cells increases the severity of pancreatitis in mice. Gastroenterology. 2013;144(1):202-10.

34

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

35

ACCEPTED MANUSCRIPT

Table 1. Overview of Pancreatic Cancer What is pancreatic cancer?

RI PT

Malignant Neuorendocrine Mesenchymal Lymphomas Secondary

e.g. serous cystadenoma e.g PanIN, mucinous cystic neoplasm (MCN), intraductal papillary mucinous neoplasm (IPMN) e.g. pancreatic ductal adenocarcinoma (PDAC) e.g. Carcinoid, gastrinoma, glucagonoma e.g. lypoma

SC

Classification Epithelial tumors Benign Premalignant

Higher risk:

M AN U

Most common form is pancreatic ductal adenocarcinoma (PDAC) PDAC is difficult to detect early, resistant to treatment and has a 28% one year survival rate 1.5% of Americans will be diagnosed with pancreatic cancer in their lifetime Who is at risk for pancreatic cancer? (Risk factors)

Individuals with a strong family history and/or a germline mutation Individuals who are older in age

Smokers Lower risk:

Alcoholics Obese

EP

Diabetes mellitus

TE D

Chronic pancreatitis

When does pancreatic cancer occur?

PDAC is most frequently diagnosed among individuals 65-74 years

AC C

The median age of diagnosis is 71 years Why does pancreatic cancer occur in the pancreas? In mice, pancreas-targeting promoters direct cancer-generating mutations to the pancreas

Pancreatitis may trigger the oncogenic process in susceptible individuals How does pancreatic cancer develop? An activating KRASD12G mutation is necessary, but not sufficient to case PDAC Triggers (e.g. inflammation) may initiate the pathogenic process Toxins and inflammation may cause DNA damage that is inadequately repaired.

Somatic cancer mutations accumulate in pancreatic cells (CDKN2A, TP53, SMAD3, BRCA2)

ACCEPTED MANUSCRIPT

Table 2: Modifying Risk Factors for Pancreatic Cancer. Relative Risk ~2

Risk modifying interactions Alcohol, recent-onset diabetes, family history

RI PT

Factor Tobacco

Minor alleles for XRCC2 (120), CAPN10, (121) and EPHX1 (122); At least one A allele in CTLA-4 (123)

SC

In women, variants in CYP1A2 and NAT1 (124) Protective: XPD N312N (125) 1.02 – 1.14 per 5 unit increase in BMI

IGF-1 variant and BMI ≥ 25 (126)

M AN U

Obesity

FTO or ADIPOQ variants and BMI ≥ 25 (127) Protective: FTO or ADIPOQ variants and BMI < 25 (127)

Diet: Fruits, vegetables, whole grains

Increased

AC C

EP

TE D

Red and processed meat, fructose, saturated fatty acids

Decreased

Alcohol

No minor CAT alleles for rs12807961 and high grain intake (128)

≥ 1 minor GAA allele (rs3816257) and low intake of deep-yellow vegetables or other starches (128) SOD2 1221 G>A and low vitamin E intake (129) In men: NAT1 slow metabolizers and high dietary mutagen intake of 2-amino-1methyl-6-phenylimidazo[4,5-b]pyrmidine and benzo[a]pyrene (124) Protective: overexpression of SOD2, SOD2 1221 G>A and high vitamin E intake,

1.22 – 1.38

ADH1B*1 and CTLA-4 49G>A (130)

IGF2R L252V and IRS1 A512P (131)

Complex disorders involve risk potentiation through gene-environment interactions. Multiple modifiable risk factors are linked to non-modifiable germline genetic risk (right column) that potential the relative risk of the environmental factor. Adapted from Jansen et al (132).

ACCEPTED MANUSCRIPT

Gene

Syndrome

Familial Adenomatous Polyposis (FAP) Lynch syndrome

Peutz-Jeghers Syndrome

2-fold(57)

BRCA1

3 to 9-fold(139, 140)

BRCA2

5-fold(59)

9 to 11-fold(61, 62)

Up to 132-fold (63)

7-fold (65)

0 to 20% ((136-138))

AC C

Hereditary Pancreatitis

ATM carrier (Ataxia Telangiectasia)

MajorAssociated Cancers

Melanoma

0 to 6% ((9, 44, 137))

Breast

0 to 6% ((9, 44, 137))

Ovary

APC

Unknown

Colon

MLH1

<1% ((44))

Colon

MSH2

<1% ((44))

Endometrial

MSH6

<1% ((44))

PMS2

Unknown

STK11/LKB1

0% (138)

p53

GI Breast

<1% (44)

EP

Li-Fraumeni syndrome

CDKN2A

M AN U

Familial Breast and Ovarian

13 to 39-fold(133135)

TE D

Familial Atypical Multiple Mole Melanoma (FAMMM)

Yield of testing in FPC kindreds who do not meet criteria for known syndromes

SC

Relative Risk of PC

RI PT

Table 3. Hereditary Syndromes Associated with Pancreatic Adenocarcinoma

Sarcomas Breast Brain Adrenocortical

53 to 70-fold (17, 21)

PRSS1

Unknown

Pancreas

3-fold (68)

ATM

1 to 2.4% (44, 69)

Breast Colon Pancreas

PALB2 carrier (Fanconi Anemia)

Unknown

PALB2

0 to 5% (137, 138, 141)

Breast Pancreas

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

Figure 1. Percentage of new cases of PDAC by age group. (Modified from (38))

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 2. Pancreatic cancer – Mortality, ASR (age-standardized Rate), both sexes

AC C

EP

TE D

Source: Simple maps - (http://globocan.iarc.fr/Pages/Map.aspx#) (IARC, 150 Cours Albert Thomas, 69372 Lyon CEDEX 08, France - Tel: +33 (0)4 72 73 84 85 - Fax: +33 (0)4 72 73 85 75) -:

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Figure 3. PDAC Carcinogenesis Model. Initial risk factors include inherited genes (green box), DNA-damaging toxins and inflammation (yellow cloud). Inflammation itself can cause acinarductal reprograming. The KRASG12D mutation (yellow box) is a key early event. Continued inflammation activates KRAS and leads to loss or silencing of CDKN2A (orange box) and the reprograming becomes irreversible metaplasia. The process of oncogenesis becomes independent of external risk as a vicious cycle of progressive loss of tumor suppressor genes (purple box) and ability to repair DNA or trigger apoptosis is lost (Blue box). Eventually, PDAC “Stem Cells” emerge, with the capacity of self-replication and production of daughter clones (Red circle). Subclones acquire the capacity of invasion and metastasis (purple circle), leading to recognized features of PDAC. The dashed lines represent germline mutations that accelerate the rate of oncogenesis at various stages and pathways. CIS, carcinoma in situ.

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

Figure 4. Manhattan plot of GWAS data from PDAC association studies. Loci previously associated with pancreatic cancer in individuals of European ancestry are shown in black, 2p13.3 is shown in blue and new loci are shown in red. The red line indicates the cutoff for statistical significance (5 × 10–8). The blue line indicates the cutoff for suggestive evidence of association (1 × 10−6) From (110).

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

Figure 5. Relationship between of the minor allele frequency and effect size (odds ratio) in studies of complex genetic disorders. Family studies focus on highly penetrant rare alleles with large effect (blue) while GWAS focus on common alleles that tend to have small effects (pink). Thus, most statistical approaches rely on identifying associations with characteristics shown within diagonal dotted lines, but do not explain the majority of genetic risk. Here we argue that the missing hereditary for complex disorders is in the combinations of rare, low frequency and common variants of individual low-effect may interact to exert a high combined effect (arrows A and B) that cannot be easily detected by global statistics (green box). Arrow A: various combinations of low-effect alleles generate high-effect sizes through epistatic mechanisms. Arrow B: state-dependent risk alleles are down-stream of susceptibility factors and accelerate disease progression. Some common alleles may have strong effects in specific contexts, such as modifier variants. (Figure modified from (111))

ACCEPTED MANUSCRIPT

Supplemental materials

Genetically Engineered Mice (GEMs)

RI PT

Strong experimental evidence to complement molecular pathology in defining the mechanism of commonly mutated genes in human PDAC comes from studies in GEM (6, 42, 43, 142-146). Continual efforts to develop and refine animal models now provide a clear picture of the

SC

fundamentals of pancreatic cancer development and progression.

M AN U

Early animal models of PDAC using toxins had limited success. An exception was the Syrian hamster following injection with N -Nitrosobis(2-oxopropyl)amine (BOP) (147). This model did develop many of the features and genetic variants seen in humans, but manipulation of the

TE D

system in hamsters is limited and BOP treatment does not generate PDAC in mice.

The first genetically engineered mouse linked the activating mutation of KRAS pG12D

EP

(KRASG12D) with an elastase 1 promoter (142, 148). Although PanIN lesions and PDAC developed, the GEM was limited because constitutively active KRAS did not reflect the human

AC C

condition (142). A “second generation” of GEMs added conditional activation of the pathogenic KRASG12D using a “Cre-lox” activation system (143, 145). This highly versatile GEM became the primary tool to study PDAC oncogenesis for a decade. The key variant was KRASG12D with additional genetic variants altering the rate of progression to pancreatic cancer and other phenotypic variants. But once KRASG12D expression was turned on, it was impossible to turn the oncogene off. The “third generation” of GEMs have inducible-KRASG12D, allowing it

ACCEPTED MANUSCRIPT

to be turned on and off as needed using a doxycycline-dependent transgene (149, 150). The model demonstrated that KRASG12D expression in adult mice with addition of doxycycline can

RI PT

cause PanINs and PDAC development. More importantly, withdrawal of doxycycline results in disappearance of PanIN lesions and even regression of metastatic cancers in these GEMs (142).

SC

GEMs also provide insights into the link between inflammation and PDAC oncogenesis. Cells with KRASG12D mutations generally behave normally until the KRAS molecule is activated by

M AN U

persistent inflammation as occurs with repeated cerulein injections (32). Active KRASG12D drives further inflammation through the NFκB pathway, as well as other downstream targets to generated a feed-forward loop of continued inflammation, inhibited apoptosis, altered metabolism, cell proliferation and other pro-neoplastic events (31, 41, 49, 150-152). Studies of

TE D

inflammation in GEMs have also provided insights into acinar-to-ductal reprograming / transdifferentiation (153), acinar-ductal metaplasia and epithelial to mesenchymal transformation (48), a possible role for sensory nerves and neuroinflammation (154), and

AC C

GWAS

EP

increased susceptibility to DNA damage during inflammation-induced cell proliferation (47).

A genome wide association study (GWAS) is a statistical approach to discovering germline alleles associated with disease. The approach is to identify patients with well-defined disease phenotypes (cases) and collect DNA from these patients and unaffected individuals (controls) from the same population. Cases and controls are genotyped for hundreds of thousands to millions of single nucleotide polymorphisms (SNPs) / variants (SNVs) across the genome at regular intervals. Particular SNPS are selected because they define sections of DNA with groups

ACCEPTED MANUSCRIPT

of SNPs that tend to be inherited together to define haplotypes (e.g. tag SNP is linked to an allele where the other SNPs in the section are not inherited randomly and are therefore in

RI PT

disequilibrium). Other SNPs are common (e.g. >1% of the population) and in the coding region of the DNA such as exons, and these SNPs are predicted to alter the protein coded by the gene. The frequency of the common (major) and less common (minor) variant allele at each location

SC

in each person is measured, and the differences between the expected frequency of the minor allele in the cases is compared with the controls. Locations of SNPs that show a difference

M AN U

favoring risk for the disease phenotype are considered causative quantitative trait loci (QTL). The probability that any difference in allele frequency is caused by random selection of the cases or controls is calculated as a p value. When the exponent of the p value is recorded (e.g. a p value of 1 x 10-5 is a log of the odds (LOD of 5), and graphed according to the location on the

TE D

genome from the beginning of chromosome 1 to the end of chromosome 23, the result is a Manhattan plot. However, with so many tests being performed, there is a high statistical chance for a difference in risk caused by chance, leading to false discovery that a genetic locus

EP

contains a disease-associated mutation. To guard against this possibility several approaches are commonly taken. First, the p value needed to generate genome-wide significance is set at

AC C

1x10-8 or more, with a suggestive association with a p value of 1X10-6 or more. To obtain such a high p value when the minor allele is uncommon (e.g. 5% of alleles) and not the only causative factor requires thousands of cases and controls. Secondly, to further guard against false discovery, a second GWAS should be performed in a similar population. If they are similar, the results can be combined. Thus, GWAS projects are massive, expensive and often challenging.

ACCEPTED MANUSCRIPT

15-00702 References cont. (online-only)

Brand RE, Lerch MM, Rubinstein WS, Neoptolemos JP, Whitcomb DC, Hruban RH, et al. Advances in counselling and surveillance of patients at risk for pancreatic cancer. Gut. 2007;56(10):1460-9.

52.

Syngal S, Brand RE, Church JM, Giardiello FM, Hampel HL, Burt RW, et al. ACG clinical guideline: Genetic testing and management of hereditary gastrointestinal cancer syndromes. The American journal of gastroenterology. 2015;110(2):223-62; quiz 63.

53.

Chen S, Parmigiani G. Meta-analysis of BRCA1 and BRCA2 penetrance. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2007;25(11):1329-33.

54.

Boulton SJ. Cellular functions of the BRCA tumour-suppressor proteins. Biochemical Society transactions. 2006;34(Pt 5):633-45.

55.

Brose MS, Rebbeck TR, Calzone KA, Stopfer JE, Nathanson KL, Weber BL. Cancer risk estimates for BRCA1 mutation carriers identified in a risk evaluation program. Journal of the National Cancer Institute. 2002;94(18):1365-72.

56.

Iqbal J, Ragone A, Lubinski J, Lynch HT, Moller P, Ghadirian P, et al. The incidence of pancreatic cancer in BRCA1 and BRCA2 mutation carriers. British journal of cancer. 2012;107(12):2005-9.

57.

Thompson D, Easton DF, Breast Cancer Linkage C. Cancer Incidence in BRCA1 mutation carriers. Journal of the National Cancer Institute. 2002;94(18):1358-65.

58.

Breast Cancer Linkage C. Cancer risks in BRCA2 mutation carriers. Journal of the National Cancer Institute. 1999;91(15):1310-6.

59.

Giardiello FM, Offerhaus GJ, Lee DH, Krush AJ, Tersmette AC, Booker SV, et al. Increased risk of thyroid and pancreatic carcinoma in familial adenomatous polyposis. Gut. 1993;34(10):1394-6.

61.

62.

SC

M AN U

TE D

EP

AC C

60.

RI PT

51.

Jasperson KW, Tuohy TM, Neklason DW, Burt RW. Hereditary and familial colon cancer. Gastroenterology. 2010;138(6):2044-58. Win AK, Young JP, Lindor NM, Tucker KM, Ahnen DJ, Young GP, et al. Colorectal and other cancer risks for carriers and noncarriers from families with a DNA mismatch repair gene mutation: a prospective cohort study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2012;30(9):958-64. Kastrinos F, Mukherjee B, Tayob N, Wang F, Sparr J, Raymond VM, et al. Risk of pancreatic cancer in families with Lynch syndrome. JAMA : the journal of the American Medical Association. 2009;302(16):1790-5.

ACCEPTED MANUSCRIPT

Giardiello FM, Brensinger JD, Tersmette AC, Goodman SN, Petersen GM, Booker SV, et al. Very high risk of cancer in familial Peutz-Jeghers syndrome. Gastroenterology. 2000;119(6):1447-53.

64.

Syngal S, Brand RE, Church JM, Giardiello FM, Hampel HL, Burt RW. ACG Clinical Guideline: Genetic Testing and Management of Hereditary Gastrointestinal Cancer Syndromes. Am J Gastroenterol. 2015;110(2):223-62.

65.

Ruijs MW, Verhoef S, Rookus MA, Pruntel R, van der Hout AH, Hogervorst FB, et al. TP53 germline mutation testing in 180 families suspected of Li-Fraumeni syndrome: mutation detection rate and relative frequency of cancers in different familial phenotypes. J Med Genet. 2010;47(6):421-8.

66.

Fernandes PH, Saam J, Peterson J, Hughes E, Kaldate R, Cummings S, et al. Comprehensive sequencing of PALB2 in patients with breast cancer suggests PALB2 mutations explain a subset of hereditary breast cancer. Cancer. 2014;120(7):963-7.

67.

Antoniou AC, Casadei S, Heikkinen T, Barrowdale D, Pylkas K, Roberts J, et al. Breastcancer risk in families with mutations in PALB2. N Engl J Med. 2014;371(6):497-506.

68.

Geoffroy-Perez B, Janin N, Ossian K, Lauge A, Croquette MF, Griscelli C, et al. Cancer risk in heterozygotes for ataxia-telangiectasia. International journal of cancer Journal international du cancer. 2001;93(2):288-93.

69.

Roberts NJ, Jiao Y, Yu J, Kopelovich L, Petersen GM, Bondy ML, et al. ATM mutations in patients with hereditary pancreatic cancer. Cancer discovery. 2012;2(1):41-6.

70.

Pogue-Geile KL, Chen R, Bronner MP, Crnogorac-Jurcevic T, Moyes KW, Dowen S, et al. Palladin mutation causes familial pancreatic cancer and suggests a new cancer mechanism. PLoS Med. 2006;3(12):e516.

71.

Otey CA, Rachlin A, Moza M, Arneman D, Carpen O. The palladin/myotilin/myopalladin family of actin-associated scaffolds. International review of cytology. 2005;246:31-58.

72.

Slater E, Amrillaeva V, Fendrich V, Bartsch D, Earl J, Vitone LJ, et al. Palladin mutation causes familial pancreatic cancer: absence in European families. PLoS medicine. 2007;4(4):e164.

74.

SC

M AN U

TE D

EP

AC C

73.

RI PT

63.

Klein AP, Borges M, Griffith M, Brune K, Hong SM, Omura N, et al. Absence of deleterious palladin mutations in patients with familial pancreatic cancer. Cancer Epidemiol Biomarkers Prev. 2009;18(4):1328-30. Mounzer R, Whitcomb DC. Genetics of acute and chronic pancreatitis. Curr Opin Gastroenterol. 2013;29(5):544-51.

ACCEPTED MANUSCRIPT

Ulrich II CD. Pancreatic cancer in hereditary pancreatitis – Consensus guidelines for prevention, screening, and treatment. Pancreatology. 2001;1(5):416-22.

76.

Bellin MD, Freeman ML, Gelrud A, Slivka A, Clavel A, Humar A, et al. Total pancreatectomy and islet autotransplantation in chronic pancreatitis: Recommendations from PancreasFest. Pancreatology. 2014;14(1):27-35.

77.

Schneider A, Larusch J, Sun X, Aloe A, Lamb J, Hawes R, et al. Combined Bicarbonate Conductance-Impairing Variants in CFTR and SPINK1 Variants Are Associated With Chronic Pancreatitis in Patients Without Cystic Fibrosis. Gastroenterology. 2011;140(1):162-71.

78.

LaRusch J, Jung J, General IJ, Lewis MD, Park HW, Brand RE, et al. Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis. PLoS Genetics. 2014;10(7):e1004376.

79.

LaRusch J, Jung J, General IJ, Lewis MD, Park HW, Brand RE, et al. Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis. PLoS genetics. 2014;10(7):e1004376.

80.

Rosendahl J, Landt O, Bernadova J, Kovacs P, Teich N, Bodeker H, et al. CFTR, SPINK1, CTRC and PRSS1 variants in chronic pancreatitis: is the role of mutated CFTR overestimated? Gut. 2013;62(4):582-92.

81.

Maisonneuve P, Marshall BC, Lowenfels AB. Risk of pancreatic cancer in patients with cystic fibrosis. Gut. 2007;56(9):1327-8.

82.

Whitcomb DC. Genetic risk factors for pancreatic disorders. Gastroenterology. 2013;144(6):1292-302.

83.

Chen JM, Ferec C. Genetics and pathogenesis of chronic pancreatitis: the 2012 update. Clinics and research in hepatology and gastroenterology. 2012;36(4):334-40.

84.

Chen JM, Ferec C. Chronic pancreatitis: genetics and pathogenesis. Annu Rev Genomics Hum Genet. 2009;10:63-87.

SC

M AN U

TE D

EP

AC C

85.

RI PT

75.

Felderbauer P, Hoffmann P, Einwachter H, Bulut K, Ansorge N, Schmitz F, et al. A novel mutation of the calcium sensing receptor gene is associated with chronic pancreatitis in a family with heterozygous SPINK1 mutations. BMC Gastroenterol. 2003;3(1):34.

86.

Felderbauer P, Karakas E, Fendrich V, Bulut K, Werner I, Dekomien G, et al. Pancreatitis in primary hyperparathyroidism-related hypercalcaemia is not associated with mutations in the CASR gene. Exp Clin Endocrinol Diabetes. 2007;115(8):527-9.

87.

Muddana V, Lamb J, Greer JB, Elinoff B, Hawes RH, Cotton PB, et al. Association between calcium sensing receptor gene polymorphisms and chronic pancreatitis in a US

ACCEPTED MANUSCRIPT

population: Role of serine protease inhibitor Kazal 1type and alcohol. World J Gastroenterol. 2008;14(28):4486-91. Zenker M, Mayerle J, Lerch MM, Tagariello A, Zerres K, Durie PR, et al. Deficiency of UBR1, a ubiquitin ligase of the N-end rule pathway, causes pancreatic dysfunction, malformations and mental retardation (Johanson-Blizzard syndrome). Nat Genet. 2005;37(12):1345-50.

89.

Boocock GR, Morrison JA, Popovic M, Richards N, Ellis L, Durie PR, et al. Mutations in SBDS are associated with Shwachman-Diamond syndrome. Nat Genet. 2003;33(1):97101.

90.

Raeder H, Johansson S, Holm PI, Haldorsen IS, Mas E, Sbarra V, et al. Mutations in the CEL VNTR cause a syndrome of diabetes and pancreatic exocrine dysfunction. Nat Genet. 2006;38(1):54-62.

91.

Whitcomb DC, Larusch J, Krasinskas AM, Klei L, Smith JP, Brand RE, et al. Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis. Nature genetics. 2012;44(12):1349-54.

92.

Witt H, Beer S, Rosendahl J, Chen JM, Chandak GR, Masamune A, et al. Variants in CPA1 are strongly associated with early onset chronic pancreatitis. Nat Genet. 2013;45(10):1216-20.

93.

Brand H, Diergaarde B, O'Connell MR, Whitcomb DC, Brand RE. Variation in the gammaglutamyltransferase 1 gene and risk of chronic pancreatitis. Pancreas. 2013;42(5):83640.

94.

Shelton CA, Whitcomb DC. Genetics and treatment options for recurrent acute and chronic pancreatitis. Current treatment options in gastroenterology. 2014;12(3):359-71.

95.

Diergaarde B, Brand R, Lamb J, Cheong SY, Stello K, Barmada MM, et al. Pooling-Based Genome-Wide Association Study Implicates Gamma-Glutamyltransferase 1 (GGT1) Gene in Pancreatic Carcinogenesis. Pancreatology. 2010;10(2-3):194-200.

97.

98.

SC

M AN U

TE D

EP

AC C

96.

RI PT

88.

Brune KA, Lau B, Palmisano E, Canto M, Goggins MG, Hruban RH, et al. Importance of age of onset in pancreatic cancer kindreds. Journal of the National Cancer Institute. 2010;102(2):119-26. Klein AP, Brune KA, Petersen GM, Goggins M, Tersmette AC, Offerhaus GJ, et al. Prospective risk of pancreatic cancer in familial pancreatic cancer kindreds. Cancer research. 2004;64(7):2634-8. Brand RE, Greer JB, Zolotarevsky E, Brand R, Du H, Simeone D, et al. Pancreatic cancer patients who smoke and drink are diagnosed at younger ages. Clinical gastroenterology

ACCEPTED MANUSCRIPT

and hepatology : the official clinical practice journal of the American Gastroenterological Association. 2009;7(9):1007-12. Schenk M, Schwartz AG, O'Neal E, Kinnard M, Greenson JK, Fryzek JP, et al. Familial risk of pancreatic cancer. J Natl Cancer Inst. 2001;93(8):640-4.

100.

Arem H, Yu K, Xiong X, Moy K, Freedman ND, Mayne ST, et al. Vitamin D metabolic pathway genes and pancreatic cancer risk. PLoS One. 2015;10(3):e0117574.

101.

Wu L, Rabe KG, Petersen GM. Do variants associated with susceptibility to pancreatic cancer and type 2 diabetes reciprocally affect risk? PLoS One. 2015;10(2):e0117230.

102.

Wu L, Goldstein AM, Yu K, Yang XR, Rabe KG, Arslan AA, et al. Variants associated with susceptibility to pancreatic cancer and melanoma do not reciprocally affect risk. Cancer Epidemiol Biomarkers Prev. 2014;23(6):1121-4.

103.

Amundadottir L, Kraft P, Stolzenberg-Solomon RZ, Fuchs CS, Petersen GM, Arslan AA, et al. Genome-wide association study identifies variants in the ABO locus associated with susceptibility to pancreatic cancer. Nat Genet. 2009;41(9):986-90.

104.

Greer JB, Yazer MH, Raval JS, Barmada MM, Brand RE, Whitcomb DC. Significant association between ABO blood group and pancreatic cancer. World J Gastroenterol. 2010;16(44):5588-91.

105.

Wolpin BM, Kraft P, Gross M, Helzlsouer K, Bueno-de-Mesquita HB, Steplowski E, et al. Pancreatic cancer risk and ABO blood group alleles: results from the pancreatic cancer cohort consortium. Cancer Res. 2010;70(3):1015-23.

106.

Risch HA, Lu L, Wang J, Zhang W, Ni Q, Gao YT, et al. ABO blood group and risk of pancreatic cancer: a study in Shanghai and meta-analysis. Am J Epidemiol. 2013;177(12):1326-37.

107.

Nakao M, Matsuo K, Hosono S, Ogata S, Ito H, Watanabe M, et al. ABO blood group alleles and the risk of pancreatic cancer in a Japanese population. Cancer science. 2011;102(5):1076-80.

109.

SC

M AN U

TE D

EP

AC C

108.

RI PT

99.

Risch HA, Yu H, Lu L, Kidd MS. ABO blood group, Helicobacter pylori seropositivity, and risk of pancreatic cancer: a case-control study. J Natl Cancer Inst. 2010;102(7):502-5. Weiss FU, Schurmann C, Guenther A, Ernst F, Teumer A, Mayerle J, et al. Fucosyltransferase 2 (FUT2) non-secretor status and blood group B are associated with elevated serum lipase activity in asymptomatic subjects, and an increased risk for chronic pancreatitis: a genetic association study. Gut. 2015;64(4):646-56.

ACCEPTED MANUSCRIPT

Childs EJ, Mocci E, Campa D, Bracci PM, Gallinger S, Goggins M, et al. Common variation at 2p13.3, 3q29, 7p13 and 17q25.1 associated with susceptibility to pancreatic cancer. Nat Genet. 2015.

111.

Manolio TA, Collins FS, Cox NJ, Goldstein DB, Hindorff LA, Hunter DJ, et al. Finding the missing heritability of complex diseases. Nature. 2009;461(7265):747-53.

112.

Larusch J, Barmada MM, Solomon S, Whitcomb DC. Whole exome sequencing identifies multiple, complex etiologies in an idiopathic hereditary pancreatitis kindred. JOP : Journal of the pancreas. 2012;13(3):258-62.

113.

LaRusch J, Lozano-Leon A, Stello K, Moore A, Muddana V, O'Connell M, et al. The Common Chymotrypsinogen C (CTRC) Variant G60G (C.180T) Increases Risk of Chronic Pancreatitis But Not Recurrent Acute Pancreatitis in a North American Population. Clin Transl Gastroenterol. 2015;6:e68.

114.

Whitcomb DC. What is personalized medicine and what should it replace? Nat Rev Gastroenterol Hepatol. 2012;9(7):418-24.

115.

Beck AH. STUDENTJAMA. The Flexner report and the standardization of American medical education. JAMA. 2004;291(17):2139-40.

116.

Flexner A. Medical Education in the United States and Candida: A report to the Carnegie Foundation for the Advancement of teaching. Boston, Mass: 1910.

117.

Maeshiro R, Johnson I, Koo D, Parboosingh J, Carney JK, Gesundheit N, et al. Medical education for a healthier population: reflections on the Flexner Report from a public health perspective. Acad Med. 2010;85(2):211-9.

118.

Tang H, Wei P, Duell EJ, Risch HA, Olson SH, Bueno-de-Mesquita HB, et al. Axonal guidance signaling pathway interacting with smoking in modifying the risk of pancreatic cancer: a gene- and pathway-based interaction analysis of GWAS data. Carcinogenesis. 2014;35(5):1039-45.

119.

Atkinson AJJ, Colburn WA, DeGruttola VG, DeMets DL, Downing GJ, Hoth DF, et al. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin Pharmacol Ther. 2001;69(3):89-95.

120.

121.

AC C

EP

TE D

M AN U

SC

RI PT

110.

Jiao L, Hassan MM, Bondy ML, Wolff RA, Evans DB, Abbruzzese JL, et al. XRCC2 and XRCC3 gene polymorphism and risk of pancreatic cancer. Am J Gastroenterol. 2008;103(2):360-7.

Fong PY, Fesinmeyer MD, White E, Farin FM, Srinouanprachanh S, Afsharinejad Z, et al. Association of diabetes susceptibility gene calpain-10 with pancreatic cancer among smokers. Journal of gastrointestinal cancer. 2010;41(3):203-8.

ACCEPTED MANUSCRIPT

Jang JH, Cotterchio M, Borgida A, Gallinger S, Cleary SP. Genetic variants in carcinogenmetabolizing enzymes, cigarette smoking and pancreatic cancer risk. Carcinogenesis. 2012;33(4):818-27.

123.

Yang M, Sun T, Zhou Y, Wang L, Liu L, Zhang X, et al. The functional cytotoxic T lymphocyte-associated Protein 4 49G-to-A genetic variant and risk of pancreatic cancer. Cancer. 2012;118(19):4681-6.

124.

Suzuki H, Morris JS, Li Y, Doll MA, Hein DW, Liu J, et al. Interaction of the cytochrome P4501A2, SULT1A1 and NAT gene polymorphisms with smoking and dietary mutagen intake in modification of the risk of pancreatic cancer. Carcinogenesis. 2008;29(6):118491.

125.

Jiao L, Hassan MM, Bondy ML, Abbruzzese JL, Evans DB, Li D. The XPD Asp312Asn and Lys751Gln polymorphisms, corresponding haplotype, and pancreatic cancer risk. Cancer Lett. 2007;245(1-2):61-8.

126.

Nakao M, Hosono S, Ito H, Watanabe M, Mizuno N, Yatabe Y, et al. Interaction between IGF-1 polymorphisms and overweight for the risk of pancreatic cancer in Japanese. International journal of molecular epidemiology and genetics. 2011;2(4):354-66.

127.

Tang H, Dong X, Hassan M, Abbruzzese JL, Li D. Body mass index and obesity- and diabetes-associated genotypes and risk for pancreatic cancer. Cancer Epidemiol Biomarkers Prev. 2011;20(5):779-92.

128.

Jansen RJ, Robinson DP, Stolzenberg-Solomon RZ, Bamlet WR, Tan X, Cunningham JM, et al. Polymorphisms in metabolism/antioxidant genes may mediate the effect of dietary intake on pancreatic cancer risk. Pancreas. 2013;42(7):1043-53.

129.

Tang H, Dong X, Day RS, Hassan MM, Li D. Antioxidant genes, diabetes and dietary antioxidants in association with risk of pancreatic cancer. Carcinogenesis. 2010;31(4):607-13.

130.

Mohelnikova-Duchonova B, Vrana D, Holcatova I, Ryska M, Smerhovsky Z, Soucek P. CYP2A13, ADH1B, and ADH1C gene polymorphisms and pancreatic cancer risk. Pancreas. 2010;39(2):144-8.

132.

SC

M AN U

TE D

EP

AC C

131.

RI PT

122.

Dong X, Li Y, Tang H, Chang P, Hess KR, Abbruzzese JL, et al. Insulin-like growth factor axis gene polymorphisms modify risk of pancreatic cancer. Cancer epidemiology. 2012;36(2):206-11. Jansen RJ, Tan XL, Petersen GM. Gene-by-Environment Interactions in Pancreatic Cancer: Implications for Prevention. The Yale journal of biology and medicine. 2015;88(2):115-26.

ACCEPTED MANUSCRIPT

Goldstein AM, Fraser MC, Struewing JP, Hussussian CJ, Ranade K, Zametkin DP, et al. Increased risk of pancreatic cancer in melanoma-prone kindreds with p16INK4 mutations. The New England journal of medicine. 1995;333(15):970-4.

134.

Vasen HF, Gruis NA, Frants RR, van Der Velden PA, Hille ET, Bergman W. Risk of developing pancreatic cancer in families with familial atypical multiple mole melanoma associated with a specific 19 deletion of p16 (p16-Leiden). International journal of cancer Journal international du cancer. 2000;87(6):809-11.

135.

Borg A, Sandberg T, Nilsson K, Johannsson O, Klinker M, Masback A, et al. High frequency of multiple melanomas and breast and pancreas carcinomas in CDKN2A mutation-positive melanoma families. Journal of the National Cancer Institute. 2000;92(15):1260-6.

136.

McWilliams RR, Wieben ED, Rabe KG, Pedersen KS, Wu Y, Sicotte H, et al. Prevalence of CDKN2A mutations in pancreatic cancer patients: implications for genetic counseling. Eur J Hum Genet. 2011;19(4):472-8.

137.

Ghiorzo P, Fornarini G, Sciallero S, Battistuzzi L, Belli F, Bernard L, et al. CDKN2A is the main susceptibility gene in Italian pancreatic cancer families. J Med Genet. 2012;49(3):164-70.

138.

Schneider R, Slater EP, Sina M, Habbe N, Fendrich V, Matthai E, et al. German national case collection for familial pancreatic cancer (FaPaCa): ten years experience. Fam Cancer. 2011;10(2):323-30.

139.

Consortium TBCL. Cancer Risks in BRCA2 Mutation Carriers. Journal of the National Cancer Institute. 1999;91(15):1310-6.

140.

van Asperen CJ, Brohet RM, Meijers-Heijboer EJ, Hoogerbrugge N, Verhoef S, Vasen HF, et al. Cancer risks in BRCA2 families: estimates for sites other than breast and ovary. Journal of medical genetics. 2005;42(9):711-9.

141.

Jones S, Hruban RH, Kamiyama M, Borges M, Zhang X, Parsons DW, et al. Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene. Science. 2009;324(5924):217.

143.

SC

M AN U

TE D

EP

AC C

142.

RI PT

133.

Murtaugh LC. Pathogenesis of pancreatic cancer: lessons from animal models. Toxicologic pathology. 2014;42(1):217-28. Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell. 2003;4(6):437-50.

ACCEPTED MANUSCRIPT

Wagner M, Greten FR, Weber CK, Koschnick S, Mattfeldt T, Deppert W, et al. A murine tumor progression model for pancreatic cancer recapitulating the genetic alterations of the human disease. Genes and Development. 2001;15(3):286-93.

145.

Aguirre AJ, Bardeesy N, Sinha M, Lopez L, Tuveson DA, Horner J, et al. Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma. Genes Dev. 2003;17(24):3112-26.

146.

Hingorani SR, Wang L, Multani AS, Combs C, Deramaudt TB, Hruban RH, et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell. 2005;7(5):469-83.

147.

Pour P, Althoff J, Kruger FW, Mohr U. A potent pancreatic carcinogen in Syrian hamsters: N-nitrosobis(2-oxopropyl)amine. J Natl Cancer Inst. 1977;58(5):1449-53.

148.

Grippo PJ, Nowlin PS, Demeure MJ, Longnecker DS, Sandgren EP. Preinvasive pancreatic neoplasia of ductal phenotype induced by acinar cell targeting of mutant Kras in transgenic mice. Cancer Res. 2003;63(9):2016-9.

149.

Collins MA, Bednar F, Zhang Y, Brisset JC, Galban S, Galban CJ, et al. Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice. J Clin Invest. 2012;122(2):639-53.

150.

Ying H, Kimmelman AC, Lyssiotis CA, Hua S, Chu GC, Fletcher-Sananikone E, et al. Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism. Cell. 2012;149(3):656-70.

151.

Bryant KL, Mancias JD, Kimmelman AC, Der CJ. KRAS: feeding pancreatic cancer proliferation. Trends in biochemical sciences. 2014;39(2):91-100.

152.

Kang R, Hou W, Zhang Q, Chen R, Lee YJ, Bartlett DL, et al. RAGE is essential for oncogenic KRAS-mediated hypoxic signaling in pancreatic cancer. Cell death & disease. 2014;5:e1480.

153.

Strobel O, Dor Y, Alsina J, Stirman A, Lauwers G, Trainor A, et al. In vivo lineage tracing defines the role of acinar-to-ductal transdifferentiation in inflammatory ductal metaplasia. Gastroenterology. 2007;133(6):1999-2009.

AC C

EP

TE D

M AN U

SC

RI PT

144.

154. Stopczynski RE, Normolle DP, Hartman DJ, Ying H, DeBerry JJ, Bielefeldt K, et al. Neuroplastic changes occur early in the development of pancreatic ductal adenocarcinoma. Cancer Res. 2014;74(6):1718-27