The genetics of pancreatic cancer

The genetics of pancreatic cancer

The American Journal of Surgery 186 (2003) 279 –286 Special article The genetics of pancreatic cancer Sarah M. Cowgill, M.D., Peter Muscarella, M.D...

206KB Sizes 15 Downloads 207 Views

The American Journal of Surgery 186 (2003) 279 –286

Special article

The genetics of pancreatic cancer Sarah M. Cowgill, M.D., Peter Muscarella, M.D.* Department of Surgery, Ohio State University Medical Center and Ohio State University Comprehensive Cancer Center, N711 Doan Hall, 410 West 10th Ave., Columbus, OH 43210, USA Manuscript received April 30, 2003

Abstract The genetic basis for invasive and preoneoplastic neoplasms of the exocrine and endocrine pancreas has been the subject of a number of investigations in recent years. The purpose of this paper was to briefly review and summarize the pertinent findings. High frequency changes associated with pancreatic adenocarcinomas include mutations of the k-ras oncogene, and inactivating alterations of the p53, p16, and DPC4 tumor suppressor genes. Hereditary syndromes that have a known predisposition for pancreatic adenocarcinoma development include hereditary pancreatitis, familial atypical multiple mole melanoma (FAMM) syndrome, Peutz-Jeghers syndrome, familial breast cancer (BRCA-2), hereditary nonpolyposis colorectal cancer syndrome (HNPCC), and Li-Fraumeni syndrome. The underlying genetic defects have been identified and are currently being studied. Germline mutations of the men-1 gene are responsible for the MEN-1 syndrome, known to be associated with pancreatic endocrine tumors. It appears that somatic mutations of the gene are present in at least a subset of sporadic tumors. In addition, alterations in the Rb/p16 pathway appear to be commonly associated with pancreatic endocrine tumors. Further characterization of pancreatic tumors will result in a better understanding of the cellular pathways involved in pancreatic tumorigenesis and holds promise to identify targets for novel diagnostic and therapeutic strategies. © 2003 Excerpta Medica, Inc. All rights reserved. Keywords: Pancreatic cancer; Pancreatic endocrine tumors; p53; p16; DPC4; K-ras; MEN-1

In recent years, a considerable body of data has been accumulated regarding the molecular and genetic characterization of pancreatic cancers. Pancreatic adenocarcinoma currently represents the fifth most common cancer causing death in the United States [1], and histologically, constitutes approximately 90% of pancreatic tumors. The histopathological spectrum also includes neuroendocrine tumors, cystadenocarcinomas, and papillary mucinous tumors. Known genetic changes associated with these tumors will be briefly reviewed. Despite advances in medical science, the overall prognosis for pancreatic cancer remains grim, with current 5-year survival rates of only 4% [1]. Surgical resection remains the only hope for cure, although only 5% to 20% of patients are candidates at the time of presentation due to the biologically aggressive nature of these tumors. Even in this select group of patients, only 25% can hope to survive for 5 years. Adjuvant chemotherapy and radiation may allow for * Corresponding author. Tel.: ⫹1-614-293-5815; fax: ⫹1-614-2934030. E-mail address: [email protected]

marginal increases in survival and remain under investigation. Clearly, novel diagnostic and therapeutic strategies are needed, and a cohesive understanding of the genetic regulatory pathways involved in tumorigenesis may provide the necessary targets. Although encountered with considerably lower frequencies in the clinical setting, pancreatic endocrine neoplasms have been studied through various welldescribed syndromes and may provide insight into the genetic profiles of all pathologic types of pancreatic cancer. Pancreatic tumorigenesis appears to conform to the Vogelstein model, as classically described for colorectal cancers [2]. Multiple checkpoints in cell proliferation and differentiation must be affected in order for a normal cell to differentiate into a neoplasm (Fig. 1). Key genetic foci contributing to tumor development may be broadly classified as tumor suppressor genes or oncogenes. Generally speaking, tumor suppressor genes express proteins that halt cell proliferation while oncogenes encode proteins that promote cell growth. Both are critical elements in growth regulation. Multiple genetic abnormalities have been identified in pancreatic cancer; the more well characterized findings are discussed here.

0002-9610/03/$ – see front matter © 2003 Excerpta Medica, Inc. All rights reserved. doi:10.1016/S0002-9610(03)00226-5


S.M. Cowgill and P. Muscarella / The American Journal of Surgery 186 (2003) 279 –286

Fig. 1. Regulatory pathways involved in cancer initiation and progression. (Reproduced with permission from Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.)

Pancreatic ductal adenocarcinoma An accumulation of inherited and acquired genetic defects results in the neoplastic transformation and progression of pancreatic cancers. The oncogene k-ras is almost universally mutated in pancreatic cancer, but does not appear to be a particularly specific finding for cancer. In contrast, knowledge of tumor suppressor gene involvement in pancreatic ductal adenocarcinoma tumorigenesis continues to grow (Table 1), and it appears that changes in a number of their regulatory pathways is extremely important for neoplastic growth. K-ras K-ras mutations are found in approximately 90% of pancreatic cancers, but have also been identified in a number of benign conditions, including chronic pancreatitis [3–5]. The ras group of proteins function as a part of the membrane-associated, signal-transduction pathway of GTPbinding proteins. Once activated, these proteins code for key mediators in a number of pathways regulating cell growth and differentiation. Mutations in the oncogene have

been found in all degrees of ductal anaplasia and alterations are considered to be early events in tumorigenesis [6]. Studies of k-ras mutations in chemically induced hamster ductal adenocarcinomas and preneoplastic lesions confirm this conclusion [7]. Most commonly, k-ras is present as a single mutation in cancer clones, but multiple oncogenic mutations have been described. In general, k-ras status has not been found to correlate with changes in survival, although specific mutations may be associated with prognosis [8,9]. p16 Abrogation of the Rb/p16 tumor-suppressive pathway is thought to be an important mechanism in the development of many human cancers [10 –12]. The pathway is a well described component of global cell cycle regulation and inactivation may occur through alterations of Rb, CDK4, cyclin D, or p16. These alterations occur almost universally exclusively of each other, indicating that only one event is necessary for abrogation of the pathway. The p16 gene product is a 16 Kd protein that inhibits formation of cyclin D/CDK4 complexes. Loss of p16 function results in the

S.M. Cowgill and P. Muscarella / The American Journal of Surgery 186 (2003) 279 –286


Table 1 Summary of genetic alterations in pancreatic tumors according to histopathological type Adenocarcinoma

Sporadic k-ras p53 p16 DPC4 Hereditary PRSS1 FAMM (p16) STK11/LKB1 BRCA-2

Pancreatic endocrine tumors

men-1 p16 p27 Cyclin D DPC4

Intraductal papillary mucinous tumors k-ras p53 p16 — Fig. 2. Tumor suppressor gene p16 deletion analysis in hamster cell lines by multiplex polymerase chain reaction. POT2 and HCPC represent hamster cheek pouch carcinoma cell lines; KL5B and H2T represent hamster pancreatic carcinoma cell lines; KL5N represent a nontumorigenic precursor cell line to KL5B. Note the absence of p16 amplification for cell lines HCPC, KL5B, and H2T indicating the presence of homozygous p16 deletion in these specimens.

men-1 Von Hippel-Lindau (VHL) Von Recklinghausen’s disease (NF-1) Tuberous sclerosis (TSC1,TSC2)

HNPCC Li-Fraumeni syndrome (p53)

release of activated transcription factors and progression of the cell cycle through the G1/S checkpoint. Frequent alterations of p16 have been described in a number of human malignancies and mechanisms of inactivation include homozygous deletion, mutation, and aberrant methylation of 5' CpG islands. A number of studies suggest a significant role for p16 inactivation in pancreatic cancer development (Table 2). Liu et al [13] initially identified p16 mutations in 50%, and homozygous deletions in 30%, of 10 human pancreatic cancer cell lines. The identification of frequent deletions (41%) and mutations (38%) in primary pancreatic tumor specimens indicated that these changes were more likely associated with carcinogenesis than immortalization in cell culture. Subsequent studies have confirmed the presence of p16 inactivation, by homozygous deletion or mutation, in 27% to 82% of primary tumors [14,15]. When 5' CpG island methylation is considered, 98% (49 of 50) of pancreatic cancers exhibit abrogation of the Rb/p16 tumor-suppressive pathway by potentially inac-

tivating p16 gene alterations [16]. Immunohistochemical confirmation of loss of p16 expression in affected tumors indicates that these genetic events have functional significance. A significantly increased risk of pancreatic cancer in melanoma-prone kindred families with p16 mutations lends further support to the role of p16 inactivation in human pancreatic carcinogenesis [17]. A number of recent studies have demonstrated that transfection of wild-type p16 into human pancreatic cancer cells results in decreased tumor cell proliferation in vitro and in vivo [18,19]. Studies performed in our laboratory at Ohio State University indicate that 93.3% of chemically induced hamster pancreatic tumors demonstrate inactivating alterations of p16 (Fig. 2). These findings suggest that this model may be useful for evaluating specific therapies targeting p16. Recent studies indicate that tumor suppressor gene alterations may be associated with prognosis. Gerdes et al [20] divided two groups of patients with pancreatic adenocarcinoma into short- and long-term survivors, and evaluated p16 and p53 status in both groups. Noting no significant differences in the size or stage of the pancreatic ductal adenocarcinomas between the groups, a significantly greater frequency of p16 alterations were found in the short-term

Table 2 Summary of studies evaluating p16 inactivation in pancreatic tumor cell lines and primary tumors. Study



Homozygous deletion



Caldas et al

PCR Direct sequencing Multiplex PCR SSCP RT-PCR Direct sequencing PCR Direct sequencing

Cell lines Tumors Cell lines Tumors Cell lines Tumors Cell lines

5/10 (50%) 10/27 (37%) 5/18 (28%) 3/30 (10%) 7/19 (37%) 3/3 (100%) 5/9 (56%)

3/10 (30%) 11/27 (41%) 7/18 (39%) 5/30 (17%) 5/19 (26%) 0/3 (0%) 3/9 (33%)

8/10 (80%) 21/27 (78%) 12/18 (67%) 8/30 (27%) 12/19 (63%) 3/3 (100%) 8/9 (89%)

Huang et al Naumann et al Liu et al

PCR ⫽ polymerase chain reaction; SSCP ⫽ single-strand conformation polymorphism; RT-PCR ⫽ reverse transcription-polymerase chain reaction.


S.M. Cowgill and P. Muscarella / The American Journal of Surgery 186 (2003) 279 –286

survivors when compared to the long-term survivors (85% versus 50%). Molecular characterization of tumors may provide clinically useful information for guiding further therapy after surgical resection. p53 The p53 tumor suppressor gene is ubiquitous in the discussion of genetic profiles of human carcinomas and is inactivated in 40% to 75% of pancreatic cancers [21,22]. The protein expressed by p53 responds to cellular DNA damage. Like p16, it may block the progression of cells through the G1 phase of the cell cycle. The protein is also capable of mediating cell death, or apoptosis, by detecting irreversible DNA damage within a cell. Many of the upstream controls and downstream actions of p53 are poorly understood, but interactions with many regulatory pathways have been identified and appear to be highly complex. In pancreatic cancer, the primary mechanism of p53 inactivation appears to be mutation, as homozygous deletions have not been observed. Mutated p53 proteins have been shown to inhibit wild-type p53, and bi-allelic inactivation may not be necessary for loss of function. Alterations of p53 are associated with k-ras mutations suggesting a cooperative effect in tumorigenesis [6,21,23]. Germline mutation of p53 is known to be the underlying genetic defect in the LiFraumeni syndrome of childhood malignancies, bone and soft-tissue sarcomas, premenopausal breast carcinoma, brain tumors, adrenocortical carcinoma, and leukemias. Pancreatic adenocarcinoma is the only common, adult epithelial malignancy that has been proven to be associated with Li-Fraumeni syndrome, other than breast cancer. This identifies an additional form of hereditary pancreatitis. DPC4 The DPC4 (deleted in pancreatic cancer locus 4) tumor suppressor gene is deleted in approximately 50% of pancreatic cancers [24]. Up to 90% of pancreatic adenocarcinomas may harbor loss of heterozygosity. The gene product, Smad 4, is a component of the TGF-␤ signal transduction pathway [25–28]. The functional role of DPC4 in pancreatic cancer has yet to be clarified, but it has been shown that inactivation of DPC4 in murine intestinal neoplasms is associated with more invasive growth [29]. Germline mutations have also been identified in approximately 30% of patients with benign juvenile intestinal polyposis [30].

Multiple genetic abnormalities No single genetic change has been identified as the catalyst for tumorigenesis in pancreatic ductal adenocarcinoma, but several groups have attempted to evaluate the cummulative effects of various alterations. Of 42 pancreatic tumors specimens analyzed by Rozenblum et al [22], 100%

were found to have point mutations in k-ras, consistent with other studies. Mutations in p16, p53 and DPC4 were found in 82%, 76%, and 53%, respectively. Interestingly, alterations in all three tumor suppressors and k-ras were found in 38%, or 15 of 39 tumors. Furthermore, DPC4 inactivation was always accompanied by inactivation of p16, while the reverse was not consistently true. This would suggest that interactions among the identified pathways may confer selective growth advantages for neoplastic or affected subclonal populations

Familial pancreatic cancer Hereditary pancreatitis Chronic hereditary pancreatitis is a autosomal dominant syndrome with variable expression and a penetrance of approximately 80%. The genetic defect is found on chromosome seven and encodes a defective form of the cationic trypsinogen gene (PRSS1). The risk of developing pancreatic adenocarcinoma in patients with chronic pancreatitis may be increased by up to 20-fold over the normal population [31]. Lowenfels et al [32] studied the frequency of pancreatic adenocarcinoma in patients with known hereditary pancreatitis and noted that, compared with an expected number of 0.15, 8 patients developed pancreatic cancers. While the estimated cumulative risk of pancreatic cancer at age 70 is 40%, with paternal inheritance patterns, the risk rises to 75%. Familial atypical multiple mole melanoma syndrome Familial atypical multiple mole melanoma (FAMMM) syndrome was described in 1975 as a syndrome associated with dysplastic nevi and melanomas, and was linked to development of pancreatic cancer [33,34]. Again, germline mutation of the p16 tumor suppressor gene is the underlying etiology of this syndrome. Goldstein et al [17] identified a 22-fold increased risk for developing pancreatic cancer in FAMM family members. Of 10 kindreds with a history of invasive melanoma and germline p16 mutations, four had at least one family member with a pancreatic cancer. None of the 9 kindreds with known invasive melanoma and wildtype p16 had any pancreatic cancer in their families [17]. Peutz-Jeghers syndrome Peutz-Jeghers syndrome is an autosomal dominant syndrome with variable penetrance. Affected individuals manifest hamartomatous gastrointestinal polyps and perioral pigmented spots. The genetic defect in this syndrome is a germline mutation in the tumor suppressor gene STK11/ LKB1. The syndrome confers a significant predisposition to other gastrointestinal cancers, including biliary and pancreatic malignancies. Giardiello et al [35] estimated the relative

S.M. Cowgill and P. Muscarella / The American Journal of Surgery 186 (2003) 279 –286

risk of pancreatic cancer in Peutz-Jeghers syndrome to be 132 . STK11/LKB1 inactivation has been identified in 4.6% of sporadic pancreatic and bilary cancers [36]. BRCA2 The function of BRCA2 has not been established, but the gene product appears to be a tumor suppressor participating in DNA damage repair. Mutations in BRCA2 have been linked to hereditary breast and ovarian cancers, but recent studies suggest a significantly increased risk of pancreatic cancer development in patients with germline BRCA2 mutations. Goggins et al [37] evaluated 41 pancreatic adenocarcinomas and 15 demonstrated loss of heterozygosity at BRCA2, while four harbored mutations. Germline BRCA2 mutations were responsible for 7.3% of inactivating events, indicating that both germline and sporadic events may play a role in pancreatic cancer tumorigenesis [37]. Hereditary nonpolyposis colorectal cancer syndrome Hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is a result of germline mutations in DNA mismatch repair genes. The disease is characterized by the development of colorectal cancers and is associated with an increased propensity for the development of other types of cancer. The most common extracolonic cancer associated with HNPCC is endometrial cancer, but other cancers have also been linked, including pancreatic. Intraductal papillary-mucinous tumors Intraductal papillary-mucinous tumors (IPMT) of the pancreas are a recently defined histopathologic entity of pancreatic malignancy. Characteristic features include intraductal papillary projections and dilated, mucin-filled ducts. In 1996 the World Health Organization defined this entity of pancreatic disease and classified them into four groups including benign adenomas, borderline tumors with moderate dysplasia, and invasive and noninvasive carcinomas [38]. IPMTs are considered to be less aggressive than ductal adenocarcinomas and the genetic profiles of these tumors appear to be distinct. In a recent study, 65% of patients with IPMTs harbored k-ras mutations, as compared with 92% of patients with invasive adenocarcinomas. Interestingly, loss of heterozygosity of p16 and p53 was significantly more frequent in invasive IPMTs than noninvasive IPMTs with 100% of invasive carcinomas demonstrating loss of heterozygosity of both p16 and p53 [39]. Pancreatic intraepithelial neoplasia Because pancreatic ductal adenocarcinoma carries such a grim prognosis and is typically detected and treated once the


lesion has spread beyond the boundaries of the pancreatic parenchyma, effort has been put forth to characterize a group of ductal lesions that may be precursors to pancreatic adenocarcinoma. A relatively new classification system has been proposed for these precancerous lesions based on the degree of dysplasia [40]. Three categories have been established: PanIN 1A, PanIN 2, and PanIN 3. These lesions currently have minimal clinical significance, as the retroperitoneal location of the pancreas is inaccessible for routine evaluation and early lesions are typically asymptomatic. The majority of genetic studies rely upon the evaluation of histologically identified precursor lesions found adjacent to invasive cancers in surgically resected specimens. While the role of k-ras in noninvasive pancreatic duct lesions has yet to be determined, several groups have studied this with various results. Mutations in k-ras appear to occur early and most papillary hyperplasias contain some form of mutation. DPC4 expression appears to be unaffected in PanIN 1A, 1B and 2, but only 31% of those classified as PanIN 3 demonstrated DPC4 expression, suggesting that loss of functional DPC4 occurs late in the tumor progression [41]. The same may be true for BRCA2, as few low grade pancreatic intraepithelial neoplastic lesions demonstrate inactivation [42].

Pancreatic endocrine tumors Pancreatic endocrine tumors are extremely rare with an estimated incidence of one to five cases per million population annually. They account for less than 1% of all pancreatic neoplasms. It can be expected that a single physician will care for no more than one, or perhaps two, patients with pancreatic endocrine tumors in his or her lifetime. At the current time, no significant environmental risk factors have been identified as being associated with the development of these tumors. This is most likely due to the rare nature of these tumors. Four genetic syndromes, however, have been reported to be associated with pancreatic endocrine tumor development. These include the multiple endocrine neoplasia type I syndrome (MEN-1), von Hippel-Lindau (VHL) disease, von Recklinghausen’s disease (NF-1), and tuberous sclerosis [43– 46]. The MEN-1 syndrome of parathyroid hyperplasia, pancreatic neuroendocrine tumors, and pituitary adenomas has the strongest association with the development of pancreatic endocrine tumors and forms the basis for classification of these tumors into sporadic and familial variants. Approximately 30% to 50% of MEN-1 patients develop symptomatic pancreatic endocrine tumors, and up to 100% of patients have been found to harbor small, nonfunctioning pancreatic endocrine tumors in pathology studies [46,47]. Twenty-five percent of gastrinoma patients have MEN-1 syndrome, but only 4% to 5% of insulinoma patients are diagnosed with MEN-1. Loss of heterozygosity (LOH) involving a region


S.M. Cowgill and P. Muscarella / The American Journal of Surgery 186 (2003) 279 –286

Fig. 3. Men-1 mutation analysis of genomic DNA extracted from a sporadic gastrinoma specimen (right) and a normal control (left). Direct sequence analysis demonstrates a novel one-base pair insertion at nucleotide 643, producing a frameshift mutation and a truncated, nonfunctional protein.

lying on the short arm of chromosome 11 was initially reported to occur frequently in tumors harvested from MEN-1 patients [47– 49]. These data suggested the presence of a possible tumor suppressor gene. The responsible gene was subsequently cloned and the gene product termed Menin [50]. Germline mutations have also been identified, confirming that abnormalities of this gene are responsible for the clinical syndrome. Further studies indicate that Menin is a nuclear protein, although the exact function of this protein remains unclear [51]. It is crucial that all patients with PET’s be evaluated clinically for the MEN-1 syndrome. Genetic testing for MEN-1 gene mutations is currently tedious and is not widely available. Knowledge of other genetic changes associated with the initiation and progression of pancreatic endocrine tumors is limited, but appears to be growing. Several recent advances in the molecular characterization of these tumors deserve mention. The association between familial pancreatic endocrine tumors and men-1 mutation/LOH has previously been discussed. Studies of sporadic gastrinomas and other pancreatic endocrine tumors demonstrate allelic deletion of men-1 in 58.4% of specimens and mutation in 23.2% (Fig. 3) [52]. The frequency of men-1 gene inactivation is variable among tumor types. Allelic deletions occur in up to 93% of gastrinomas and 50% of insulinomas, while mutations are present in 33% of gastrinomas and 17% of insulinomas [53]. VIPomas and somatostatinomas demonstrate remarkably high frequencies of men-1 inactivating alterations in small sample groups [54]. In a recent study of 25 PET’s, DPC4/Smad4 mutations or deletions were identified in 55% of nonfunctioning pancreatic endocrine tumors, while no abnormalities were identified in 16 functional tumors [55]. These results indicated that DPC4 might be an important target in the development of nonfunctioning tumors. Based on a study of 11 gastrinomas, Evers et al [56] have concluded that amplification of

the HER-2/neu proto-oncogene may be involved in the pathogenesis of gastrinomas. These data are based on the results of semiquantitative polymerase chain reaction analyses of genomic DNA. There are no published data, however, that demonstrate elevated levels of HER-2/neu messenger RNA or protein in gastrinoma specimens. Immunohistochemical analysis of 20 gastrinomas and nonfunctioning pancreatic endocrine tumors, performed in our laboratory, failed to demonstrate membranous staining with antibody to the HER-2/neu gene product. This suggests that genomic amplification of HER-2/neu may not have functional significance. Subsequent studies have confirmed these observations [57]. K-ras and p53 mutations do not appear to play a role in the development of endocrine tumors of the pancreas [56,58,59]. Recently, there have been a number of studies evaluating the role of Rb pathway alterations in the development of pancreatic endocrine tumors. No instances of Rb tumor suppressor gene allelic loss were identified in a study of 46 tumors [60]. Our laboratory initially identified the presence of p16 inactivating events, by homozygous deletion (42%) and 5⬘CpG island methylation (58%), in a study of 12 gastrinomas and nonfunctioning pancreatic endocrine tumors [61]. Overall, 92% of the specimens demonstrated inactivating p16 alterations. These data suggested that transcriptional silencing of the p16 gene could be an important event in the development of gastrointestinal endocrine tumors. A later study confirmed the finding of frequent p16/ MTS1 methylation in pancreatic endocrine tumor specimens, but failed to identify further deletions [62]. Inactivating p16/MTS1 mutations and deletions have been identified in insulinomas, but appear to occur with lower frequencies (17%) [63]. Another recent study failed to demonstrate any instances of p16/MTS1 inactivation in a large number of pancreatic endocrine tumors [64]. In the same study, no homozygous deletions were identified in 34 pancreatic ductal cancer specimens. This would seem to contradict previous studies that typically report a much higher rate of p16 deletional inactivation in exocrine pancreatic tumors [13–16,65]. RNA expression analysis of 9p21 locus tumor suppressor genes demonstrates that loss of p14, p15, or p16 occurs frequently in neuroendocrine gastroenteropancreatic tumors. Loss of expression of at least one of the three tumor suppressors was identified in 57% of nonfunctioning pancreatic endocrine tumor, 44% of small intestinal carcinoids, 30% of insulinomas and 22% of gastrinomas [66]. Interestingly, paradoxical overexpression of the CDK inhibitor p27 has been identified in a study of pancreatic endocrine tumors and endocrine tumor cell lines [67]. Finally, Chung et al [68] have identified over-expression of cyclin D in 43% of 64 pancreatic endocrine tumors. At the current time, it would appear that further investigations evaluating the role of Rb pathway involvement in pancreatic endocrine tumor pathogenesis are warranted.

S.M. Cowgill and P. Muscarella / The American Journal of Surgery 186 (2003) 279 –286

Comments Pancreatic adenocarcinoma continues to be a devastating disease with almost universally fatal consequences. Recent advances in molecular biology have resulted in a significant increase in current knowledge regarding the genetic events associated with pancreatic tumorigenesis. High frequency changes include alterations in the k-ras oncogene and the p53, p16, and DPC4 tumor suppressor genes. A number of other low frequency changes and associated hereditary syndromes have been described. Further characterization of the molecular changes associated with tumor initiation and progression will shed light on the essential cellular pathways responsible for maintaining noncancerous states. Potential targets for novel therapeutic and diagnostic strategies have been identified and more will almost certainly be recognized. The molecular basis for endocrine pancreatic tumor development has been poorly characterized until recently. Men-1 and Rb-pathway alterations appear to play a significant role and represent targets for intervention. The genetic characterization of preneoplastic lesions and intraductal papillary mucinous tumors will also undoubtedly continue, as they become increasingly recognized. Doctor Zollinger’s keen interest in the basic science of pancreatic malignancies was clearly a priority for him and this is proven by his invaluable contributions to our understanding of their pathophysiology. Specimens collected by Zollinger continue to be studied as part of our gastrinoma clinical database and tissue bank. Undoubtedly, the genetics of pancreatic cancer would be a major focus of interest for him if he were here with us today.













References [1] Jemal A, Murray T, Samuels A, et al. Cancer statistics 2003. CA Cancer J Clin 2003;53:5–26. [2] Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319:525– 32. [3] Almoguera C, Shibata D, Forrester K, et al. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53: 549 –54. [4] Caldas C, Hahn SA, Hruban RH, et al. Detection of K-ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia. Cancer Res 1994;54:3568 –73. [5] DiGiuseppe JA, Hruban RH, Offerhaus GJ, et al. Detection of K-ras mutations in mucinous pancreatic duct hyperplasia from a patient with a family history of pancreatic carcinoma. Am J Pathol 1994; 144:889 –95. [6] Kalthoff H, Schmiegel W, Roeder C, et al. p53 and K-RAS alterations in pancreatic epithelial cell lesions. Oncogene 1993;8:289 –98. [7] Cerny WL, Mangold KA, Scarpelli DG. K-ras mutation is an early event in pancreatic duct carcinogenesis in the Syrian golden hamster. Cancer Res 1992;52:4507–13. [8] Hruban RH, van Mansfeld AD, Offerhaus GJ, et al. K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase


[22] [23]







chain reaction analysis and allele-specific oligonucleotide hybridization. Am J Pathol 1993;143:545–54. Kawesha A, Ghaneh P, Andren-Sandberg A, et al. K-ras oncogene subtype mutations are associated with survival but not expression of p53, p16(INK4A), p21(WAF-1), cyclin D1, erbB-2 and erbB-3 in resected pancreatic ductal adenocarcinoma. Int J Cancer 2000;89: 469 –74. Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994; 264:436 – 40. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cellcycle control causing specific inhibition of cyclin D/CDK4. Nature 193;366:704 –7. Nobori T, Miura K, Wu DJ, et al. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 1994;368: 753– 6. Liu Q, Yan YX, McClure M, et al. MTS-1 (CDKN2) tumor suppressor gene deletions are a frequent event in esophagus squamous cancer and pancreatic adenocarcinoma cell lines. Oncogene 1995;10:619 – 22. Huang L, Goodrow TL, Zhang SY, et al. Deletion and mutation analyses of the P16/MTS-1 tumor suppressor gene in human ductal pancreatic cancer reveals a higher frequency of abnormalities in tumor-derived cell lines than in primary ductal adenocarcinomas. Cancer Res 1996;56:1137– 41. Caldas C, Hahn SA, da Costa LT, et al. Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nature Genet 1994;8:27–32. Schutte M, Hruban RH, Geradts J, et al. Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carcinomas. Cancer Res 1997;57:3126 –30. Goldstein AM, Fraser MC, Struewing JP, et al. Increased risk of pancreatic cancer in melanoma-prone kindreds with p16INK4 mutations. N Engl J Med 1995;333:970 – 4. Kobayashi S, Shirasawa H, Sashiyama H, et al. P16INK4a expression adenovirus vector to suppress pancreas cancer cell proliferation. Clin Cancer Res 1999;5:4182–5. Ghaneh P, Greenhalf W, Humphreys M, et al. Adenovirus-mediated transfer of p53 and p16(INK4a) results in pancreatic cancer regression in vitro and in vivo. Gene Ther 2001;8:199 –208. Gerdes B, Ramaswamy A, Ziegler A, et al. p16INK4a is a prognostic marker in resected ductal pancreatic cancer: an analysis of p16INK4a, p53, MDM2, and Rb. Ann Surg 2002;235:51–9. Pellegata NS, Sessa F, Renault B, et al. K-ras and p53 gene mutations in pancreatic cancer: ductal and nonductal tumors progress through different genetic lesions. Cancer Res 1994;54:1556 – 60. Rozenblum E, Schutte M, Goggins M, et al. Tumor-suppressive pathways in pancreatic carcinoma. Cancer Res 1997;57:1731– 4. Scarpa A, Capelli P, Mukai K, et al. Pancreatic adenocarcinomas frequently show p53 gene mutations. Am J Pathol 1993;142:1534 – 43. Hahn SA, Schutte M, Hoque AT, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996;271: 350 –3. Zhou S, Buckhaults P, Zawel L, et al. Targeted deletion of Smad4 shows it is required for transforming growth factor beta and activin signaling in colorectal cancer cells. Proc Natl Acad Sci USA 1998; 95:2412–16. Lagna G, Hata A, Hemmati-Brivanlou A, et al. Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature 1996;383:832– 6. Zhang Y, Feng X, We R, et al. Receptor-associated Mad homologues synergize as effectors of the TGF-beta response. Nature 1996;383: 168 –72. Moskaluk CA, Kern SE. Cancer gets Mad: DPC4 and other TGFbeta pathway genes in human cancer. Biochim Biophys Acta 1996;1288: M31–3.


S.M. Cowgill and P. Muscarella / The American Journal of Surgery 186 (2003) 279 –286

[29] Takaku K, Oshima M, Miyoshi H, et al. Intestinal tumorigenesis in compound mutant mice of both Dpc4 (Smad4) and Apc genes. Cell 1998;92:645–56. [30] Howe JR, Roth S, Ringold JC, et al. Mutations in the SMAD4/DPC4 gene in juvenile polyposis. Science 1998;280:1086 – 8. [31] Lynch HT, Brand RE, Lynch JF, et al. Hereditary factors in pancreatic cancer. J Hepatobiliary Pancreat Surg 2002;9:12–31. [32] Lowenfels AB, Maisonneuve P, DiMagno EP, et al. Hereditary pancreatitis and the risk of pancreatic cancer. International Hereditary Pancreatitis Study Group. J Natl Cancer Inst 1997;89:442– 6. [33] Lynch HT, Frichot BC, Lynch P, et al. Family studies of malignant melanoma and associated cancer. Surg Gynecol Obstet 1975;141: 517–22. [34] Bergman W, Gruis N. Familial melanoma and pancreatic cancer. N Engl J Med 1996;334:471–2. [35] Giardiello FM, Brensinger JD, Tersmette AC, et al. Very high risk of cancer in familial Peutz-Jeghers syndrome. Gastroenterology 2000; 119:1447–53. [36] Su GH, Hruban RH, Bansal RK, et al. Germline and somatic mutations of the STK11/LKB1 Peutz-Jeghers gene in pancreatic and biliary cancers. Am J Pathol 1999;154:1835– 40. [37] Goggins M, Schutte M, Lu J, et al. Germline BRCA2 gene mutations in patients with apparently sporadic pancreatic carcinomas. Cancer Res 1996;56:5360 – 4. [38] Kloppel G, Solcia E, Longnecker DS, et al, editors. Histological typing of tumours of the exocrine pancreas. WHO. World Health Organization international histological classification of tumours. 2nd ed. Berlin: Springer-Verlag, 1998. [39] Wada K. p16 and p53 gene alterations and accumulations in the malignant evolution of intraductal papillary-mucinous tumors of the pancreas. J Hepatobiliary Pancreat Surg 2002;9:76 – 85. [40] Hruban RH, Adsay NV, Albores-Saavedra J, et al. Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions. Am J Surg Pathol 2001;25:579 – 86. [41] Wilentz RE, Iacobuzio-Donahue CA, Argani P, et al. Loss of expression of Dpc4 in pancreatic intraepithelial neoplasia: evidence that DPC4 inactivation occurs late in neoplastic progression. Cancer Res 2000;60:2002– 6. [42] Goggins M, Hruban RH, Kern SE. BRCA2 is inactivated late in the development of pancreatic intraepithelial neoplasia: evidence and implications. Am J Pathol 2000;156:1767–71. [43] Kondo K, Kaelin WG. The von Hippel-Lindau tumor suppressor gene. Exp Cell Res 2001;264:117–25. [44] Lubensky IA, Pack S, Ault D, et al. Multiple neuroendocrine tumors of the pancreas in von Hippel-Lindau disease patients: histopathological and molecular genetic analysis. Am J Pathol 1998;153:223–31. [45] Verhoef S, van Diemen-Steenvoorde R, Akkersdijk WL, et al. Malignant pancreatic tumour within the spectrum of tuberous sclerosis complex in childhood. Eur J Pediatr 1999;158:284 –7. [46] Jensen RT. Pancreatic endocrine tumors: recent advances. Ann Oncol 1999;10(suppl 4):170 – 6. [47] Radford DM, Ashley SW, Wells SA, et al. Loss of heterozygosity of markers on chromosome 11 in tumors from patients with multiple endocrine neoplasia syndrome type 1. Cancer Res 1990;50:6529 –33. [48] Nakamura Y, Larsson C, Julier C, et al. Localization of the genetic defect in multiple endocrine neoplasia type 1 within a small region of chromosome 11. Am J Hum Genet 1989;44:751–5. [49] Bale SJ, Bale AE, Stewart K, et al. Linkage analysis of multiple endocrine neoplasia type 1 with INT2 and other markers on chromosome 11. Genomics 1989;4:320 –2.

[50] Chandrasekharappa SC, Guru SC, Manickam P, et al. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 1997;276:404 –7. [51] Guru SC, Goldsmith PK, Burns AL, et al. Menin, the product of the MEN1 gene, is a nuclear protein. Proc Natl Acad Sci USA 1998;95: 1630 – 4. [52] Komminoth P. Review: multiple endocrine neoplasia type 1, sporadic neuroendocrine tumors and MENIN. Diagn Molec Pathol 1999;8: 107–12. [53] Zhuang Z, Vortmeyer AO, Pack S, et al. Somatic mutations of the MEN1 tumor suppressor gene in sporadic gastrinomas and insulinomas. Cancer Res 1997;57:4682– 6. [54] Gortz B, Roth J, Krahenmann A, et al. Mutations and allelic deletions of the MEN1 gene are associated with a subset of sporadic endocrine pancreatic and neuroendocrine tumors and not restricted to foregut neoplasms. Am J Pathol 1999;154:429 –36. [55] Bartsch D, Hahn SA, Danichevski KD, et al. Mutations of the DPC4/ Smad4 gene in neuroendocrine pancreatic tumors. Oncogene 1999; 18:2367–71. [56] Evers BM, Rady PL, Tyring SK, et al. Amplification of the HER-2/ neu protooncogene in human endocrine tumors. Surgery 1992;112: 211–18. [57] Goebel SU, Iwamoto M, Raffeld M, et al. Her-2/neu expression and gene amplification in gastrinomas: correlations with tumor biology, growth, and aggressiveness. Cancer Res 2002;62:3702–10. [58] Yoshimoto K, Iwahana H, Fukuda A, et al. ras mutations in endocrine tumors: mutation detection by polymerase chain reaction-single strand conformation polymorphism. Jpn J Cancer Res 1992;83:1057– 62. [59] Yashiro T, Fulton N, Hara H, et al. Comparison of mutations of ras oncogene in human pancreatic exocrine and endocrine tumors. Surgery 1993;114:758 – 64. [60] Chung DC, Smith AP, Louis DN, et al. Analysis of the retinoblastoma tumour suppressor gene in pancreatic endocrine tumours. Clin Endocrinol (Oxf) 1997;47:523– 8. [61] Muscarella P, Melvin WS, Fisher WE, et al. Genetic alterations in gastrinomas and nonfunctioning pancreatic neuroendocrine tumors: an analysis of p16/MTS1 tumor suppressor gene inactivation. Cancer Res 1998;58:237– 40. [62] Serrano J, Goebel SU, Peghini PL, et al. Alterations in the p16INK4a/ CDKN2A tumor suppressor gene in gastrinomas. J Clin Endocrinol Metab 2000;85:4146 –56. [63] Bartsch DK, Kersting M, Wild A, et al. Low frequency of p16(INK4a) alterations in insulinomas. Digestion 2000;62:171–7. [64] Moore PS, Orlandini S, Zamboni G, et al. Pancreatic tumours: molecular pathways implicated in ductal cancer are involved in ampullary but not in exocrine nonductal or endocrine tumorigenesis. Br J Cancer 2001;84:253– 62. [65] Naumann M, Savitskaia N, Eilert C, et al. Frequent codeletion of p16/MTS1 and p15/MTS2 and genetic alterations in p16/MTS1 in pancreatic tumors. Gastroenterology 1996;110:1215–24. [66] Lubomierski N, Kersting M, Bert T, et al. Tumor suppressor genes in the 9p21 gene cluster are selective targets of inactivation in neuroendocrine gastroenteropancreatic tumors. Cancer Res 2001;61:5905–10. [67] Guo SS, Wu X, Shimoide AT, et al. Anomalous overexpression of p27(Kip1) in sporadic pancreatic endocrine tumors. J Surg Res 2001; 96:284 – 8. [68] Chung DC, Brown SB, Graeme-Cook F, et al. Overexpression of cyclin D1 occurs frequently in human pancreatic endocrine tumors. J Clin Endocrinol Metab 2000;85:4373– 8.