Genetics of Pancreatic Cancer

Genetics of Pancreatic Cancer

1055-3207/98 $8.00+ .OO PANCREATIC CANCER GENETICS OF PANCREATIC CANCER From Genes to Families Ralph H. Hruban, MD, Gloria M. Petersen, PhD, Patrick...

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1055-3207/98 $8.00+ .OO


GENETICS OF PANCREATIC CANCER From Genes to Families Ralph H. Hruban, MD, Gloria M. Petersen, PhD, Patrick K. Ha, BA, a n d Scott E. Kern, M D

The last 10 years have brought remarkable advances in our understanding of human genetics. Indeed, in a few short years, the human genome project is likely to identify and sequence all human genes. Some of the genes that already have been identified are associated with the development of cancer, and it is clear that cancer, including pancreatic cancer, is a disease of inherited and acquired mutat i o n ~At . ~the ~ genetic level, cancer of the pancreas is currently among the better characterized neoplasms, and the development of pancreatic carcinoma has been shown to be associated with the activation of an oncogene, K-uas, and the inactivation of multiple tumor suppressor genes, including p53, DPC4, p16, and BRCA2.* These genetic changes are not only of interest to research scientists but they also have direct clinical relevance. For example, genetic changes will form the basis of new screening and diagnostic tests for the early detection of pancreatic neoplasms; they are used to identify patients at risk for familial forms of pancreatic cancer; and they can be used to characterize even the most subtle pathologic changes, thereby advancing our understanding of early pancreatic neoplasia. Finally, and perhaps most importantly, an understanding of the genetic changes associated with the development of pancreatic neoplasms may form a foundation for developing novel, rational, gene-based therapies for pancreatic carcinoma.

"References 14, 15,23,24,33, 39,40,43, 78, 86, 91,94. Supported in part by NIH P50-CA62924.

From the Departments of Pathology (RHH, SEK) and Oncology (RHH, GMP, SEK), The Johns Hopkins Medical Institutions; and the Department of Epidemiology (GMP),The Johns Hopkins School of Public Health; and The Johns Hopkins School of Medicine (PKH), Baltimore, Maryland SURGICAL ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 7. NUMBER 1. JANUARY 1998




This article focuses on the most common type of cancer in the pancreas, ductal adenocarcinoma. The article reviews the genetic alterations that have been identified in sporadic pancreas cancers, the timing of these genetic alterations in the development of these cancers, and the familial forms of pancreatic cancer, including a description of the familial pancreas cancer kindreds enrolled in the National Familial Pancreas Tumor Registry (NFPTR). GENETIC ALTERATIONS AND PANCREATIC CANCER

A large number of resected pancreatic cancers have been investigated for genetic alterations in cancer-causing genes. In general, these genes can be divided into three broad groups. Tumor suppressor genes are genes that normally function as genetic barriers to control cellular proliferation. When they are inactivated by genetic events such as mutation, deletion, chromosome rearrangements, or mitotic recombination, their growth suppressor function can be lost, resulting in disregulated growth.'jOThus, the analogy has been made between a mutated tumor suppressor gene and a failed brake pedal in a car. The second major type of cancercausing genes is oncogenes. Oncogenes are derived from normal cellular genes called proto-oncogenes, and they encode for proteins that, when overexpressed or activated by mutation, possess transforming properties. In contrast to tumor suppressor genes, which are inactivated by mutation, oncogenes are activated by mutation. Activated oncogenes are therefore likened to the accelerator pedal of a car becoming stuck in the down or "on" position. The third broad class of cancer causing genes is the DNA mismatch repair genes. These genes normally function to ensure the fidelity of DNA replication. Because the human genome contains approximately 100,000 genes spread over 3 billion base pairs, it is not surprising that errors (spontaneous mutations) occur when cells replicate. When DNA repair genes are dysfunctional, these errors are not repaired. Among the genes that may be mutated by failed DNA repair are tumor suppressor genes and oncogenes, so that the former are inactivated and the latter activated. Thus, the analogy can be made between a mutated DNA repair gene and failing to get a car properly serviced. DNA repair genes are only rarely (-4%) inactivated in pancreatic cancer; therefore, this article focuses on tumor suppressor genes and oncogenes. Karyotyping

The identity of specific chromosomes lost or gained in a cancer type can be determined by karyotyping metaphase spreads obtained from fresh cancers (Fig. 1). The occurrence of consistent chromosome abnormalities in a cancer in turn can provide clues to specific genes involved in the development of that neoplasm. For example, the consistent loss of a chromosome arm in many tumors of the same type suggests that a tumor suppressor gene related to the development of that have karyotyped more tumor is located on that chromosome arm. Griffin et a136,37 than 62 primary pancreatic cancers and have reported clonally abnormal karyotypes in 44 (71%)of these neoplasms. In these 44 cancers, the most frequent losses occurred on chromosomes 18 (22 tumors), 13 (16 tumors), 12 (13 tumors), 17 (13 tumors), and 6 (12 tumors). The most frequent gains were of chromosomes 12 (8 tumors) and 7 (7 tumors), and the most common recurrent structural abnormalities involved chromosome arms lp, 6q, 7q, 17p, lq, 3p, l l p , and 19q.36,37 These genetic changes, detected by karyotyping the cancers, can be used not only to suggest the location of specific tumor suppressor genes involved in the development of pancreatic neoplasia but also to suggest a mechanism by which many of these genes are inactivated-loss of whole chromosomes. For example,



Figure 1. Metaphase karyotype of a pancreatic cancer. Arrows indicate clonal abnormalities. In this cancer entire copies of chromosomes 2, 5, 6, 12, 13, 17, 18, 19, and 21 were lost. (From Brat DJ, Hahn SA, Griffin CA, et al: Structural basis of molecular genetic deletions:

Integration of classical cytogenetic and molecular analyses in pancreatic adenocarcinoma. Am J Pathol 150:383-391, 1997; with permission.)

the recently discovered DPC4 tumor suppressor gene resides on chromosome 18q, and loss of 18q was identified in half of the cancers with a clonally abnormal k a r y ~ t y p eSimilarly, .~~ the p53 tumor suppressor gene resides on 17p, and chromosome 17 was lost in 13 of the 44 cancers e ~ a m i n e d .Similar ~~,~~ results have been and Bardi et al.3Taken together, obtained by Gorunova et al,35Johansson et these results suggest that both DPC4 and p53 are frequently lost in pancreatic carcinomas. Karyotyping also can be used to suggest loci of yet undiscovered tumor suppressor genes. For example, chromosome 6q24 to 6q25 frequently was suggesting that this locus may harbor lost in the series reported by Griffin et a1,36,37 an uncharacterized tumor suppressor gene. Comparative Genomic Hybridization

Comparative genomic hybridization (CGH) is a technique that, like karyotyping, can be used to screen the entire genome for genetic abnormalities. In CGH,



DNA is prepared from tumor cells and labeled with one fluorochrome, whereas DNA that is prepared from normal lymphocytes is labeled with a different fluorochrome. The two are then used as probes for fluorescence in situ hybridization Comparison of signal intensities of the with normal metaphase chromosomes.lO~ tumor and the normal DNA probes allows one to detect gains and losses of chromosomal material in the cancer.102When Solinas-Toldo et allo2applied CGH to a panel of 27 pancreatic carcinomas, they found that gains of chromosomal material were much more frequent than losses. The most common gains (or overrepresentations) were observed on chromosomes 16p, 20q, 22q, and 17q.lo2Losses (or underrepresentations) were identified at 9p. The p16 (or MTSI) tumor suppressor gene resides on 9p, and the identification of the loss of genetic material on 9p suggests that the p16 tumor suppressor gene is also frequently lost in pancreatic carcinoma^.'^ Similarly, Morsberger et a177analyzed a series of pancreatic cancer cell lines using CGH and were able to demonstrate frequent loss of 6q, 9p, 17p, and 18q. These data support the results of classical karyotyping and suggest that the p16 tumor suppressor gene on 9p, the p53 tumor suppressor gene on 17p, and the DPC4 tumor suppressor gene on 18q are all lost frequently in pancreatic carcinoma. Allelotyping

Losses of genetic material can be localized more precisely by using a technique called allelotyping. In this approach, DNA prepared from a cancer is probed using a panel of molecular markers that is specific for a variety of different loci on each chromosome (Fig. 2). Hahn et al4I and Seymour et a198extensively allelotyped a series of pancreatic xenografts and found high frequencies of loss at chromosome arms lp, 9p, 17p, and 18q. These results confirm those obtained by karyotyping and CGH. In addition, the high frequency of loss in l p suggests that another, yet undetermined, tumor suppressor gene may be located on this chromosome arm. Of note, slightly lower frequencies of loss were seen at 3p, 6p, 8p, 10q, 12q, 13q, 18p, 21q, and 22q,41,98and Achille et a12 recently reported a high frequency of loss at 7q. Although allelotyping and karyotyping rely on entirely different methodologies, Brat et all2 recently showed a high degree of correlation between the two techniques. They compared the chromosomal abnormalities identified by karyotyping primary adenocarcinomas of the pancreas with the molecular changes identified by allelotyping these same cancers.12In 14 cancers with abnormal karyotypes, 65% (123 of 188) of the chromosome arms with molecular evidence of loss of heterozygosity (LOH) were associated with karyotypically detected structural abnormalities. Karyotypic changes accounting for these molecular losses included 83 chromosome losses, 18 partial deletions, 9 isochromosomes, 8 additions, and 5 translocations. Chromosome structural abnormalities detectable by karyotyping the cancers therefore could account for two thirds of the molecular losses of heterozygosity identified.I2 These data not only validate both techniques, but also demonstrate the mechanisms by which allelic losses can occur. Thus, based on the karyotyping, CGH, and allelotyping data, the p16, p53, and DPC4 loci are all common sites of loss in pancreatic carcinomas. However, in order to establish that the function of these genes is indeed inactivated in these cancers, one must demonstrate the inactivation of both copies of these genes (each autosomal gene is inherited in pairs, one maternal and the other paternal). The two most common mechanisms by which tumor suppressor genes are inactivated are loss of one allele with mutation of a second, and loss of both copies of a gene. This latter mechanism is also known as homozygous deletion.



Figure 2. Allelotype of pancreatic cancer. Representative electrophoretic gel lanes of pancreatic adenocarcinoma (T) and normal (N) for nine cases. Loss of heterozygosity (LOH) is demonstrated in cases 1, 3, 5, 7, and 9. Case 2 shows retained heterozygous alleles,cases 4 and 8 are uninformative, and case 6 has the mutator phenotype. (From Brat DJ, Hahn SA, Griffin CA, et al: Structural basis of molecular genetic deletions: Integration of classical cytogenetic and molecular analyses in pancreatic adenocarcinoma. Am J Pathol 150:383-391, 1997;with permission.)


As discussed previously, tumor suppressor genes are genes that normally function as genetic barriers to control cellular proliferation. The tumor suppressor genes p53, pl6, and DPC4 are inactivated frequently in sporadic carcinomas of the pancreas. p53 is probably the best characterized of the tumor suppressor genes. p53 function appears to be inactivated in 50% to 75% of all pancreatic carcinomas This inactivation almost always occurs by mutation (Table 1).16,21,23,61,s4,86,93,104,114,121 p53 is a of one copy (allele) of the p53 gene and loss of the other.16,84,86,91,93,104,114 nuclear DNA-binding protein that acts as both a cell cycle (Gl/S) checkpoint and ~ , ~ ~ , ~ ~ ~of p53 function in panas an inducer of cell death ( a p o p t o s i ~ ) . Inactivation creatic cancer therefore leads to the loss of two important controls of cell growth: the regulation of proliferation and the induction of cell death.45The loss of p53 function in most pancreatic cancers helps us understand the cellular events responsible for the development of pancreatic cancer, and p53 loss also may provide prognostic information and suggest avenues for the development of novel treat~ ~ ~example, ,'~' Weyrer et aln4have correlated the presence of ment ~ t r a t e g i e s . ~ For p53 mutations in pancreatic cancers with distant metastases ( P < 0.05), and gene therapy techniques have been developed that can deliver wild type p53 and thereby restore p53 function to cancer cells.56

Table 1. GENETIC ALTERATIONS IN PANCREATIC CANCER Genes Chromosome Mechanism of Inactivation tumor suppressor ~ 1 6 9P LOH + IM Homozygous deletion hypermethylation ~ 5 3 17P LOH + IM DPC4 18q Homozygous deletion LOH + IM

Frequency (%) 40 40 15 50-75 30 20



germline mutation




mutation/small deletion



point mutations condons l2,13

oncogenes K-ras LOH

80-1 00

+ IM = loss of heterozygosity and intragenic mutation.

The cyclin-Cdk complexes and their inhibitors also play an important role in the control of the cell cycle. The p16 gene encodes a protein that binds cyclin DCdk4 c~mplexes.l~,~O When p16 binds to these complexes, it inhibits the phosphorylation of a number of growth and regulatory proteins, including Rb. Hypophosphorylated Rb binds to and sequesters transcription factors that would otherwise promote the Gl/S tran~ition.~O In contrast, hyperphosphorylated Rb, as seen in p16-deficient cells, releases these factors, allowing the cell to progress through the Gl/S c h e c k p ~ i n tInactivation .~~ of p16 therefore inactivates another important cell cycle checkpoint. The p16 gene resides on chromosome 9p. As described earlier, 9p is a frequent site of allelic loss in pancreatic cancer^.^^,^^ The second allele of p16 is inactivated by point mutations in 40% of pancreatic cancers, and in 40% the second allele is In another 15% of pancreatic cancers, p16 may be inacdeleted (Table 1).5,14,46,82,91 tivated by a third mechanism, hypermethylation of a CpG island in the p16 proThese data suggest that the cell cycle checkpoint, p16, is lost in more moter.76,87,95 than 90% of pancreatic cancers. These data also demonstrate that homozygous deletions play an important role in the development of pancreatic carcinoma and that hypermethylation of CpG islands is a mechanism for inactivating the expression of some tumor suppressor genes. p16 is part of a complex pathway that controls the cell cycle, and Huang et a147recently reported that another member of that pathway, Rb, also can be inactivated in pancreatic cancer. They analyzed 80 pancreatic cancers (62 cancers and 18 cell lines) for the expression of the RBI protein.47These cases were analyzed immunohistochemically or by Western blotting or both using a panel of antibodies, and complete loss of expression of RBI was found in 6 (7.5%)of the 80 cases.47 These data, taken together with the p16 data, suggest that the p16-Rb pathway that controls the cell cycle is a frequent target of inactivation in pancreatic cancer.

The demonstration that p53 and p16 are frequently inactivated in pancreatic cancer provides insight into the molecular genetics associated with the development of cancer of the pancreas. However, inactivation of these genes is not specific



to pancreatic cancer. Recently, Kern et a140identified a new tumor suppressor gene called DPC4 and, in contrast to p53 and p16, inactivation of DPC4 appears to be more specific to pancreatic cancer.96 DPC4 resides on chromosome 18q and, as noted earlier, allelic loss studies have demonstrated that 18q is lost in nearly 90% of pancreatic carcinomas (Table 1).41,98 Based on this high frequency of allelic loss, Hahn et a140performed detailed genomic scanning on a panel of pancreatic carcinomas. They identified a small consensus region of homozygous deletion; it was in this consensus region of loss that the DPC4 (deleted in pancreatic carcinoma, locus 4) gene was dis~overed.39,~o DPC4 has homology to a family of proteins called the Mad proteins, and the Mad proteins play a role in signal transduction from the transforming growth factor-p (TGF-p) family of cell surface receptor^.^^,^^,^^ TGF-P can downregulate the growth of epithelial cells and stimulate differentiation and apoptosis. Therefore, it is reasonable to expect that the loss of DPC4 would promote cell growth. Hahn et a140examined the status of DPC4 in 84 pancreatic cancers (xenografts and cell lines) and found that DPC4 was inactivated in 30% of the cancers by homozygous deletion and in another 20% by point mutation of one allele and loss of the second allele (Table 1).DPC4 is therefore inactivated in approximately half of all pancreatic cancers. The inactivation of DPC4 appears to be relatively specific to pancreatic cancer. Schutte et a196examined a panel of more than 300 tumors from various sites and found that DPC4 is only rarely inactivated (
In addition to p16, p53, and DPC4, a variety of other loci may harbor tumor suppressor genes associated with the development of pancreatic neoplasia. For example, molecular analyses have revealed high frequencies of unexplained allelic loss on chromosomes l p (70%) and 7q (80%),and karyotyping studies have identified a consensus region of loss at 6q24, suggesting that all of these loci may harbor yet undiscovered tumor suppressor genes (Table 1).2,41,98 In addition, patients with Peutz-Jeghers syndrome are at increased risk for developing pancreatic can~er.3~ The gene responsible for this syndrome has been localized recently to the distal It is tempting to speculate that the gene responsible for Peutzportion of 19p.32,42 Jeghers syndrome also may play a role in the development of sporadic pancreatic cancer. Summary of Tumor Suppressor Genes

A variety of tumor suppressor genes are inactivated in sporadic pancreatic cancers, including p53, p16, and DPC4. Whereas p16 and p53 are inactivated in a number of different types of tumors, some aspects of the inactivation of tumor suppressor genes are relatively unique to cancer of the pancreas. First, multiple tumor suppressor genes are inactivated frequently in a single cancer.91For example, Rozenblum et a19' demonstrated coinactivation of p16, p53, and DPC4 in



37% of 40 pancreatic cancers that they examined. Furthermore, one tumor they studied had a germline mutation in the breast cancer gene (BRCA2) and eight additional mutations in other genes.91Thus, pancreatic cancers have accumulated numerous mutations by the time they are resected. In addition, homozygous deletions are common in pancreatic cancer. As described previously, inactivating of homozygous deletions has been identified at p16 in 40% of pancreatic cancers and at DPC4 in 30%.14,40 ONCOGENES

Oncogenes encode proteins that when overexpressed or activated by mutation, possess transforming properties. To date, the most common genetic alteration identified in a cancer causing gene in pancreatic cancer is in an oncogene called K-ras. Point mutations in codons 12,13, or 61 of the K-ras gene impair the intrinsic GTPase activity of its gene product, resulting in a protein that is constitutively Mutant K-ras, therefore, has transforming propactive in signal transd~ction."~ erties. A large number of cancers already have been analyzed for these mutations, and 80% to 100% of pancreatic cancers have been reported to harbor activating point mutations in K-ras (Table 1).38,43,75,80,81,84,101,107,114 Most of these mutation are in codon 12 of the K-ras gene and most are guanine to adenine transitions or guanine to thymine trans version^.^^ These mutations are relative easy to detect, making K-ras a potential target for the development of molecular-based screening tests for pancreatic cancer.* K-ras is an attractive candidate for molecular-based screening tests for pancreatic cancer for four reason^.^^,^^ First, as noted previously, K-ras is mutated in most cancers of the pancreas, suggesting that it would be a sensitive marker.38,43,75,80,81,84,101,107 Second, K-ras mutations occur early in the development of these cancers, suggesting that K-ras could be used to detect early and therefore curable pancreatic cancers.9,15,22,44,106,119 Third, most of the mutations in K-ras in cancers of the pancreas are restricted to a single codon of that gene, so a limited number of probes can be used to detect these mutations, greatly simplifying these analyses.*,45 Finally, molecular-based tests already have been developed that can detect rare mutant copies of K-ras, even when they are admixed with many more copies of the wild type (normal) gene.15,M,106 Indeed, mutant K-ras already has been demonstrated in samples of pancreatic juice, duodenal fluid, stool, and blood obFor example, tained from patients with cancer of the pancreas.9,1315,48,57,105,106,112,117 Caldas et all5 were able to detect mutant K-ras in 6 (55%) of 11 stool samples obtained from patients with pancreatic cancer. However, in this same study, mutant K-ras also was detected in one of three stool samples obtained from patients with chronic pancreatitis.15These results suggest that tests for mutant K-ras may lack the specificity needed for a population-based screening test. Although mutations in K-ras may lack the specificity needed for widespread population-based screening tests for pancreatic cancer, the results obtained from screening a variety of different samples from patients with pancreatic cancer for mutant K-ras have demonstrated the potential usefulness of gene-based tests. FAMILIAL PANCREATIC CANCER

Cancer of the pancreas occasionally clusters in families, and several reports of families in which multiple members have been affected have been published. These families are important because they provide a unique opportunity to study the genetics of cancer of the pancreas and because, as our understanding of the



genetics of cancer of the pancreas advances, these families may benefit from new gene based tests and preventive interventions for this disease. In general, the familial aggregation of cancer can be divided into two broad groups: those associated with known syndromes and those without such an a s s ~ c i a t i o n . ~ ~ Genetic Syndromes Associated with Pancreatic Cancer

The importance of genetic alterations in the development of cancer of the pancreas is perhaps most apparent in the genetic syndromes associated with an increased risk of developing pancreatic cancer. To date, at least six such syndromes have been identified (Table 2), including hereditary pancreatitis, ataxia-telangiectasia, hereditary nonpolyposis colorectal cancer (HNPCC),familial atypicalmultiple mole-melanoma, Peutz-Jeghers, and familial breast c a n ~ e r . ~ ~ , ~ ~ HNPCC

HNPCC strikes as many as 1 in 200 individuals and is associated with an increased risk of developing a cancer of the colon, endometrium, stomach, and ~ ~ a r y . ~HNPCC ~ , ~ is~ caused , ~ ~by, germline ~ ~ , ~mutations ~ in one of the DNA mismatch repair genes. These genes include MSH2 located on chromosome 2p, MLH1 on chromosome 3p, PMS2 on chromosome 7p, and PMS1 on 2q.54HNPCC is produced by germline mutations in any of these genes, and at the molecular level, patients with HNPCC develop characteristic multiple replication errors in dinucleotide repeats.' This phenotype is called the mutator phenotype.54 Recently, Lynch et aF9z70reported the development of pancreatic cancer in several kindreds with HNPCC. However, when large numbers of patients with pancreatic cancer have been examined, only 2% to 3% have been found to have . ~ ~ HNPCC can predispose to the development of the mutator p h e n ~ t y p e Thus, pancreatic cancer in some families, but the overall contribution of this syndrome to the development of pancreatic cancer is small. Of interest, we have found that pancreas cancers with the mutator phenotype frequently have a similar histologic appearance-a syncytial growth pattern, pushing boarders, and poor differentiation (unpublished observation).

The second breast cancer gene to be discovered is called BRCA2. BRCA2 is located on chromosome 13q12-13 and accounts for a significant minority of inherited breast cancers.8~18~58~85~103,"8 In addition, germline mutations of BRCA2 predispose to breast cancer in men.18 For example, 40% of all men diagnosed with breast cancer in Iceland over the last 40 years have been shown to harbor the same germline BRCA2 mutation."O Germline BRCA2 mutations also are associated with an increased risk of other malignancies, including cancers of the ovaries, prostate, colon, and p a r ~ c r e a s . ~ , ~ ~ , ~ ~ In fact, the discovery of BRCA2 was aided greatly by the localization of the BRCA2 locus in a pancreatic ~ a n c e r .Using ~ ~ , ~a ~technique called representational difference analysis, Schutte et a194identified a homozygous deletion in a pancreatic cancer that mapped to a 180-kb region on chromosome 13q. The mapping of this deletion facilitated the discovery of the BRCA2 gene, provided the first evidence that BRCA2 was a tumor suppressor gene as opposed to a proto-oncogene, and demonstrated that BRCA2 is inactivated in some pancreatic cancer^.^^,^^^,"^ The association of germline BRCA2 mutations with pancreatic cancer is remarkable because these mutations are associated with the development of pancreatic cancer



hereditary pancreatitis











MLHl PMS2 PMS 1 ~ 1 6




familial breast cancer BRCAP



AD cancer.




autosomal dominant, AR

autosomal recessive, FAMMM

Chromosome Locus



recurrent episodes of severe pancreatitis occurring at an early age over two generations cerebellar ataxia, telangiectasia, thymic hypoplasia colon and other cancers including breast, endometrial and ovarian; mutator phenotype

multiple nevi, atypical nevi, and melanomas hamartomatous polyps of the gastrointestinal tract and mucocutaneous melanin deposits female and male breast cancers, other cancers =

familial atypical multiple mole-melanoma syndrome, HNPCC


59,116 67,92 1,30,66,68-70,72,113

34,65,70,115 11,32,42 8,18,33,85,110,118

hereditary nonpolyposis colorectal



even in patients without a strong family history of breast cancer. For example, Goggins et aP3 screened an unselected series of patients with pancreatic cancer and found that 7% had germline BRCA2 mutations. Of note, none of the patients in this series came from a cIinically recognizable hereditary breast or pancreatic cancer family. These results suggest that germline BRCA2 mutations cause a small but significant fraction of all pancreatic cancers and importantly, that because of the low penetrance of the BRCA2 gene, these patients may not be recognizable without screening for BRCA2 mutations. Peutz-Jeghers Syndrome

Peutz-Jeghers syndrome is characterized by hamartomatous polyps of the gastrointestinal tract and by mucocutaneous melanin deposits.",32These patients are also at an increased risk of developing cancer. For example, Giardiello et a132 recently followed 31 patients with Peutz-Jeghers syndrome and found that 48% of these patients developed a cancer. Remarkably, four of these cancers were pancreatic cancer.32This observation represents a 100-fold excess of pancreatic cancer compared with that expected. The gene responsible for Peutz-Jeghers syndrome recently has been localized to a region on chromosome 19p.42Its role, if any, in pancreatic cancer can be established once the gene is definitively identified and sequenced. Ataxia-Telangiectasia

Ataxia-telangiectasia is an autosomal recessive inherited disorder characterized by disabling cerebellar ataxia, oculocutaneous telangiectasias, and cellular and humoral immune d e f i c i e n c i e ~ .The ~ ~ ,gene ~ ~ responsible for ataxia-telangiectasia, called ATM, encodes a protein believed to play a role in mitogenic signal transduction, meiotic recombination, and cell-cycle contr01.9~ Ataxia-telangiectasia is associated with an increased risk of neoplasia in homozygous patients, including leukemia and lymphoma. Heterozygous relatives (ATM carriers) are at increased risk for developing cancers of the breast, ovaries, biliary tract, stomach, and, rarely, the pancreas.'j7 Familial Atypical Multiple Mole-Melanoma

The familial atypicaI multiple mole-melanoma (FAMMM) syndrome is characterized by multiple nevi, multiple atypical nevi, and multiple melanom a ~ . ~ An ~ ,increased ~ ~ , ~risk ~ ,of~pancreatic ~ cancer has been reported in some kindreds with FAMMM. The increased risk of pancreatic cancer in these kindreds segregates with germline mutations in the p16 tumor suppressor gene.34,"5For example, Goldstein et a134compared 10 melanoma-prone kindreds with p16 mutations that impaired the function of the p16 gene product with 9 melanoma-prone kindreds with p16 mutations that did not and found that the risk of pancreatic cancer was increased 13-fold in the kindreds with mutations that impaired p16 function. Furthermore, as is true for pancreatic cancer and the BRCA2 syndrome, not all patients with germline p16 mutations come from families with the classical FAMMM syndrome. For example, Moskaluk et a179identified a germline p16 mutation that segregated with the development of pancreatic cancer in 1 of 21 kindreds with familial pancreatic cancer. None of these 21 kindreds were recognized to have the FAMMM syndrome, demonstrating that once the genetic basis of a cancer is identified, it may be found to contribute to the development of that cancer even among kindreds without the defining clinical phenotype of that syndrome. The demonstration that germline mutations in p l 6 predispose to pancreatic


HRUBAN et a1

cancer illustrates another important principal of cancer genetics: the same genes are frequently responsible for both the familial and for sporadic forms of a cancer.55 Inherited mutations in a gene lead to the familial form of a cancer, whereas acquired mutation of the same gene are associated with the sporadic forms of that cancer. For example, the Li-Fraumeni syndrome (a cancer syndrome characterized by breast cancer in young women, childhood sarcomas, and other neoplasms) has been shown to be caused by inherited point mutations in the p53 tumor suppressor gene,10,74 and inactivating mutations in p53 have been identified in sporadic breast cancers and in sarcomas.z7Cancer of the pancreas follows the same pattern. Germline mutations of p16 are associated with the development of pancreatic cancer in some families, and p16 is inactivated in more than 80% of sporadic pancreatic cancers.5r14,46.79r82,91

Hereditary Pancreatitis

Hereditary pancreatitis is a rare disease characterized by the early onset of recurrent episodes of severe chronic pan~reatitis."~ Affected individuals from kindreds with hereditary pancreatitis are at increased risk for developing pancreatic pseudocysts, pancreatic exocrine failure, diabetes mellitus, and pancreatic cancer.5z Recently, Whitcomb et alU6reported that an Arg-His substitution at residue 117 of the cationic trypsinogen gene is associated with the hereditary pancreatitis phenotype in some kindreds. Mutations at this site block the inactivation of trypsin, resulting in autodigestion of the pancreas. Thus, hereditary pancreatitis is an example of how the identification of an inherited disease can lead to the discovery of the gene responsible for that disease and how an understanding of the function of the gene involved can, in turn, uncover the mechanisms by which changes in that gene cause the disease phenotype. Unfortunately, finding the gene involved does not always lead to an understanding of how it causes disease. For example, the mechanism by which mutations in the trypsinogen gene cause pancreatitis is clear; however, the mechanisms by which hereditary pancreatitis causes pancreatic cancer are less clear. Perhaps the increased risk of pancreatic cancer observed in patients with hereditary pancreatitis is due to chronic epithelial regeneration from the pancreatitis i t ~ e l f . ' ~ , ~ ~ Summary of Genetic Syndromes

The study of genetic syndromes associated with the development of pancreatic cancer not only has led to the discovery of several important genes but also suggests that familial and sporadic cancers often involve the same genes, that the identification of a gene associated with a disease can lead to an understanding of the mechanisms underlying the development of that disease, and that germline mutations can play a role in the development of cancer of the pancreas in patients without a recognized genetic syndrome. The following section therefore examines families that are not part of known genetic syndromes but in which there is an aggregation of pancreatic cancer. Familial Cancer

For a number of years, anecdotal case reports have suggested that cancer of Although the aggregation the pancreas clusters in some families.19~25~51~6244~70~71~73~88 of a cancer in a family may occur by chance or because the affected family members share a common nongenetic environmental exposure (such as cigarette smok-



ing), a growing body of evidence currently suggests that the familial aggregation of cancer of the pancreas is genetically based. For example, a case control study conducted by Fernandez et a129examined the relationship between family history and the risk of liver, gallbladder, and pancreatic cancer in Northern Italy. The authors found that patients with a family history of pancreatic cancer were more cancer themselves than were controls (relative risk = likely to develop 3.0, 95% confidence interval = 1.4-6.6).29Similarly Ghadirian et al3l performed a population-based case control study of cancer of the pancreas in Montreal, Canada. They compared 179 patients with pancreatic cancer with 179 controls selected by random digit dialing and matched for age, gender, and language (French speaking).31Remarkably, 7.8% of the patients with pancreatic cancer had a family history of pancreatic cancer compared with 0.6% of the controls. This represents a 13-fold difference between cases and controls.31Furthermore, no differences in environmental exposures were documented between the familial and nonfamilial cases; thus, the aggregation of pancreatic cancer in the families could not be explained by shared environmental risks.31These case-control studies confirm the anecdotal observations that cancer of the pancreas can aggregate in families. Families in which an aggregation of pancreatic cancer exists are important because they provide a unique opportunity to study efficiently the clinicalpatterns and genetics of cancer of the pancreas and because members of these families may benefit most from a better understanding of this disease. Because of the relative rareness of these families, we have established the National Familial Pancreas Tumor Registry (NFPTR).60As of November 1, 1997, 84 families in which two or more first-degree relatives have been diagnosed with pancreatic cancer have joined the NFPTR, as have almost 165 families with sporadic pancreatic cancer. The NFPTR contains numerous families, including families in which multiple siblings are affected (Fig. 3), families in which members from several generations are affected (Fig. 4), and families in which there is an aggregation of both a pancreatic and breast cancer (Fig. 5). Table 3 provides a detailed description of the familial pancreatic cancer kindreds in the NFPTR. These include 38 families in which one generation is affected, 69 families in which two generations are affected, and 7 families in which three generations are affected. This research-based registry is designed to examine (1) the possible contribution of environmental risk factors, such as smoking, to the development of familial pancreatic cancer, (2) the patterns of inheritance of pancreatic cancer (autosomal dominant, autosomal recessive or X linked, and the degree of penetrance), and (3) the types and prevalence of other tumor types (such as melanoma and breast cancer). Finally, as new genes are identifi;dand found to play a role in sporadic pancreatic cancer, affectedmembers in the NFPTR can be tested to determine if germline mutations in these same genes cause the familial aggregation of pancreatic cancer. Persons who wish to enroll patients or themselves in the NFPTR are urged to contact the registry at:




Colon ca @ 66 Pancreas ca @ 67 Pancreas ca @ 72 Uterlne ca



115 '

Pancreas ca @66


Pancreas ca @ 67 Pancreas ca @ 67 K~dneyca

Figure 3. Pedigree of National Familial Pancreas Tumor Registry (NFPTR) family 1932showing five siblings with pancreas cancer.


HRUBAN et a1

Pancreas ca

Lung ca

Pancreas ca Prostate ca


Pancreas ca @ 67

Figure 4. Pedigree of National Familial Pancreas Tumor Registry (NFPTR) family 1062 showing pancreas cancer in three generations.

The National Familial Pancreas Tumor Registry Meyer 7-181 Department of Pathology 600 N. Wolfe Street Baltimore, MD 21287 Phone: (410) 955-9132 Fax: (410) 955-0115

We examined data from the first 212 kindred enrolled in the NFPTR to identify similarities and differences between familial and sporadic cases of pancreatic cancer. Sporadic pancreatic cancer cases were defined as those patients with pancreatic cancer who had no first-degree relatives with pancreatic cancer. Familial pancreatic cancer cases were defined as those patients with pancreatic cancer who had at least one first-degree relative with pancreatic cancer. We then compared these two groups for the ages at which the pancreatic cancers were diagnosed in the index cases, for the other types of cancer that the index cases developed, and for the cancers that occurred in other family members. As seen in Table 4, this analysis included 80 familial and 132 sporadic pancreatic cancer cases. In contrast to familial breast cancer and familial colon cancer, which are characterized by younger age at onset, no difference in the age at which pancreatic cancer was diagnosed was observed between the familial cases and sporadic cases (both groups have a mean age of 65 years). A nonsignificant, slightly increased proportion of familial pancreatic cancer index cases had a second primary cancer (23.8%versus 18.9%),with the main types including cancers of the bladder, colon, lung, and prostate, and melanoma. Among sporadic pancreatic cancer cases, the main second primaries were cancers of the breast, colon, liver, and prostate. When we examined the risk of cancer in the relatives of the index cases, we found that the risk of cancer was higher in first-degree relatives of familial pancreatic cancer cases [174/618 (28.2%)]than it was in first-degree relatives of sporadic pancreatic cancer cases [133/894 (14.9%)](Table 5). As expected from the definition of familial pancreatic cancer, this increase was primarily due to pancreatic cancer in the first-degree relatives of the familial pancreatic cancer cases, because the risk of cancer other than pancreatic cancer was similar for the two groups of first-degree relatives (11% and 14.9%).However, the increased risk of pancreatic cancer in familial pancreatic cancer kindreds extended to second-degree relatives.


Pancreas ca


Pancreas ca Breast ca

Pancreas ca Breast ca

Breast ca

Breast ca

Pancreas ca

Breast ca Bladder ca

Pancreas ca

IV:5 Thyro~dca

Ovarian ca Uterine ca

Breast ca




IV:7 Breast ca

Figure 5. Pedigree of NFPTR family 556 showing the aggregation of pancreas and breast cancer. Five family members developed pancreas cancer and seven developed breast cancer.


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Relationship of Affected Persons One Generation 2 siblings 3 siblings 4 siblings 5 siblings 2 siblings and one third-degree relative 2 siblings and two third-degree relatives Two Generations parent, 1 offspring parent, 2 offspring parent, 3 offspring parent, 1 offspring, one sibling parent, 1 offspring, three siblings parent, 2 offspring, one sibling parent, offspring, sibling, one second-degree relative parent, offspring, sibling, one third-degree relative parent, offspring, one second-degree relative parent, 1 offspring, one third-degree relative Three Generations grandparent, parent, grandchild grandparent, parent, grandchild, one firstdegree relative grandparent, parent, grandchild, two firstdegree relatives grandparent, parent, grandchild, one thirddegree relative TOTAL

No. of Kindreds

Total No. of Pancreas Cancer Cases

22 8 2 1 4 1

44 24 8 5 9 3

45 5 3 7 1 1 1

90 15 12 21 5 4 4



2 3

6 9

3 2

9 8







A significantly increased proportion of pancreatic cancer existed in second-degree relatives of familial cases compared with sporadic pancreatic cancer cases [12/324 (3.7%)versus 4/702 (0.6%),P < 0.00011. This difference was also true for all cancer types in second-degree relatives of the familial pancreatic cancer cases (27.2% versus 12.1%, P < 0.0001). The most common primaries to occur in relatives of pancreatic cancer patients, both sporadic and familial, were breast cancer, lung cancer, and colon cancer. Although these may represent coincidental occurrences in these families because these cancers are relatively common, there have been reports of an aggregation of pancreatic and colon cancer and pancreatic and breast cancer in epidemiologic s t u d i e ~ . ' ~ ~ , ~ ~ ' Because of the possibility that the definition of familial pancreatic cancer that we employed in our analyses might be too broad, we defined a highly familial subset of familial pancreatic cancer as those patients with pancreatic cancer who had two or more first-degree relatives with pancreas cancer. Nineteen cases met these criteria, and the mean age at diagnosis for this subset of patients with familial pancreatic cancer was only slightly lower (64 years 2 11.1),but the proportion of index cases who had a second primary cancer was increased to 36.8% (7 of 19). Taken together, -theseresults demonstrate that a familial aggregation of pan-




mean age at diagnosis of pancreas cancer range patients (% of total N) with additional primary cancer type of primary*

Familial Pancreas Cancer

Sporadic Pancreas Cancer

bladder, colon, lung, melanoma, prostate, breast, laryngeal, liver, brain, urethra, testes

breast, colon, liver, prostate, gallbladder, lung, melanoma, ovary, uterus, cervix, tongue, kidney, cheek, arm, brain, lymphoma, adrenal

'Most commonly reported primary cancers are shown in bold print. No single cancer type exceeds 3% of the total.

creatic cancer exists and suggest that the age at diagnosis cannot be used to distinguish familial pancreatic cancer cases from sporadic pancreatic cancer cases. Unlike the experience of hereditary colorectal cancer, in which the Amsterdam criteria for HNPCC7 have had reasonable discriminatory power to identify a homogeneous group of high-risk families, criteria based on other clinical or molecular studies need to be developed to further characterize familial pancreatic cancer. Summary of Familial Pancreatic Cancer

Cancer of the pancreas rarely aggregates in families, but evidence exist that some forms of familial pancreatic cancer have a genetic basis. Further studies are needed to establish the patterns of inheritance in these families and to identify the genes responsible for this aggregation. An improved understanding of the genetics of cancer of the pancreas in turn will provide a rational strategy to screen family members for cancer of the pancreas.


Cancer of the pancreas is a genetic disease. To date, more than 80% of resected pancreatic cancers have been found to harbor activating point mutations in K-uas. In addition, the tumor suppressor genes pl6, p53, and DPC4 are all frequently inactivated in this cancer. Many of the same genes responsible for the development of sporadic cancers of the pancreas also play a role in familial forms of this disease. In familial forms, patients inherit a germline mutation in one allele of a cancercausing gene and acquire the second hit to the second allele of this gene later in life. In sporadic forms of pancreatic cancer, both "hits" are acquired. An improved understanding of the genetics of this disease should lead to rational gene-based screening tests and therapies for cancer of the pancreas. Important steps toward this understanding already have been taken.


Sporadic Pancreas Cancer (SPC)

Second-Degree Relatives

First-Degree Relatives

Second-Degree Relatives

N number of relatives with pancreas cancer (%) number of relatives with other cancers (%) total number of relatives with any cancer type of primary observed in relativest

breast, liver, lung, stomach, colon, bladder, brain, esophageal, kidney, laryngeal, leukemia, lymphoma, melanoma, ovary, prostate, thyroid, uterus

breast, colon, lung, prostate, stomach, bladder, bone, brain, liver, gallbladder, leukemia, liver, lymphoma, melanoma, ovary, uterus, cervix, throat, thyroid, tonQue,kidney

'P < 0.0001 for FPC vs. SPC second-degree relatives. tMost commonly reported primary cancers are shown in bold print. No single cancer type exceeds 3% of the total



ACKNOWLEDGMENTS The authors would like to thank Michele Heffler and Flo Falatko for their hard work and dedication. To learn more about pancreas cancer visit our Website at

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