Pancreatic cancer is the fourth leading cause of cancer death in the United States, with an estimated 38,000 American deaths in 2013. Worldwide it causes approximately 227,000 deaths each year and is the eighth most common cause of cancer-related death. The major type, pancreatic ductal adenocarcinoma, is a nearly uniformly metastatic and fatal disease, and the mortality rate for pancreatic ductal adenocarcinoma closely follows that of the incidence. Pancreatic cancer presents clinically with pain, with symptoms related to obstruction of the biliary or pancreatic ducts, or with protean symptoms such as weight loss and cachexia. Pancreatic neuroendocrine tumor is a separate major category of pancreatic neoplasms, often has endocrine clinical sequelae, and is less frequently metastatic and fatal.
Although most carcinomas of the pancreas appear to be sporadic, studies suggest that 3-10% of all cases of pancreatic ductal adenocarcinoma are hereditary. Some of the genes responsible for the familial aggregation of pancreatic cancer are known, and they most convincingly include germline mutations in the BRCA2, PALB2, CDKN2A, PRSS1, ATM, STK11/LKB1, and DNA mismatch-repair genes.
The profile of somatic genetic mutations in pancreatic cancer is distinct from other neoplasms. The KRAS oncogene is commonly activated by somatic mutations in pancreatic cancer, whereas three tumor-suppressor genes are commonly inactivated. Ninety percent or more of pancreatic cancers harbor activating point mutations in codon 12 of KRAS. The CDKN2A tumor-suppressor gene is inactivated in 90-100% of pancreatic cancers, TP53 in 75%, and SMAD4 in 50%. In addition, recurrent somatic mutations of the MKK4, STK11/LKB1, TGFβ receptors, Smad and Fanconi anemia pathways, ATM, and ARID1A genes have also been reported. Various loci of gene amplifications each affect a minority of carcinomas.
Inactivation of the SMAD4 gene may be rather specific for pancreatic ductal adenocarcinoma. SMAD4 is inactivated in as few as 15% of metastatic colorectal cancers and in less than 10% of other major cancer types. SMAD4 belongs to a class of proteins that mediate signals of the TGF-β superfamily.
Microsatellite instability (RER+) is seen in a small minority (~ 4%) of pancreatic cancers. These RER+ cancers have a characteristic histologic appearance (medullary histology) and frequently have wild-type KRAS gene.
The diagnosis of pancreatic cancer is suspected based on clinical findings, and often can be confirmed with radiologic and endoscopic techniques. Effective screening tests are not available yet. However, DNA-based testing appears promising.
Recently, the exomes of the four most common cystic neoplasms of the pancreas have been sequenced, and each cyst type has its own unique mutational profile. Serous cystic neoplasms are characterized by VHL mutations, solid-pseudopapillary neoplasms harbor CTNNB1mutations, intraductal papillary mucinous neoplasms have GNAS, RNF43, KRAS, and, in advanced cases, TP53 and SMAD4 gene mutations, and mucinous cystic neoplasms have RNF43, KRAS, and, in advanced cases, TP53 and SMAD4 alterations.
DAXX, ATRX, MEN-1 and mTOR pathway genes are mutated in pancreatic neuroendocrine tumors.
Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is one of the most aggressive human malignancies. It typically presents clinically late in the course of the disease, with nonspecific symptoms. As a result, patients with pancreatic cancer have an extremely poor prognosis, with an overall 5-year survival rate of 6%.1 However, those patients with early, surgically resectable carcinomas have a substantially improved prognosis. Early detection of the neoplasm, while it is still a noninvasive precursor lesion or before a cancer has spread beyond the pancreas, is the key to the successful treatment of patients with pancreatic cancer. A better understanding of the molecular genetic alterations in pancreatic cancer and its precursors may lead to the development of new tests to detect this cancer earlier.
Our understanding of the genetics of pancreatic cancer was developed later than that of some other cancer types. In large part, this was because of difficulties presented by the carcinomas themselves. Pancreatic cancers induce a prominent non-neoplastic reaction. As a result, the neoplastic cells constitute a minority of the cells in the tumor. This problem of low neoplastic cellularity has hampered the molecular analyses of pancreatic cancer. One can overcome this obstacle by selectively enriching for neoplastic cells, through propagating the cancers in tissue culture or in immunodeficient mice, and by deep sequencing. Once a mutation is identified in these enriched populations, it can be confirmed by a sensitive assay of the original primary tumor. Indeed, these techniques are a major advance in our ability to analyze pancreatic cancers, and pancreatic cancer and variant tumors of the pancreas are now among the best characterized neoplasms at the genetic level. Whereas much of what is known about pancreatic cancer has been learned by the study of sporadic pancreatic cancers, an understanding of the genetic basis for the development of inherited forms of pancreatic cancer is also emerging.
In this chapter we will focus on the advances in our understanding of the molecular genetic alterations in human PDAC, as this tumor type accounts for the majority of pancreatic neoplasms, followed by brief description of genetic aberrations in other forms of pancreatic neoplasia.
Clinical Aspects of Pancreatic Ductal Adenocarcinoma
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States.1 In 2013, 45,220 new cases of pancreas cancer will be diagnosed in the United States and nearly the same number of patients will die from it.1 These patients mostly will be elderly. The incidence of pancreatic cancer increases steadily with age, and approximately 54% of pancreatic cancers occur between 65 and 84 years of age.2,3 Pancreatic cancer is uncommon before the age of 40, although cases have been reported in children.2 Pancreatic cancer appears to occur slightly more commonly in men than in women, and, in the United States, in African-Americans more than in whites.1 – 3 The incidence of pancreatic cancer is higher among Ashkenazi Jews than among non-Jews, and it is higher in Western industrialized countries than it is in the third world.3,4 In the United States, there was a 1.4% increase in the incidence of pancreatic cancer in men and women between 2000 and 2009, and the mortality rate increased 0.5% between 2001 and 2009.2
A variety of environmental factors have been studied as possible etiologic agents in the development of pancreatic cancer, and cigarette smoking has the highest association with pancreatic cancer.5,6 It has been estimated that cigarette smoking causes 20% of all pancreatic cancer.6,7 The risk of developing pancreatic cancer increases in relation to the duration of smoking and the number of cigarettes smoked.6 – 8 In addition, pancreatic cancers resected from smokers have been reported to have more genetic alterations than do pancreatic cancers resected from non-smokers.9The excess risk associated with smoking persists for at least 10 years after smoking cessation.6,8 Smoking results in a younger age at diagnosis, as compared to non-smokers, especially in patients with familial pancreatic cancer.10
Long-term diabetes mellitus, chronic pancreatitis, diets high in meat and fat and low in fiber, heavy alcohol consumption, obesity, and non-O blood types have been associated with modest increased risk for pancreas cancer.3,5,11 – 22 Based on an early well-publicized study, coffee was once proposed to be a possible risk factor for the development of pancreatic cancer. This study, however, had serious methodological flaws and coffee is now not felt to be a significant risk factor.22,23 Thus, age and cigarette smoking remain the greatest risk factors for developing pancreatic cancer.
Familial Patterns of Pancreatic Cancer
Almost all cancers show a tendency to aggregate in families, but the fraction of cancer that is hereditary varies substantially among different cancer types.24 It has been suggested that as many as 10% of the cases of pancreatic cancer are hereditary.23 For example, Ghadirian et al interviewed 179 patients with pancreas cancer and 179 controls matched for gender and age, and they reported that 7.8% of the patients with pancreatic cancer had a family history of the disease, compared to only 0.6% of control patients without pancreatic cancer.8,25 Fernandez et al conducted a case-control study in northern Italy of 362 patients with histologically confirmed pancreatic cancer and 1408 hospital controls. Significantly more of the patients with pancreatic cancer had a family history of pancreatic cancer than did the controls (risk ratio = 3.0).26 From their data they estimated that 3% of newly diagnosed pancreatic cancers were familial.26 Similarly, we analyzed 1718 kindreds enrolled in the National Familial Pancreas Tumor Registry (NFPTR) (*see footnote). “Familial” pancreatic kindreds were defined as those families in which there had been at least two first-degree relatives diagnosed with pancreatic cancer, and we found that relatives of patients with familial pancreatic cancer have an increased risk for developing pancreatic cancer (standard incidence ratio, SIR=6.8) as compared with the general population.27 In addition, the risk was higher among members of familial pancreatic cancer kindreds with a young-onset case in the kindred than those without a young-onset case in the kindred (SIR=9.3 versus 6.3).27
Thus anecdotal reports, and several case-control and prospective studies all suggest that between 3–10% of pancreatic cancers are caused by inherited factors and relatives of patients with pancreatic cancer are at increased risk for developing the disease.
Diagnosing Pancreatic Cancer
Although the pancreas is located deep in the retroperitoneal space, it can be visualized with sophisticated imaging techniques. Multidetector computed tomography (CT) is the most commonly utilized modality in diagnosis and staging of pancreatic cancer, and other commonly used techniques include magnetic resonance imaging (MRI), positron emission tomography, and endoscopic ultrasonography(EUS).28 Despite improvements in these new techniques, the death rate for pancreatic cancer has not changed significantly. This is not surprising since the biggest determinant of patient outcome is stage at diagnosis, and these tools do not currently influence the timing of patient presentation. The survival rate for pancreatic cancer will not improve significantly until new tests are developed to screen for the disease before the patients become symptomatic.
Although all of the imaging techniques are capable of revealing a suspicious pancreatic mass, the gold standard for diagnosing pancreatic cancer remains histopathology. Tissue for microscopic examination can be obtained by EUS-guided fine needle aspiration (FNA) or tissue needle core biopsy.29 Again, as was true for imaging, the need for biopsy is likely to be apparent only after the disease has advanced.
Pathology of Pancreatic Cancer
The most common type of exocrine pancreatic cancer is the ductal adenocarcinoma (PDAC). The majority of these cancers arise in the head of the pancreas (60-70%) and the remainder in the body (5-15%) or tail (10-15%) or infiltrate diffusely throughout the gland (5-15%).30 By light microscopy, PDAC are composed of neoplastic glands infiltrating a dense non-neoplastic stroma (Figs. 50-1 A and B). Numerous inflammatory cells, including lymphocytes, also are frequently admixed with the neoplastic cells. This non-neoplastic host response is characteristic of pancreatic cancers and it must be considered when conducting molecular analyses, since nucleic acids and proteins isolated from most pancreatic cancers will originate primarily from admixed non-neoplastic cells. Perineural (Fig. 50-2) and vascular invasion are frequently present in pancreatic cancers, as is infiltration of adjacent structures and metastases to regional lymph nodes (Fig. 50-3).
Infiltrating pancreatic ductal adenocarcinoma (PDAC). Note the haphazard arrangement of markedly atypical glands (A, hematoxylin and eosin, original magnification 40X) and the intense non-neoplastic inflammatory and fibroblastic response elicited by the carcinoma (B, hematoxylin and eosin, original magnification 200X).
Infiltrating pancreatic ductal adenocarcinoma growing along a nerve (hematoxylin and eosin, original magnification 200X). Pain is a common symptom of pancreatic cancer.
Metastatic pancreatic ductal adenocarcinoma in a lymph node. Note the glandular structures in the lymph node (hematoxylin and eosin, original magnification 40X).
PDAC is frequently accompanied by pancreatic intraepithelial neoplasia (PanIN), believed to be the most common precursor of pancreatic cancer. PanIN represents a microscopic noninvasive neoplastic epithelial proliferation (<0.5 cm) of the pancreatic ductal system.31 PanINs are graded into PanIN-1, PanIN-2, and PanIN-3 based on degrees of cytologic and architectural atypia.31 PanIN-1lesions are characterized by mucin-containing columnar epithelium with basally oriented, round, uniform nuclei with no to little atypia. They can be either flat (PanIN-1A, Fig. 50-4A)) or papillary (PanIN-1B, Fig. 50-4B). PanIN-2 lesions have some nuclear atypia, such as nuclear crowding, enlargement, hyperchromasia, and pseudostratification (Fig. 50-5). PanIN-3 has marked nuclear atypia and architectural complex, including micropapillae, budding, and cribriform formation (Fig. 50-6).
Pancreatic intraepithelial neoplasia 1(PanIN1) lesions from pancreata with cancer. Note flat PanIN1A lesion (A) and papillary PanIN1B lesion (B) with no to little nuclear atypia. (A,B both hematoxylin and eosin, original magnification 200X).
Pancreatic intraepithelial neoplasia 2 (PanIN2) from a pancreas with cancer. Note PanIN2 with some nuclear atypia including hyperchromatin, increased nuclear to cytoplasmic ratio, and pseudostratification (hematoxylin and eosin, original magnification 100X).
Pancreatic intraepithelial neoplasia 3 (PanIN3) from a pancreas with cancer. Note PanIN3 with cribriform formation, budding and marked nuclear atypia. (A, B both hematoxylin and eosin, original magnification 40X for A and 200X for B).
PanIN lesions are more common in pancreata with PDAC than they are in pancreata without cancer.32 – 35 For example, Cubilla and Fitzgerald compared the duct changes in 227 pancreata having pancreatic cancer with the duct changes in 100 age- and sex-matched controls lacking pancreatic cancer.36 They found that PanINs were three times more common in pancreata obtained from patients with pancreatic cancer than they were in pancreata obtained from patients without pancreatic cancer and that high-grade lesions were seen only in pancreata containing cancer.32 These findings were confirmed by Kozuka et al and Pour et al34,35 Furukawa et al, using three-dimensional mapping techniques, demonstrated a stepwise progression from mild dysplasia to severe dysplasia in pancreatic ductal lesions.33 These results suggested that infiltrating cancers of the pancreas arose from precursors in the pancreatic ducts, that there was a progression in the pancreas from PanIN-1 to PanIN-2, then to PanIN-3, and finally to infiltrating adenocarcinoma (Fig. 50-7).37 – 39 This hypothesis was, however, based on observations of fixed static specimens. Serial samples taken over time are needed to demonstrate that, in fact, these lesions in pancreatic ducts can progress to infiltrating cancer.37 Brat et al have done just that in a small series of patients followed after partial pancreatectomy. Brat et al studied three patients in which papillary duct lesions with atypia were documented 17 months, nine years, and ten years before the development of an infiltrating cancer of the pancreas.37,40 These morphologic observations therefore strongly suggested that, just as there was a progression from adenoma to infiltrating cancer in the colorectum, so too was there a progression from PanIN to infiltrating cancer in the pancreas.41 Furthermore, genetically engineered mouse models helped confirm a progression of PanIN to invasive carcinoma.42 Of interest, patients with a strong family history of pancreatic cancer have more PanIN lesions than do patients without a family history.43
Invasive adenocarcinoma arising from a pancreatic intraepithelial neoplasia 3 (PanIN3). Note infiltrating cancerous glands (black arrows) surrounding a PanIN3 lesion with a very complex architecture and marked nuclear atypia (hematoxylin and eosin, original magnification 40X).
Other precursor lesions for pancreatic cancer include the intraductal papillary mucinous neoplasm (IPMN) and the mucinous cystic neoplasm (MCN).44 IPMNs are grossly visible, mucin-producing cystic lesions (≥ 1.0 cm) involving the main pancreatic duct or branch duct. They are the second most-common precursors for pancreatic cancer, representing 20-33% of pancreatic cysts.45,46 IPMNs mainly occur in individuals older than 60 years of age with male slightly more common than female. Most IPMNs involve the pancreatic head. Microscopically, IPMNs frequently have prominent papillary architecture. Like PanINs, IPMNs can be graded into low- (Fig. 50-8), intermediate- (Fig. 50-9), and high-grade dysplasia (Fig. 50-10) based on cytoarchitectural atypia. Associated invasive carcinoma can be seen in up to 50% of resected IPMNs, most with high-grade dysplasia.46 – 52
Intraductal papillary mucinous neoplasm with low-grade dysplasia. Note cyst wall lined with mucin-containing columnar epithelial with minimal atypia (hematoxylin and eosin, original magnification 40X).
Intraductal papillary mucinous neoplasm with intermediate-grade dysplasia. Note prominent papillary hyperplasia of columnar cells with some nuclear atypia very similar to villous adenoma of the colon (hematoxylin and eosin, original magnfication 40X).
Intraductal papillary mucinous neoplasm with high-grade dysplasia. Note a complex architecture Including cribriform and budding as well as marked cytological atypia. (A, B both hematoxylin and eosin, original magnification 20X for A and 200X for B).
MCNs are the least-common precursors for pancreatic cancer. Like IPMNs, MCNs are mucin-producing cystic lesions; unlike IPMN, however, MCNs usually do not communicate with the pancreatic ductal system.45 In addition, most MCNs occur in middle-aged women. Approximately 90% MCNs are found in the body and tail of the pancreas. Microscopically, they are characterized by an ovarian-type stroma (Fig. 50-11). Similar to IPMNs, MCNs are graded into low-, intermediate-, and high-grade dysplasia. Approximately one-third of resected MCNs are associated with an invasive carcinoma.47,53
Mucinous cystic neoplasm. Note the cyst wall composed of ovarian stroma and the lining epithelium from low-grade dysplasia with minimal atypia to moderate dysplasia with some nuclear atypia (A) to high-grade dysplasia with a complex architecture and marked nuclear atypia (B). (A, B both hematoxylin and eosine, original magnification 100X)
Genetic Loci Causing Hereditary Pancreatic Cancer
It has been difficult to perform classical genetic linkage studies in families with pancreatic cancer due to the small size of most kindreds and the short life expectancy of patients with pancreatic cancer. Nonetheless, analyses of families in which there is an aggregation of pancreatic cancer may provide clues as to which genes are responsible.
Families with an aggregation of pancreatic cancer can be divided into two general groups: (1) those associated with known syndromes; and (2) those in which there is an aggregation of pancreatic cancers, but not part of a known syndrome.
Syndromes Associated with Pancreatic Cancer
A number of well-characterized genetic syndromes have been shown to predispose affected family members to the development of pancreatic cancer.3,54 – 58 These include hereditary breast and ovarian cancer, hereditary pancreatitis, Lynch syndrome, the Peutz-Jeghers syndrome, familial atypical mole-multiple melanoma (FAMMM) syndrome, and ataxia telangiectasia (Table 50-1).3,54,56 – 61
Table 50-1 Hereditary Syndromes Associated with Pancreatic Cancer |Favorite Table|Download (.pdf) Table 50-1 Hereditary Syndromes Associated with Pancreatic Cancer
| Syndrome || Mode of Inheritance || Gene || Chromosome Locus || Manifestation |
|Hereditary pancreatitis ||AD || PRSS1 ||7q ||Recurrent episodes of severe pancreatitis occurring at an early age |
|Lynch syndrome ||AD || MSH2 ||2p ||Colonic, endometrial, and stomach cancers. Mutator phenotype |
| || || MLH1 ||3p || |
| || || PMS2 ||7p || |
| || || PMS1 ||2q || |
|FAMMM ||AD || p16 ||9p ||Multiple nevi, atypical nevi, melanomas |
|Peutz-Jeghers ||AD || LKB1 ||19p ||Hamartomatous polyps of the gastrointestinal tract, mucocutaneous melanin macules |
|Familial Breast Cancer – 2 ||AD || BRCA2 ||13q ||Breast, ovarian, and pancreatic cancer |
|Familial Breast Cancer-1 ||AD || BRCA1 ||17q ||Breast, ovarian, and pancreatic cancer |
|Familial Breast Cancer ||AD || PALB2 ||16p ||Breast and pancreatic cancer |
|Ataxia - telangiectasia ||AR || ATM ||11q ||Cerebellar ataxia, oculocutaneous telangiectasia, thymic hypoplasia |
Hereditary Breast and Ovarian Cancer
An increased risk of pancreatic cancer has been reported in breast cancer families having BRCA2 mutations. In addition to increased risks of breast and ovarian cancers, mutations in BRCA2 may be associated with several other cancers including pancreatic cancer.36 BRCA2, a genome-maintenance gene, resides at chromosomal location 13q13.1.62 It encodes for protein aiding double-strand break repair and homologous recombination.63 Murphy et al analyzed samples from patients with pancreatic cancer enrolled in the NFPTR and identified BRAC2 mutations in 17%, 64 while a recent study including additional familial pancreatic cancer families from the NFPTR estimated that BRCA2 mutations accounted for 6% of familial pancreatic cancer.65 BRCA2 mutations have a 5-10 fold increased risk of pancreatic cancer, 3 and BRCA2 mutation carriers have a 5-7% lifetime for developing pancreatic cancer.36
PALB2, the partner and localizer of Brca2 and a breast cancer susceptibility gene in its own right, has been identified as a familial pancreatic cancer susceptibility gene.66 PALB2 resides at chromosomal location 16p12.2. Like BRCA2, PALB2 codes for a protein that is involved in double-strand break repair. Approximately 3% of familial pancreatic cancer patients have a germ-line mutation in PALB2.66 PALB2 mutations appeared associated with a 6-fold increased risk for pancreatic cancer as determined in relatives of wild-type BRCA1/2 familial breast cancer patients.67
Germline BRCA1 gene mutations have also been reported in some, but not all, studies to associate with the occurrence of pancreatic cancer, but any increased risk is less than for BRCA2 mutations.68 – 71
Hereditary pancreatitis is an autosomal dominant disorder with incomplete penetrance characterized by recurrent episodes of severe pancreatitis.72 – 75 There is often an early age of onset of the pancreatitis. Men are affected at the same rate as women. Most cases of familial pancreatitis were found by Whitcomb et al to be caused by mutations in the cationic trypsinogen gene (PPSS1) on 7q35.72,73 Deleterious mutations in PRSS1 block the inactivation of trypsin, resulting in autodigestion of the pancreas. Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 (SPINK1), a pancreatic trypsin inhibitor, can also cause familial pancreatitis.74 Mutations impair SPINK1 function, preventing the inactivation of trypsin.
Patients with hereditary pancreatitis have up to an 80-fold increased risk of pancreatic cancer, with nearly 40% lifetime risk of developing PDAC.76 The mechanism by which pancreatitis leads to the development of pancreatic cancer is not clear, however, some have suggested that the increased risk of pancreatic cancer observed in patients with chronic pancreatitis is secondary to chronic injury and regeneration from the pancreatitis itself.77,78
Lynch syndrome is another syndrome that predisposes affected individuals to pancreatic cancers.79 – 81 This syndrome, also known as hereditary nonpolyposis colorectal cancer (source: NIH) (see chapter 32), is characterized by the autosomal dominant transmission of a predisposition to colonic cancer in association with other cancers, including endometrial, stomach, and pancreatic cancer.82 Lynch syndrome is caused by germline mutations in one of the DNA mismatch repair genes.83 – 87 These genes include MLH1, MSH2, MSH6 and PMS2, and they code for proteins which repair single basepair changes and small insertions/deletions that occur during DNA replication. When one of these genes is inactivated in a neoplasm, the neoplastic cells accumulate mutations in DNA sequences, but at highest rates in repeated short sequences called “microsatellite repeats”, resulting in changes in length of these repeats, a phenotype called “microsatellite instability.” Of note, replication errors, such as those found in Lynch syndrome, are found in approximately 4% of pancreatic cancers88 – 90 and, as noted previously, pancreatic cancer has been reported in some Lynch syndrome kindreds.82 It has been estimated that Lynch syndrome mutation carriers have a 3.68% lifetime risk of pancreatic cancer.81
Peutz-Jeghers syndrome is a hereditary disease with an autosomal dominant pattern of inheritance characterized by hamartomatous polyps of the gastrointestinal tract and by mucocutaneous melanocytic macules (see chapter 34). Germline mutations in the STK11/LKB1 gene on 19p have been identified as the cause of Peutz-Jeghers syndrome.91,92 STK11 has homology to a cytoplasmic Xenopus serine threonine protein kinase, XEEK1. Peutz-Jeghers was therefore the first cancer susceptibility syndrome attributable to inactivating mutations in a protein kinase. Su et al demonstrated loss of the wild-type STK11 allele in a pancreatic cancer from a patient with a germline STK11 mutation and Peutz-Jeghers syndrome.93 Forty-eight percent of thirty-one patients with Peutz-Jeghers syndrome followed by Giardiello et al developed cancer and four of these cancers were pancreatic cancer.94 This represents a 100-fold excess of pancreatic cancer compared to that expected. A systematic review of the literature estimated that patients with Peutz-Jeghers syndrome had a 132-fold increased risk of pancreatic cancer, and the cumulative risk of pancreatic cancer was 11-36% by age 65-70 years.95
Familial Atypical Multiple Mole Melanoma (Fammm) Syndrome
A subset of patients with the FAMMM syndrome appear to be at increased risk for developing pancreatic cancer.80,96 The FAMMM syndrome is inherited in an autosomal dominant fashion, and characterized by multiple nevi, multiple atypical nevi, and multiple cutaneous malignant melanomas. Germline mutations in CDKN2A with disruption of p16INK4 have been shown to segregate with the increased risks of pancreatic cancer in some kindreds with the FAMMM syndrome.97,98 Of interest, although the risk of pancreatic cancer is increased in these kindreds (32-fold), it is not a highly penetrant trait. In these families there may be a tendency for mutations at the C-terminal end of p16INK4 to be associated with a higher penetrance for pancreatic cancer.97 McWilliams et al reported that germline mutations in CDKN2A among unselected pancreatic cancer patients are uncommon (0.6%) and the estimated cumulative risk of pancreatic cancer in the mutation carriers is 58% by age 80.99
Ataxia-Telangiectasia (Source: Nih)
Patients with ATM gene mutations may also be at increased risk for developing pancreatic cancer.80,100 Ataxia-telangiectasia syndrome is characterized by progressive cerebellar-ataxia with degeneration of Purkinje cells, telangiectasias (primarily conjuctival), thymic hypoplasia with cellular and humoral immunodeficiencies, and oculomotor apraxia (see chapter 29). Ataxia-telangiectasia syndrome (source: NIH) is inherited in an autosomal recessive pattern, in which syndromic patients have one ATM gene mutation from each parent. The ATM gene resides at chromosomal location 11q22–23 and encodes a protein similar to yeast and mammalian phosphatidylinositol 3´ kinases involved in mitogenic signal transduction, meiotic recombination, and cell-cycle control.101 Patients with ataxia-telangiectasia are at increased risk for developing a number of neoplasms, including ovarian cancer, biliary cancer, gastric cancer, leukemia, lymphoma, and pancreatic cancer.80,100,101 Using whole-exome sequencing, Roberts et al identified two familial pancreatic cancer kindreds in which germline inactivating mutations in ATM segregated with pancreatic cancer.59 Because the individuals had inherited only one ATM mutation, this was not a setting of syndromic disease. In one of the patients they were able to demonstrate a second hit (loss of the wild type allele) in the patient’s pancreatic cancer.
Families with an Aggregation of Pancreatic Cancer
Although a number of well-characterized syndromes have been associated with an increased risk of pancreatic cancer, the majority of pancreatic cancers cannot be explained in this way. In many families, pancreatic cancer occurs absent the diagnosis of a known syndrome. In these instances, environmental exposures and yet-to-be-discovered familial pancreatic cancer genes need to be considered. A multi-center collaborative effort was established to hunt for new familial pancreatic cancer genes and to define the contribution of environmental factors in the clustering of pancreatic cancer.102
The National Familial Pancreas Tumor Registry (NFPTR) was established at Johns Hopkins in 1994. This is now one of the largest registries of families in which more than one family member is affected with pancreatic cancer (see footnote)*. More than 4,500 families, of which more than 1,400 have two or more first-degree relatives with PDAC, were enrolled in this registry. As of 2013 the average age at diagnosis for patients with pancreatic cancer in these families (65.5 years) did not appear to differ from the age of onset of pancreatic carcinoma that are apparently sporadic.41 The kindreds enrolled in the NFPTR included more than 135 in which four or more family members had PDAC, and more than 60 in which five or more had been diagnosed with the disease. Studies of the kindreds enrolled in the NFPTR indicated that individuals with a strong past family history of pancreatic cancer had an increased personal risk of pancreatic cancer, as well as an increased risk of extra-pancreatic malignancies. As discussed previously, analyses of the kindreds enrolled in the Registry found that relatives of patients from kindreds with familial pancreatic cancer were at increased risk for developing pancreatic cancer when compared to relatives of patients from families in which only one first-degree relative developed pancreatic cancer (3.7% versus 0.6%, p<0.0001).41 Nonpancreatic cancers were also increased in relatives of the familial pancreatic cancer cases (27.2% versus 12.1%, p<0.0001). The other types of cancer which developed in these patients included breast cancer, lung cancer, and colon cancer.41
Members of these registries, as well as those of registries created for other cancer types, are an important resource that can be used to determine the contribution of environmental risk factors, the patterns of inheritance of pancreatic cancer, and the types and prevalence of other tumor types (such as melanoma, breast cancer, and ovarian cancer) in familial pancreatic cancer.103 The results of these analyses should provide a basis for counseling families with a familial aggregation of pancreatic cancer.
Genetic Loci Involved in Sporadic Pancreatic Cancer
Four genes were identified as commonly mutated in PDAC using classical cancer genetic approaches. These include the oncogene KRAS and three tumor-suppressor genes, CDKN2A, TP53, and SMAD4.
High-throughput sequencing was extensively used to identify genetic alterations in many cancers. In pancreatic cancer, whole-exome sequencing has reaffirmed frequent mutations of KRAS, CDKN2A, TP53, and SMAD4.104 In addition, elevated rates of mutations of other genes were detected in pancreatic cancer but at much lower frequencies. Exome sequencing selectively sequences the coding regions of known human genes. Exome sequencing covers only 1-2% of the genome, and most of translocations and other large structural alterations are missed. Whole-genome sequencing provides genome-wide coverage at a single base-pair resolution. In addition, structural variants, including deletions, amplifications, and chromosomal translocations, are identified by whole-genome sequencing or by hybridization arrays. Chromosome rearrangements can also be detected by transcriptome sequencing.
Specific Genes Involved in Hereditary Pancreatic Cancer
The short life expectancy of patients with pancreatic cancer has made it difficult to perform classical linkage studies on families with pancreatic cancer: it is extremely unusual to find more than one patient alive with the disease at a time in any family. Because of these difficulties, most studies of familial pancreatic cancer have relied on the candidate gene approach. In this approach, a recessive gene which is known to be inactivated in sporadic pancreatic cancers is selected as the candidate gene, and then germline tissues from affected individuals of families in which there is an aggregation of pancreatic cancer can be tested for mutations in the candidate gene. This approach is possible because familial and sporadic forms of a given type of cancer often involve the same genes.105 For example, familial adenomatous polyposis (FAP) is caused by inherited mutations in the APC gene, and inactivation of APC is a common and early event in sporadic adenocarcinomas of the colon.106,107 Similarly, missense germline mutations in the RET proto-oncogene are responsible for the multiple endocrine neoplasia-2 (MEN-2) syndrome, and the same mutations occur in sporadic medullary carcinomas.108,109
The candidate gene approach has been applied to familial pancreatic cancer. In some families the observed cancers appeared to be caused by germline mutations in the BRCA2, STK11, or CDKN2A genes. In contrast, a number of these families have been examined for germline mutations in SMAD4, but, to date, none have been found.110 Based upon whole-genome and whole-exome sequence analysis, ATM and PALB2 were recently identified as hereditary pancreatic cancer-susceptibility genes.55,59,66,103
Thus, although several susceptibility genes for pancreatic cancer have been identified using the candidate gene approach, the genetic basis of approximately 90% of familial pancreatic cancer remains unknown.
Germline Brca2 Mutations in Pancreatic Cancer Families and in Apparently Sporadic Cancers
Goggins et al screened a panel of 41 unselected, apparently sporadic PDACs for BRCA2 mutations.111 Four of the 41 cancers had both a loss of one allele of BRCA2 and a mutation in the second allele. Three of these four mutations were present in the germline. The three germline mutations identified included two germline 6174 delT mutations, a common mutation in Ashkenazi Jews.111 A larger cross-sectional population was then evaluated. Normal tissues from 245 consecutive surgical patients with PDAC were screened near the 6174 nucleotide. Sequence analysis of this limited region of the BRCA2 gene revealed two additional germline mutations, a 6174 delT mutation and a second nearby 6158 insT mutation. Thus, a total of 5 germline mutations in BRCA2 were identified. Remarkably, only one of the 5 patients with germline mutations had a relative with breast cancer, and one had a relative with prostate cancer. None had a family history of pancreatic cancer.111 Ozcelik et al confirmed these findings by Goggins and estimated that 10% of pancreatic cancers in individuals of Ashkenazi Jewish decent might be caused by germline BRCA2 6174 delT mutations and suggested that carriers of the 6174 delT mutation had a 10-fold increased risk of developing pancreatic cancer.112
Couch et al screened affected probands from 151 high-risk families for germline BRCA2 mutations and identified five truncating mutations (1983delGAAAA, 1242insA, 5950delCT, 390delCAAC, and 4065delT).65 In addition, a polymorphic stop codon in the coding region of BRCA2 (K3326X) was shown to be more prevalent in individuals with familial than those with sporadic pancreatic cancer.113 Thus, BRCA2 is one of the best characterized familial pancreatic cancer genes. Inactivating mutations of other genes of the Fanconi anemia DNA-repair pathway, such as BRCA1 and FANCC, are occasionally found in apparently sporadic pancreatic cancers, some of which are inherited mutations, found in the germline.114 – 116
Germline Palb2 Mutations in Pancreatic Cancer Families
PALB2 is a recently identified pancreatic cancer susceptibility gene. A germline truncating PALB2 mutation (TTGT at c.172-175) was first identified by Jones et al in a pancreatic cancer patient.66 The tumor harbored a somatically acquired “second hit” mutation (C to T) at a canonical splice site for exon 10 (IVS10+2). Further screening of 96 familial pancreatic cancer patients66 identified truncating mutations in additional three patients, each producing a different stop codon.66 The prevalence of truncating PALB2 mutations ranges from 2% to 5% in familial pancreatic cancer patients from North America and Europe117 – 119
Germline Stk11/Lkb1 Mutations in Pancreatic Cancer Families
As noted previously, Peutz-Jeghers syndrome is associated with an increased risk of pancreatic cancer (see chapter 34, and refs.94,120,121 Su et al demonstrated loss of the wild-type STK11 allele in a pancreatic cancer obtained from a Peutz-Jeghers syndrome patient having a germline STK11 mutation.93 Germline mutations in STK11 were reported in 70% of patients with Peutz-Jeghers syndrome. These mutations were scattered throughout all exons with the exception of exon 9, with 60% predicted to truncate the protein.122
Germline Cdkn2a Mutations in Pancreatic Cancer Families
The CDKN2A gene, which encodes the cell cycle regulator p16, is somatically inactivated in approximately 15% of sporadic adenocarcinomas of the pancreas.90 In 40% of pancreatic cancers, CDKN2A inactivation is caused by homozygous deletions, in another 40% by intragenic mutations in one allele coupled with loss of the second allele, and in 15% by methylation of the CDKN2A promoter associated with gene silencing.123,124 CDKN2A therefore would appear to be a good candidate to examine in patients with familial pancreatic cancers. Gruis et al examined a pancreatic cancer having occurred in the context of FAMMM, finding loss of the wildtype allele.125 Moskaluk et al analyzed 21 kindreds with familial pancreatic cancer but lacking FAMMM syndrome for germline mutations in CDKN2A.126 A germline CDKN2A mutation was identified in only one family (5%). The mutation was present in two individuals affected with pancreas cancer in this family, and the alteration destroyed the donor splice site of intron 2.126 Of interest, one of the two carriers in this kindred also had a melanoma, suggesting that this kindred may have indeed had an unrecognized FAMMM syndrome.
Goldstein et al examined mutations in CDKN2A, the alternative reading frame (ARF) of the CDKN2A locusand, CDK4 in 466 families (2,137 patients) with at least three melanoma patients from 17 GenoMEL centers.127 Forty-one percent of the families had mutations, with 38% involving CDKN2A and 2-3% ARF and CDK4. Similar to the previous report, a strong association with pancreatic cancer was only seen in families with CDKN2A mutations.127 The mutations in CDKN2A included missense or nonsense mutations (70%), insertions or deletions (23%), splicing mutations (5%) and regulatory mutations (2%). Among these mutations, two (p. L32P and p.V126D) had pancreatic cancer reported in more than 40% of observed families with those mutations.127 In Dutch families suspected of FAMMM, a 19 bp deletion in exon 2 of CDKN2A was associated with 17% chance of developing pancreatic cancer by age 75.128 Germline mutations in CDKN2A should therefore be suspected when there is an aggregation of both melanoma and pancreatic cancer in a family.97,98
Germline Atm Mutations in Pancreatic Cancer Families
ATM was recently associated with familial pancreatic cancer. As noted above, Roberts et al identified ATM mutations in two patients with hereditary pancreatic cancer.59 Subsequent analysis of an additional 166 familial pancreatic cancer probands identified four additional patients with deleterious germline mutations in the ATM gene. All six variants, including four missense mutations, one intronic mutation, and one deletion, have been previously observed in patients with syndromic ataxia-telangiectasia.59 Supporting a causative role for the germline ATM mutation in the development of pancreatic cancer, Roberts et al reported loss of the second unaffected allele in a pancreatic cancer that arose in one of the germline ATM mutation carriers.
Germline Prss1 Mutations in Pancreatic Cancer Families
As described previously, individuals with hereditary pancreatitis have an increased risk for pancreatic cancer. Germline gain-of-function mutations in PRSS1are responsible for most cases of hereditary pancreatitis.72,75 Whitcomb et al demonstrated that an Arg-His substitution at residue 122 of this gene segregates with the hereditary pancreatitis in some families.72,73 Mutations at this site block the inactivation of trypsin, resulting in autodigestion of the pancreas. Later, a second mutation, p.N29I, in the PRSS1 gene was identified in some patients with hereditary pancreatitis by the same group.129
Absence of Germline Smad4, Kras and Tp53 Mutations in Pancreatic Cancer Families
The SMAD4 gene was an obvious candidate gene to study in families with pancreatic cancer.130 SMAD4 was identified in a locus of consensus homozygous deletions in sporadic pancreatic carcinomas. It is biallelically inactivated in almost 50% of pancreatic carcinomas.130 Moskaluk et al therefore sequenced the complete SMAD4 coding sequence of 25 individuals from 11 separate kindred with a familial aggregation of pancreatic carcinoma, but no germline mutations were found.110 Similarly, the KRAS oncogene frequently is activated and the TP53 tumor-suppressor frequently inactivated in pancreatic carcinomas, but, to date, germline mutations have not been identified in either of these two genes in patients with familial or sporadic cancer.130,131
In summary, the gene or genes responsible for the majority of cases of familial pancreatic cancer have not yet been identified. Germline mutations in BRCA2 predispose to the development of pancreatic cancer, and, because of their low penetrance, these mutations appear responsible for some cases of pancreatic cancer that appear to be sporadic. A small minority of familial pancreatic cancers are caused by germline mutations in CDKN2A, particularly in cases in which there is a family history of melanoma. Germline alterations in LKB1, PALB2, BRCA1, ATM, PRSS1, and the DNA mismatch repair genes also increase the risk of pancreatic cancer, to varying degrees.
Specific Genes Involved in Sporadic Pancreatic Cancer (Table 2)
The development of PDAC is complex and involves the accumulation of mutations in the KRAS oncogene and in numerous recessive genes. Comprehensive genetic analysis of the exomes of 24 pancreatic cancers found an average of 63 genetic alterations per tumor.104 As is the case in other epithelial tumors, the genetic alterations in pancreatic cancer can be conceptualized as mountains (high-frequency mutations) and hills (low-frequency mutations), with the hills dominating the genetic landscape.104
Table 50-2 Genetic Alterations In Apparently Sporadic Pancreatic Carcinomas |Favorite Table|Download (.pdf) Table 50-2 Genetic Alterations In Apparently Sporadic Pancreatic Carcinomas
| Genes || Chromosome Locus || Mechanism of Inactivation || Frequency (%) |
| Oncogenes |
| KRAS ||12p ||Point mutations codons 12, 13 ||80-100 |
| Tumor Suppressor Genes |
| CDKN2A ||9p ||Homozygous deletion, LOH and IM, hypermethylation ||95 |
| TP53 ||17p ||LOH and IM ||50-75 |
| SMAD4 ||18q ||Homozygous deletion, LOH and IM ||50 |
| BRCA2 ||13q ||Germline IM and acquired LOH ||4-7 |
| MKK4 ||17p ||Homozygous deletion, LOH and IM ||4 |
| STK11 ||19p ||Homozygous deletion, LOH and IM ||5 |
| RB ||13q ||Mutation/small deletion ||0-7 |
| ATM ||11q ||Mutation ||8% |
| ARID1A ||1p ||Mutation/small deletion/insertion ||8% |
| Genome Maintenance Genes |
| hMSH2, hMLH1, hPMS1, hPMS2, hMSHG/GTBP, hMSH3 ||multiple ||Often undetermined - gives phenotype of microsatellite instability ||4 |
In normal cells, KRAS is a proto-oncogene encoding a G protein that transduces signals related to growth control.132 Point mutations in codons 12, 13, or 61 of KRAS activate its function133 by impairing the intrinsic GTPase activity of this protein, leaving it constitutively active in signal transduction.132 KRAS is the most frequently mutated gene in pancreatic cancer, with reported mutation rates of above 90%.104,131,134,135 This is the highest reported prevalence of KRAS mutations in any tumor type. The vast majority of these mutations occur in codon 12 of KRAS. These mutations appear to be early events in the development of pancreatic neoplasia. This has been demonstrated by studies of the noninvasive duct lesions that are found in the pancreata with and without cancer. KRAS mutations were detected by conventional methods in 36-44% of PanIN-1 and 87% of PanIN-2-3 lesions.15 Using more sensitive technologies, Kanda et al identified KRAS codon 12 mutations in 92% of PanIN-1, 93% of PanIN-2 and 95% of PanIN-3 lesions.136 In the same study, they also observed that in the earliest PanIN lesions, these mutations are generally present in only fraction of the cells comprising the lesion, and the percentage of mutant KRAS cells in PanIN lesions progressively increases with PanIN grade.136 Evidence suggests that oncogenic KRAS mutations are an essential early step in most PanIN lesions. In addition, the early role of KRAS mutations in the development of human pancreatic cancer can be appreciated in several genetically engineered mouse models. Activating KrasG12D expression in the mouse pancreas results in mouse PanINs and sometimes PDAC.137 – 139
Amplification of Oncogenes
Amplification on chromosome 19q, occurring in about 10% of cases, is the most common amplicon and was also the first to be identified. Originally found as the amplification of the PD-1 gene, the leading current candidates to explain the advantage provided by the amplification events on chromosome 19q are the AKT2 gene.140,141 and the neighboring cyclin E gene142 In other pancreatic cancers, the mutational inactivation of the FBXW7 gene leads to deficient destruction and thus accumulation of cyclin E.142 Other sites of amplification exist, although at much lower prevalence rates.104,143
Tumor-suppressor genes differ from oncogenes in that tumor-suppressor genes normally function to restrict the expansion of cell populations. Their loss, by deletion or mutation, leads to dysregulated cell growth.
The TP53 tumor-suppressor gene is inactivated in approximately 75% of all PDACs.104,144 – 151 In almost all of these cancers this inactivation occurs by loss of one allele coupled with an intragenic mutation of the other. The majority of mutations reported have been transitions (pyrimidine to pyrimidine or purine to purine) in the conserved regions of the gene. Redston et al and Rozenblum et al also noted a high prevalence of small frameshift mutations in the TP53 gene in pancreatic cancers.144,148 TP53 is a nuclear DNA binding protein that acts as a G1/S regulator, and it also plays a role in the induction of apoptosis.152 – 156 Inactivation of TP53 in pancreatic cancers therefore results in the loss of two important controls of cell number, the initiation of replication and the induction of cell death. TP53 mutations are one of the late events in neoplastic progression, as inactivating mutations are primarily observed in high-grade precursors (PanIN-3 or IPMN with high-grade dysplasia) and in invasive carcinomas.
CDKN2A is a tumor-suppressor gene that is inactivated in a variety of tumors.157 CDKN2A resides on chromosome 9p,a frequent site of allelic loss in pancreatic cancers.89,158 Caldas et al demonstrated loss of one allele of CDKN2A accompanied by sequence changes in CDKN2A in the second allele in 40% of tumors.89,90,158 – 163 Furthermore, Caldas et al demonstrated homozygous deletions of CDKN2A in nearly 40% of the tumors. Therefore, CDKN2A is genetically inactivated in nearly 80% of pancreatic cancers. In addition, Schutte et al demonstrated that CDKN2A is inactivated by hypermethylation of its promoter in most of the remaining cancers.123,164 CDKN2A inhibits the progression of the cell cycle by competing with cyclin D in binding to CDK4, preventing CDK4 from phosphorylating the RB protein, which activates its growth-suppressive function. Therefore, the inactivation of CDKN2A in approximately 95% of pancreatic cancers dysregulates an important cell cycle regulator. Only approximately 10% of PanIN1/2 lesions harbor CDKN2A mutations, inactivation of CDKN2A is therefore not the earliest event of pancreatic carcinogenesis.136
One of the most frequently lost chromosome arms in both the allelotypes and karyotypes of pancreatic cancer is 18q.31,158,165,166 Based on this observation, Hahn et al performed detailed genome scanning of 18q on a panel of pancreatic carcinomas.130 These analyses not only confirmed the high frequency of loss of heterozygosity (LOH) on 18q, but they also revealed a consensus locus of homozygous deletions. Further positional cloning of the locus led to the discovery of the SMAD4 gene. This tumor-suppressor gene, originally known as DPC4, is biallelically inactivated in almost 50% of pancreatic carcinomas.130 In 30% this inactivation occurs by homozygous deletion, and in 20%, by loss of one allele coupled with an intragenic mutation of the other.130 Remarkably, although SMAD4 appears to be a common target of inactivation in pancreatic cancer, it is only infrequently inactivated in other neoplasms.167
SMAD4 is a member of the Smad family of proteins. Smads play a role in signal transduction from TGF-β superfamily cell surface receptors.168 – 171 Normally, TGF-β provides a growth inhibitory signal to epithelial cells.172 When TGF-β binds to the TGFβ receptor, it promotes the dimerization of the TGFβ receptors type I and type II, which in turn activate the kinase activity of the TGFβ type I receptor.172 The signal is transferred to Smad proteins by phosphorylation. These proteins then complex with SMAD4, and relocalization to the nucleus occurs.173
The importance of SMAD4 in TGFβ signaling was proven conclusively by Zhou et al, 174 who homozygously deleted the SMAD4 gene of cultured human colonic cancer cells using homologous recombination and demonstrated that this deletion abrogated signaling from TGFβ, as well as from the TGFβ family member activin.174 Takaku et al demonstrated the importance of SMAD4 inactivation in tumorigenesis.175 They made mice that were compound heterozygotes in mutant APC and SMAD4 alleles. Because these genes lie close on the same chromosome in mice, an LOH event results in a tumor lacking functional copies of both genes.175 The intestinal neoplasms which developed in these mice were unusually invasive and had a greater stromal response than did neoplasms which developed in simple APC mutant mice.175 Therefore, by extension, the inactivation of SMAD4 in pancreatic cancer may lead to the loss of an important pathway in TGFβ-related signaling and it may play a significant role in the malignant progression of these tumors. SMAD4 mutations do not appear until late in the neoplastic progression in the pancreas, and are detected in PanIN-3 and invasive carcinoma.176 In addition, loss of SMAD4 expression in human is associated with a poor prognosis177 – 179 and widespread metastasis.180
Other Tumor-Suppressor Genes
A number of other tumor-suppressor genes appear to be inactivated in only a small minority of pancreatic carcinomas. For example the MKK4 gene on 17p encodes for the mitogen activated protein kinase 4 (MKK4) protein. MKK4 is an important component of a stress and cytokine-induced signal transduction pathway involving the mitogen-activated protein kinase (MAPK) proteins.181 Su et al confirmed the findings of Teng et al, that MKK4 is inactivated in about 4% of pancreatic carcinomas.181 Two percent of the carcinomas harbored an intragenic mutation coupled with loss of heterozygosity (LOH) and in 2% MKK4 was inactivated by homozygous deletion.181 Some of the allelic loss patterns did not extend to the TP53 locus on 17p, and inactivation of MKK4 may therefore explain some of the LOH seen on 17p in pancreatic adenocarcinomas.
Chromosome 19p is also frequently lost in pancreatic cancer and, as discussed earlier, the gene responsible for Peutz-Jeghers syndrome, STK11, has been identified at 19p13.3.91,92,182 Höglund et al karyotyped a series of pancreatic cancers and reported that structural arrangements of chromosome 19 resulting in loss of 19p were common.182 Su et al extended these studies and demonstrated, at the molecular level, that STK11 is indeed inactivated in 5% of apparently sporadic pancreatic carcinomas.93 In 1-2% of the cancers this was by homozygous deletion and in 3-4% by LOH coupled with an intragenic mutation. The inactivation of STK11 in both familial (Peutz-Jeghers syndrome) and sporadic pancreatic cancers confirms the hypothesis that the same genes are frequently responsible for the development of both sporadic and familial forms of cancer.105
High-throughput sequencing has identified several tumor-suppressor genes that are mutated in sporadic pancreatic cancer at a low frequency. For example, somatic mutations in ATM were detected in 11 (8%) of 142 sporadic pancreatic cancers by whole-genome sequencing.183 Mutations in ARID1A, another tumor-suppressor gene, were also identified in some pancreatic cancers by whole-exome squencing.184 ARID1A encodes BAF250a, a key component of the highly conserved SWI-SNF chromatin remodeling complex which is involved in regulating processes including DNA repair, cell proliferation, differentiation and development.185 Jones et al used Sanger sequencing to further analyze 119 pancreatic cancers for mutations in ARID1A and identified mutations in 10 (8%) of the 119 cases, including 5 nonsense, 4 indel, and 1 splice site mutations.184 Shain et al noted that in total, various SWI-SNF genes were mutated in nearly 30% of pancreatic cancers.186
Goggins et al screened 82 xenografted sporadic adenocarcinomas of the pancreas for DNA replication errors (RER+) using polymerase chain reaction amplification of microsatellite markers.88 Three (3.7%) of the 82 carcinomas were RER+ and contained associated mutations in the TGFBRII gene. In contrast to typical gland-forming adenocarcinomas of the pancreas, all these RER+ carcinomas were poorly differentiated and had expanding borders and a prominent syncytial growth pattern (Figure 50-12).88 Furthermore, all of the RER+ carcinomas were KRAS wild-type and the one case that was karyotyped had a near diploid pattern. These data by Goggins et al suggested that DNA replication errors occur in approximately 4% of pancreatic carcinomas and that wild-type KRAS gene status coupled with the histologic findings of medullary carcinoma with poor differentiation, a syncytial growth pattern and pushing borders, should suggest the possibility of DNA replication errors in carcinomas of the pancreas.
Pancreatic carcinoma with DNA replication errors (RER+). RER+ carcinomas of the pancreas are associated with wild-type KRAS and poor differentiation, a syncytial growth pattern and pushing borders. (A, B both hematoxylin and eosin, original magnification 40X for A and 200X for B).
Variant Tumors of the Pancreas
Intraductal Papillary Mucinous Neoplasm (Ipmn)
As described previously, the IPMN is a common precursor for PDAC. IPMNs are the most common type of the neoplastic cysts in the pancreas, accounting for 25-30% of the total pancreatic cysts. The exomes of a series of eight well-characterized IPMNs have recently been sequenced.187 IPMNs contained 27±12 somatic mutations per tumor, with three genes frequently mutated in IPMNs: KRAS, GNAS and RNF43. About two-thirds of IPMNs harbor a GNAS mutation, and 80% a KRAS mutation. Either KRAS or GNAS mutations could be identified in almost all of (96%) of IPMNs.188 GNAS encodes the stimulatory G-protein α subunit (Gsα). Gsα forms a heterotrimer with β and γ G protein subunits, which in turn couples with G protein-coupled receptor (GPCR). Upon activation, GPCR catalyzes the exchange of GTP for GDP bound to Gsα. GTP-bound Gsα then activates adenyl cyclase (AC), which activates the cAMP/protein kinase A (PKA) signaling pathway.189 The intrinsic GTP hydrolase activity of Gsα reverts the GTP-bound Gsα to its GDP-bound, inactive form.189 The point mutations in codon 201 inactivate the intrinsic activity of Gsα, consequently resulting in constitutive activation of the AC/cAMP/PKA signaling pathway.189 In addition, when a cancer arose in association with an IPMN, the same GNAS mutations was usually present in the IPMN and the matched invasive cancer, helping to establish that these cancers arose from the IPMNs.188 By contrast, GNAS mutations were only rarely identified in PDAC not arising in association with an IPMN, and GNAS was rarely mutated in PanINs.188 GNAS mutations were proposed as an early event in the development of IPMNs, as the frequency of the mutations was similar among low- and high-grade IPMNs.188 The tumor suppressor gene, RNF43, was also frequently altered in IPMNs. When targeted, both alleles of the RNF43 were inactivated in IPMNs. RNF43 resides at chromosome 17q and encodes a protein with a stem-cell E3 ubiquitin ligase activity, which was reported to reduce the Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation.190
Other genes mutated in IPMNs include PIK3CA, SMAD4 and TP53; most of these mutations occur in IPMNs having high-grade dysplasia. Somatic mutations of PIK3CA have been detected in 11% of IPMNs.191 Inactivation of TP53 is a late event, as mutations in TP53 are found less frequently in IPMNs with low- or intermediate-grade dysplasia than they are in IPMNs with high-grade dysplasia or invasive carcinomas arising in association with an IPMN.192 – 195 The expression of SMAD4 is preserved in IPMNs of all dysplastic grades, but loss of expression is seen in approximately 10% of invasive carcinomas arising in association with an IPMN.192,196 – 198
Mucinous Cystic Neoplasm (Mcn)
MCN is a less common cystic neoplasm of the pancreas (10%), but also has a potential to progress into PDAC. More than 90% of MCNs occur in the body and tail of the pancreas, and most patients with MCN are middle-aged women. Whole-exome sequencing of a series 8 of MCNs revealed 16 ± 7.6 somatic mutations per tumor. The most common mutations found in MCNs occurred in the RNF43, KRAS, TP53 and SMAD4 genes.187,192 TP53 or SMAD4 inactivation is a late event, as they are only seen in MCNs with high-grade dysplasia or invasive carcinomas arising in association with an MCN.192
SCA is the second-most-common neoplastic cyst of the pancreas. SCA has essentially minimal or no malignant potential. It is characterized by multiple small cysts lined with cuboidal glycogen-rich epithelium with centrally placed round nuclei having no atypia (Fig. 50-13). The exomes of a series of SCAs were sequenced, and the SCAs contained 10 ± 4.6 somatic mutations per tumor.187 The most common mutated gene in SCAs was the VHL (von Hippel-Lindau) gene. Four of 8 SCAs had a point mutation in VHL, with 3 of the 4 SCAs having LOH at the VHL chromosomal region. The four without detectable VHL mutations also had lost one allele of chromosome 3p within or adjacent to VHL.187 VHL is a tumor-suppressor gene involved in angiogenesis. Germline mutations in VHL lead to VHL syndrome, which is characterized by a number of “clear cell” tumors, including hemangioblastoma, endolymphatic sac tumor, pheochromocytoma, renal cell carcinoma, renal cyst, epididymal cyst, serous cystadenoma, and pancreatic neuroendocrine tumor. Serous cystadenoma are seen in 20-50% of patients with VHL syndrome.
Serous cystadenoma. Note small cysts lined with cuboidal glycogen-rich epithelium with centrally placed round nulcei with no atypia (hematoxyline and eosin, original magnification 200X).
Solid Pseudopapillary Neoplasm (Spn)
SPN is a low-grade malignancy, the least-common neoplastic cyst of the pancreas. SPNs occur mostly in young women. They are characterized by proliferation of poorly cohesive monomorphic epithelial cells forming solid and pseudopapillary structures (Fig. 50-14A). Exome sequencing of a series of SPNs revealed only 2.9 ± 2.1 somatic mutations per tumor.187 Remarkably, CTNNB1 mutations were seen in every tumor sequenced. CTNNB1 encodes for β-catenin, a key component in the Wnt signaling pathway. The mutations in CTNNB1 occurred in the region critical for regulation of the protein, which leads to inhibition of the degradation, consequently resulting in accumulation of the protein in the nuclei. It should therefore not be surprising that immunohistochemically, SPNs are characterized by β-catenin nuclear accumulation (Fig. 50-14B).
Solid pseudopapillary tumor. Note monomorphic epithelial cells forming solid and pseudopapillary structures (A, hematoxylin and eosin, original magnification 100X) and tumor cells with β-catenin nuclear accumulation (B, original magnification 200X).
Pancreatic Neuroendocrine Tumor (Pannet)
Although rare, PanNETs are the second-most-common malignancy in the pancreas. More than 90% of PanNETs are well differentiated. Endocrinologically, they can be either nonfunctional or functional. The latter are characterized by clinical syndromes secondary to the production and release of hormones by the tumor. PanNETs can be sporadic or hereditary. Hereditary syndromes associated with PanNETs include multiple endocrine neoplasia type 1 (MEN-1), VHL, neurofibromatosis and tubular sclerosis. Most PanNETs are composed of monomorphic neoplastic cells with eosinophic cytoplasm and round nuclei containing “salt-and-pepper” chromatin (Fig. 50-15). These tumors have a rich vasculature and minimal stroma. Whole-exome sequencing of a series of PanNETs identified a number of genes frequently mutated in PanNETs.199 Somatic inactivating mutations in MEN-1 were found in 44% of P.199 MEN-1 encodes menin, a component of a histone methyltransferase complex. Germline mutations in MEN-1 are responsible for Multiple Endocrine Neoplasia type 1 (MEN-1) syndrome. Patients with MEN-1 syndrome frequently develop PanNETs. In sporadic PanNETs, exome sequencing revealed somatic mutations in either DAXX (death-domain associated protein) or ATRX (alpha thalassemia/mental retardation syndrome X-linked) in 43% of the tumors.199 DAXX and ATRX encode the two subunits of a transcription/chromatin remodeling complex which plays multiple cellular roles, including chromatin remodeling at telomeres. Mutations of DAXX and ATRX have been associated with the alternative lengthening of telomeres (ALT) phenotype in PanNETs.200 Finally, mutations in genes in the mTOR (mammalian target of rapamycin) pathway were detected in 14% of the PanNETs sequenced. Among 68 PanNETs sequenced, 5 had PTEN mutations, six TSC2 mutations and one PIK3CA mutation.
Pancreatic neuroendocrine tumor. The tumor is composed of monomorphic neoplastic cells with eosinophic cytoplasm and round nuclei containing salt-and-pepper chromatin (hematoxylin and eosin, original magnification 200X).
Implications for Diagnosis
There are no molecular tests currently being used to screen for pancreatic carcinoma. However, the recent advances in our understanding of how molecular biology can be used to screen for cancer, coupled with an improved understanding of the molecular genetics of this tumor, provide several avenues for developing such a test. Probably the best example is the KRAS oncogene. KRAS is a particularly attractive target for a molecular screening test for pancreatic cancer for three reasons. First, the vast majority of PDACs harbor mutations in KRAS, suggesting that KRAS will be a sensitive genetic marker. Second, mutations in this oncogene essentially are limited to two codons, and so a limited number of probes can be employed to detect these mutations, greatly simplifying the analyses. Finally, as discussed earlier, these mutations appear to be early events in the development of pancreatic neoplasia, suggesting that KRAS could be used to detect early and therefore curable pancreatic neoplasms.201 – 204 Extensive studies have been performed to evaluate KRAS as a biomarker for detecting pancreatic cancer using body fluids and needle biopsy samples. For example, we quantified mutant KRAS levels in pancreatic secretions from patients with pancreatic cancer or chronic pancreatitis, and found that the mutation levels were substantially higher in the secretions obtained from the pancreatic cancer patients compared with chronic pancreatitis patients.204 Therefore quantification of mutant KRAS in pancreatic secretions may provide a more useful early-detection tool for pancreatic cancer.
GNAS mutations in codon 201 are frequently present in IPMN, but not in other types of cystic neoplasms such as SCA. Radiographically, SCA, especially the oligocystic form, can closely mimic IPMNs. However, SCA is a benign cystic lesion with almost no malignant potential. Therefore, it is very important to differentiate IPMN from SCA. GNAS may serve as a biomarker for that purpose. Indeed, Kanda et al demonstrated that GNAS mutations were detected in secretin-stimulated pancreatic secretions collected from the duodena of 50 of 78 patients having IPMN but in none of 57 disease controls.205
TP53 is another biomarker that can be used for early detection of pancreatic cancer. Unlike KRAS and CDKN2A, TP53 is only mutated in high-grade precursor lesions and invasive carcinomas. In contrast with SMAD4, which is commonly inactivated by homozygous deletion, TP53 is inactivated by missense mutations or by short frameshifts in pancreatic cancer. Therefore, TP53 can be a highly specific and technically feasible biomarker for pancreatic cancer. Unfortunately, TP53 is a large gene and mutations can occur throughout the gene. Therefore, extensive genetic analyses may be required to identify the complete spectrum of the mutations. Nevertheless, the presence of TP53 mutations in pancreatic secretions and in brush cytology specimens has been reported in 40% to 50% of patients with pancreatic cancers.206 Using digital high-resolution melt-curve analysis, Kanda et al detected mutant TP53 in secretin-stimulated pancreatic secretions collected from the duodena of patients having high-grade dysplasia (4 of 8 patients) and/or invasive carcinoma (29 of 43 patients).207
In addition to leading to the development of new screening tests for early pancreatic neoplasms, an improved understanding of the genetics of pancreatic cancer will lead to the discovery of additional germline mutations in cancer-causing genes that predispose to pancreatic cancer; geneticists will be able to screen at-risk individuals for these germline mutations. Carriers of germline mutations can then be clinically screened more thoroughly and may even choose prophylactic surgery, while those found not to carry germline mutations will have their anxiety relieved and can be spared unnecessary screening tests.
Information on the genetic alterations in pancreatic cancer can also be used to predict survival and stratify patients for treatment.208,209 For example, several studies have shown that inactivating mutations in SMAD4 (assessed by immunohistochemical loss of SMAD4 expression) was associated with a poor prognosis. Furthermore, SMAD4 loss in the primary tumor was highly correlated with widespread metastasis, whereas retained SMAD4 was associated with a locally destructive disease.180,210 Thus status of the SMAD4 gene in the primary tumor may help guide treatment decisions. Patients having a borderline resectable carcinoma with loss of SMAD4 expression may have a higher risk to develop widespread disease. These patients may benefit more from systemic chemotherapy than from locoregional therapy. On the other hand, patients with a borderline resectable, SMAD4 intact carcinoma may be better candidates for adjuvant chemoradiation.180,210,211
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