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Molecular Genetics of Exocrine Pancreatic Neoplasms
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PANCREATIC NEOPLASMS
MOLECULAR GENETICS OF EXOCRINE PANCREATIC NEOPLASMS Stephan A. Hahn, MD, and Scott E. Kern, MD
The development of a cancer involves an accumulation of genetic changes. Only in very recent years have many of the genetic alterations involved in carcinogenesis been determined. Adenocarcinoma of the pancreas is the fifth most common cause of death from cancer in the United States,3 but the study of its genetic make-up has been hampered by several characteristics inherent to this tumor type. There is generally a low number of primary tumors accessible for research because of the low rate of surgical resection in most centers. Furthermore, the molecular study of tumor specimens often requires tissue samples that are highly enriched for the neoplastic component. Such a high percentage of tumor cells compared with the "contaminating" normal cells is infrequent in primary pancreatic carcinoma because of a very strong desmoplastic reaction by the host tissues contacting the invading tumor.50 Despite these difficulties for the genetic studies, at least three genes are now known to be frequently altered, forming the basis for a genetic model for this cancer. Moreover, it is hoped that with increases in our knowledge about the underlying genetic alterations in pancreatic carcinoma, better approaches to diagnosis, prognostication, prevention, and treatment may emerge. This work was supported by NIH grant CA62924 and by Deutschen Krebshilfe. Dr. Kern is a McDonnell Foundation Scholar.
From the Departments of Pathology (SAH, SEK) and Oncology (SEK), The Johns Hopkins Medical Institutions, Baltimore, Maryland
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CLONAL SELECTION AND TUMORIGENESIS
One of the fundamental theorems of cancer biology is that of clonal selection. 41 This theory explains cancer as a consequence of acquired (somatic) mutations, which provide an individual cell and its progeny (a "clone" of cells) with a selective growth advantage over the surrounding cells. The mutations lie within genes involved in growth control, and mutant forms of these genes allow cells to grow in what would otherwise be limiting conditions. Clones with different mutational spectra co-exist and therefore compete. Those combinations best able to survive in the poor nutritional environment of a tumor, best able to produce progeny, or best able to invade surrounding tissue predominate. Thus the competition of the different clones at any given stage of tumor progression leads to the selection of a predominant clone for that stage (Fig. 1). The presence of a predominant clone allows the determination of the specific genetic changes harbored by it; these may identify the mutations that were critical for the production of the neoplasm. TUMOR PROGRESSION MODEL OF PANCREATIC NEOPLASMS
Macroscopic and microscopic studies of various human neoplasms demonstrate that most epithelial malignancies evolve through several stages of precursor lesions. Such a morphologic progression is seen in cervical carcinoma, ductal mammary carcinoma, colorectal carcinoma, head and neck carcinoma, and lung carcinoma and is strongly suggested in ductal adenocarcinoma of the pancreas. Owing to their extraordinary accessibility, colorectal neoplasms (carcinomas and their precursor lesions, comprising adenomas of various stages) became a productive model for the genetic study of tumor development. It is now thought that the emergence of colorectal cancer depends upon a series of genetic changes. These changes appear to occur in a preferred order, genetically defining certain stages within the progression of a colonic adenoma toward its full manifestation as a metastatic carcinomaY,56 Within a pancreas harboring a ductal adenocarcinoma, it is not unusual to find "hyperplastic" ductal lesions.?, 61 These can have either normal flat mucinous epithelium or a papillary architecture. Some le-
Figure 1. Clonal selection model for tumor progression. Sequential steps in the clonal evolution of a neoplasm are depicted schematically. Each level of evolution is a consequence of acquired (somatic) mutations, which provide one individual cell and its progeny with a selective growth advantage over the surrounding cells (neighboring clones and subclones are represented by circles of various shading). Thus, the competition among the different clones at any given stage of tumor progression will lead to the selection of a predominant clone for that stage, and eventually may lead to the evolution of a clone with malignant behavior.
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Figure 1. See legend on opposite page
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sions harbor classic signs of atypia, such as nuclear pleomorphism and hyperchromatism. A tumor progression model for the pancreas, analogous to that of epithelial neoplasms in other sites, would characterize these duct lesions as a form of pancreatic intraepithelial neoplasm (PIN). PIN occurs in persons with or without associated pancreatic disease, and each lesion carries a low risk of progression to an invasive lesion. One might then hypothesize, based on this model, that PIN and pancreatic ductal adenocarcinoma should share some clonal genetic changes, and the more histologically advanced types of PIN should be associated with a gain of certain mutations. One would hope that such an understanding would allow the detection of patients at especially high risk for cancer, before the emergence of invasive tumor.
ONCOGENES INVOLVED IN PANCREATIC ADENOCARCINOMA
Oncogenes, the activated mutated forms of normal cellular "protooncogenes," were the first gene class identified to be involved in tumorigenesis. Most oncogenes code for proteins that are thought to be major players in the control of cell growth. 2,57 One member of the ras oncogene family, K-ras, was found to be mutated in pancreatic adenocarcinomas at a frequency unprecedented among other human tumors. Various studies confirmed a mutation rate of up to 85%.16,23,26,44,47 The mutations are generally confined to codon 12 and rarely to codon 13. Mutations in the other family members, Nras and H-ras, are uncommon. The duct lesions (PIN) of the pancreas often harbor K-ras mutations. Papillary and atypical forms have the highest frequency (well Over 50%), whereas flat mucinous lesions have a much lower rate?, 61 These K-ras mutations occur with similar frequency whether they are associated with chronic pancreatitis or pancreatic cancer. The nearly universal presence of K-ras mutations in the carcinomas, combined with the high frequency of K-ras mutations in those lesions that could be viewed as precursors of the carcinoma, implies that the development of a K-ras mutation is a key rate-limiting step early in the neoplastic progression of pancreatic cancer. For technical reasons, the restriction of K-ras mutations to only codons 12 and 13 allows the development of dedicated sensitive tests for these mutations. It has been shown, for example, that K-ras mutations can be detected in stool specimen of patients with either chronic pancreatitis or pancreatic carcinoma? One limitation of this assay is that it involves sophisticated molecular techniques that are not readily applicable to clinical use. But more importantly, the very identification of K-ras mutations produces a clinical dilemma. It is likely that clinically trivial pancreatic duct lesions and small colorectal adenomas could produce a positive assay result. A considerable waste of diagnostic effort would result from the indiscriminate application of such sensitive assays. We
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still know too little about pancreatic adenocarcinoma and its precursor lesions to propose an algorithm for their rational use. Another interesting facet of ras concerns its known biochemical features. Ras encodes a guanine nucleotide-binding protein, and its function in signal transduction is rather well studied. 4. 38 One necessary step in the ras pathway involves the farnesylation of the ras protein via the farnesyl transferase enzyme. Without farnesylation, the ra:s protein cannot associate with the plasma membrane of the cell, a prerequisite for its cell-transforming activity. Recently, several farnesyl transferase inhibitors have been engineered/8. 25. 33 and studies of their effects on neoplastic cells are ongoing. The development of ras farnesyl transferase inhibitors in the form of a drug may offer some hope of a rational therapeutic option for pancreatic carcinoma, as well as other ras mutation-positive tumors. TUMOR-SUPPRESSOR GENES IN PANCREATIC ADENOCARCINOMA
The tumor-suppressor genes contribute to tumorigenesis through their loss of function, in contrast to the oncogenes, which exhibit a gain of function caused by an activating mutation. 58 Many tumor-suppressor genes can be described by a simple model: In each cell, most genes are represented by both a maternal and a paternal copy. When only one copy of a tumor-suppressor gene harbors an inactivating mutation, the remaining wild-type copy may be adequate to maintain the tumor suppressor function. 32 Therefore, mutations in tumor-suppressor genes generally involve both copies, and mutations in these genes are termed recessive. The second inactivating mutation is most often the loss of a chromosomal region containing this gene, also called allelic loss or loss of heterozygosity (LOH). Of the known tumor-suppressor genes, p53 and MTS1 have been found to be involved in pancreatic carcinogenesis. p53, MTS1, AND CELL CYCLE CONTROL
p53, the 1993 molecule of the year9 because of its importance in many types of human cancer, is located on the short (p) arm of chromosome 17,21 An allelotype (a genome-wide survey for allelic loss) in pancreatic tumors showed a high frequency of LOH for chromosome 17p, involving the area of the p53 gene. 50 Sequence analyses of the p53 gene found that 50% to 70% of pancreatic carcinomas had mutations of p53.26. 44. 47 A mutation in one copy of the p53 gene was always accompanied by the loss of the wild-type allele, as expected for a classic tumorsuppressor gene. p53 is a nuclear phosphoprotein with the ability to bind to specific sequences of DNA and to activate gene transcriptionY· 3!. 42 Small changes in the p53 amino acid sequence can prevent its binding to these DNA regulatory sites. 3!
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MTSl/CDK4I, a tumor-suppressor gene recently cloned and located on chromosome 9p,27,49 was found to have mutations in 38% of pancreatic carcinoma, again always with loss of the wild-type allele,6 The genomic analysis of MTSI in pancreatic carcinoma, as well as in other tumor types,6, 27, 40 revealed an unusual number of cases that inactivated the MTSI gene through another mechanism-the loss of both alleles. Homozygous deletions, as this second form of genomic change is termed, are thought to be the result of two steps-the loss of a larger chromosomal region and the separate loss of a smaller region that targets a tumor-suppressor gene. The total frequency of mutations and of homozygous deletion involving the MTSI gene in pancreatic carcinoma is nearly 80%, effectively rivaling the frequency of p53 mutations described in this tumor.6 MTSI and p53 have an interesting feature in common. They both contribute inhibitory signals to the control of the cell division cycle (Fig. 2)12,45,49,59; that is, they form part of the control system for movement of a cell through the cell cycle, acting to determine which cells replicate their DNA and undergo mitosis. The alternating phases of DNA replication and mitosis strongly depend on the interaction of three classes of proteins-the cyclin-dependent kinases (CDKs), the cyclins, and the CDK inhibitors. 46 The importance of this was underscored when it was found that wild-type but not mutant p53 can induce the transcription of a 21-kD protein (WAFl), a CDK inhibitor. 12,19,60 WAFI in tum can bind to cyclin-CDK complexes, inhibiting cells from progression into S phase (Fig. 2).19,60 The 16-kD product of the MTSI gene (p16) is itself a CDK inhibitor.49
p53-WAF1 Pathway Activation of Transcription
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E2F Remains in Complex with Hypophosphorylated Rb Protein
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Figure 2. Schematic model for the action of p53 and Mts1 in cell cycle control. The mutational inactivation of p53 and Mts1 in most pancreatic carcinoma removes these two key control mechanisms, presumably allowing the inappropriate entry of cells into the phases of DNA synthesis and subsequent cell division. Other suggested pathways, such as the ability of p53 to mediate apoptosis (programmed cell death), are also likely to be affected by such mutations.
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It binds CDK4, which is responsible for the phosphorylation of the Rb
protein. Because the phosphorylation of Rb allows the release of E2F, a stimulus for DNA replication, the inhibition of this pathway by p16 normally should control the frequency of cell division (Fig. 2). Thus, a common pathway through which p53 and MTSI regulate cell growth and mediate tumor-suppressive action seems to be their ability to control the cell division process. Such controls might, for example, allow cells the ability to repair damaged DNA or to turn on a program that leads these cells to undergo apoptosis.1O,28, 62 It is obvious that any mutation in these genes which loosens this set of "cell cycle brakes" may potentially support neoplastic growth. 29 OTHER GENES
Studies of allelic loss revealed a high frequency of loss (88%) of chromosome 18q (Fig. 3, unpublished observation), the site of the DDC (deleted in colorectal carcinoma) gene. Other studies suggested a low expression of the gene in pancreatic cancer. 20, 5! Owing to the incredible length and complexity of the gene (29 exons spanning 1.4 megabases),8 sequence analysis has not been possible to date in this form of carcinoma. Allelic loss studies also identified a moderate rate of loss of 13q (Fig. 3, unpublished observations), the site of the RBI (retinoblastoma) gene. Yet expression studies failed to confirm an abnormality of Rb. 50 It is possible that another tumor suppressor gene lies on chromosome 13q and is involved in pancreatic carcinoma. Chromosome 5, the site of the APC (adenomatous polyposis coli) gene, is uncommonly lost in pancreatic carcinoma (Fig. 3), and APC mutations have not been found in a well-described series from the United States50 or in a set of pancreatic carcinoma celllines. 52 A report of APC mutations in a small series from Japan22 has not been confirmed. The "mutator phenotype" describes a series of genetic changes seen at high rate in some tumors, especially those of hereditary nonpolyposis colorectal cancer (Lynch syndrome).!' 37 The increased risk for colon cancer in these families is attributable to mutations in several DNA repair genes (hMSH2, hMLHl, hPMSl, and hPMS2).5, 14, 34, 39, 43 The proteins from these genes cooperate to perform a repair function for DNA mismatch mutations. The lack of such a repair function in a cell dramatically increases the rate of mutation. By chance, some of these mutations should occasionally affect genes involved in neoplastic transformation. Another feature of this mismatch repair defect is that it produces numerous anomalies at simple base pair repeats, which provide a sensitive assay system for this defect. Detailed studies of three separate series in the United States found only the rare occurrence of a mismatch repair defect (Ref. 6 and 50, and unpublished observation). A smaller series from Japan reported finding the mutator phenotype at a high rate,17 but this has not been confirmed.
Figure 3. Frequency of allelic loss on selected chromosomal arms found in a series of 18 pancreatic adenocarcinomas. Allele-specific polymorphic markers were used in a PCRbased assay to evaluate for the individual sites of loss (unpublished data). Losses of 9p and 17p target the MTS1 and p53 genes, respectively. The suspected tumor-suppressor genes at the other sites of frequent loss have not been identified to date. Black bar = p arm; shaded bar = q arm.
ALLELIC LOSS AND A GENERAL MEASURE OF GENETIC DAMAGE Initially, surveys for genetic damage in tumor cells were done with karyotyping, but the rapid development of DNA probes has replaced karyotyping for certain kinds of studies. 55 It is now possible to do a genome-wide analysis of hundreds of sites, with an approximate resolution of 1 to 5 Mb and covering all chromosomes, through a highresolution allelotype. This allows one to map each chromosomal region individually for sites of allelic deletions in a particular tumor. The chromosomal regions showing frequent allelic loss are presumably those most likely to harbor tumor-suppressor genes. Sites with a high frequency of loss in pancreatic carcinoma include chromosomes 9, 17, and 18, mentioned above (Fig. 3). Allelic loss on chromosome 22q, including the site of the NF2 (neurofibromatosis type II) gene, was found to be present in 55% of cases (unpublished observation). Upon study, none of seven pancreatic tumors having 22q loss contained a terminating mutation of NF2, the consequence of the type of NF2 gene alteration frequently found in the syndrome (unpublished observation).24, 48, 54 Therefore, NF2 is not a likely candidate tumor suppressor gene for pancreatic carcinoma. Another potential use of an allelotype is in the calculation of the
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fractional allelic loss (FAL) value, providing a crude parameter for the estimate of the total genomic damage in a tumor. FAL is defined as the number of chromosomal arms having allelic loss, divided by the total number of chromosomal arms.3D An allelotype done with a small number of patients was able to compare the FAL value with the survival data. This study found a trend toward better survival when the FAL was below a certain threshold so and suggested that the aggressive behavior of pancreatic carcinoma may be related to the loss of multiple tumor suppressor genes. Recent data suggest that the average FAL value for pancreatic adenocarcinoma may be near 40%, the highest rate of LOH yet reported for human cancer (unpublished observation).
POTENTIAL CLINICAL APPLICATION
Insights into the molecular genetics of pancreatic carcinogenesis are beginning to form a genetic model for pancreatic cancer and its precursors, and it is reasonable to ask whether there will be a clinical utility for such an understanding. Despite progress on the molecular genetic level, the potential usefulness for clinical application is still to be defined for risk assessment, early diagnosis, prognosis, and therapeutics.
Risk Assessment
Among the risk factors attributed to pancreatic cancer are tobacco smoking and, possibly, pre-existing chronic pancreatitis. 15, 35 Families are known that have an increased frequency of pancreatic cancer, but specific genetic linkage, such as that found in familial forms of colorectal and breast carcinoma, has yet not been demonstrated. 36 These considerations currently make the identification of patients at risk difficult. It is therefore hoped that a genetic understanding of the disease, together with epidemiologic data, might eventually shape some distinctive risk groups. It will be of special interest whether molecular profiles of carcinomas and the precursor duct lesions may allow us to directly address the question of a potential link between chronic pancreatitis and pancreatic carcinoma.
Early Diagnosis
A strategy to prevent pancreatic carcinoma would be effected by the availability of a sensitive screening procedure, identifying those patients with incipient pancreatic neoplasms, at a stage when a curative treatment regimen is successful. A molecular genetic detection method might provide such a high sensitivity.7,s3 For example, a positive result
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in a stool ras mutation assay might direct clinical attention to a subset of patients? As of now, neither the sensitivity nor the specificity of such a test can be assured. Such tests await a better definition of the mutational spectra of the intraductal and invasive pancreatic neoplasms.
Prognosis It would be of great general interest to elucidate those factors in pancreatic carcinoma that account for its aggressive clinical behavior. For example, some sites of allelic loss, as identified in the allelotype of pancreatic cancer, might distinguish among patient groups with varying prognosis. An understanding of specific genetic factors involved in the prognosis of this form of cancer might provide clues useful in other malignancies as well.
Therapy
The most likely hope for curative treatment would come from earlier diagnosis. Further, better prognostication might help to discriminate patients who need more aggressive therapy. Eventually, however, it is hoped that unresectable or residual disease might be amenable to improved therapeutic strategies. Knowledge of the genetic mutations of a neoplasm might determine the biochemical alterations of the cells which might be targeted by specific drugs. Such is the promise held out by ras farnesyl transferase inhibitors, for example.
SUMMARY
Molecular genetic studies of pancreatic ductal adenocarcinoma have revealed the common co-existence of K-ras, p53, and MTSI mutations. The finding of K-ras mutations in epithelial lesions of ducts suggests them as a precursor intraepithelial neoplasm. The clinical importance of this line of work can only be anticipated at present, and a fuller understanding of genetic alterations in these neoplasms is necessary.
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32. Knudson JAG: Hereditary cancer, oncogenes, and antioncogenes. Cancer Res 45:1437, 1985 33. Kohl NE, Mosser SD, deSolms SJ, et al: Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor. Science 260:1934, 1993 34. Leach FS, Nicolaides NC, Papadopoulos N, et al: Mutation of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell 75:1215, 1993 35. Lowenfels AB, Maisonneuve P, Cavallini G, et al: Pancreatitis and the risk of pancreatic cancer. N Engl J Med 328:1433, 1993 36. Lynch HT, Fitzsimmons ML, Smyrk TC, et al: Familial pancreatic cancer: Clinicopathologic study of 18 nuclear families. Am J Gastroenterol 85:54, 1990 37. Lynch HT, Smyrk TC, Watson P, et al: Genetics, natural history, tumor spectrum, and pathology of hereditary nonpolyposis colorectal cancer-an updated review. Gastroenterology 104:1535, 1993 38. McCormick F: ras GTPase activating protein: Signal transmitter and signal terminator. Cell 56:5, 1989 39. Nicolaides NC, Papadopoulos N, Liu B, et al: Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature 371:75, 1994 40. Nobori T, Miura K, Wu DJ, et al: Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 368:753, 1994 41. Nowell PC: The clonal evolution of tumor cell population. Science 194:23, 1976 42. O'Rourke RW, Miller CW, Kato GJ, et al: A potential transcriptional activation element in the p53 protein. Oncogene 5:1829, 1990 43. Papadopoulos N, Nicolaides NC, Wei YF, et al: Mutation of a mutL homolog in hereditary colon cancer. Science 263:1625, 1994 44. 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 54:1556, 1994 45. Peters G: Stifled by inhibitions. Nature 371:204, 1994 46. Peters M, Herskowitz I: Joining the complex: Cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell 79:18i, 1994 47. Redston MS, Caldas C, Seymour AB, et al: p53 Mutations in pancreatic carcinoma and evidence of common involvement of homocopolymer tracts in DNA microdeletions. Cancer Res 54:3025, 1994 48. Rouleau GA, Merel P, Lutchman M; et al: Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2. Nature 363:515, 1993 49. Serrano M, Hannon GJ, Beach D: A new regulatory motif in cell-cycle control causing specific inhibition of cyclin DjCDK4. Nature 366:704, 1993 50. Seymour AB, Hruban RH, Redston M, et al: Allelotype of pancreatic adenocarcinoma. Cancer Res 54:2761, 1994 51. Simon B, Weinel W, Hohne M, et al: Frequent alterations of the tumor suppressor genes p53 and DCC in human pancreatic carcinoma. Gastroenterology 106:1645, 1994 52. Smith KJ, Johnson KA, Bryan TM, et al: The APC gene product in normal and tumor cells. Proc Nat! Acad Sci USA 90:2846, 1993 53. Tada M, Ornata M, Kawai S, et al: Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma. Cancer Res 53:2472, 1993 54. Trofatter JA, MacCollin MM, Rutter JL, et al: A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor. Cell 72:791, 1993 55. Vogelstein B, Fearon ER, Kern SE, et al: Allelotype of colorectal carcinomas. Science 244:207, 1989 56. Vogelstein B, Kinzler KW: The multistep nature of cancer. Trends Genet 9:138, 1993 57. Weinberg RA: Oncogenes, antioncogenes, and the molecular bases of multistep carcinogenesis. Cancer Res 49:3713, 1989 58. Weinberg RA: Tumor suppressor genes. Science 254:1138, 1991 59. White E: p53, Guardian of Rb. Nature 371:21, 1994 60. Xiong Y, Hannon GJ, Zhang H, et al: p21 is a universal inhibitor of cyclin kinases. Nature 366:701, 1993
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6l. Yanagisawa A, Ohtake K, Ohashi K, et al: Frequent c-Ki-ras oncogene activation in mucous cell hyperplasias of pancreas suffering from chronic inflammation. Cancer Res 53:953, 1993 62. Yonish-Rouach E, Resnitzky D, Lotem J, et al: Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352:345, 1991 Address reprint requests to Scott E. Kern, MD 628 Ross Research Building The Johns Hopkins University School of Medicine 720 Rutland Avenue Baltimore, MD 21205-2196
PANCREATIC NEOPLASMS
MOLECULAR GENETICS OF EXOCRINE PANCREATIC NEOPLASMS Stephan A. Hahn, MD, and Scott E. Kern, MD
The development of a cancer involves an accumulation of genetic changes. Only in very recent years have many of the genetic alterations involved in carcinogenesis been determined. Adenocarcinoma of the pancreas is the fifth most common cause of death from cancer in the United States,3 but the study of its genetic make-up has been hampered by several characteristics inherent to this tumor type. There is generally a low number of primary tumors accessible for research because of the low rate of surgical resection in most centers. Furthermore, the molecular study of tumor specimens often requires tissue samples that are highly enriched for the neoplastic component. Such a high percentage of tumor cells compared with the "contaminating" normal cells is infrequent in primary pancreatic carcinoma because of a very strong desmoplastic reaction by the host tissues contacting the invading tumor.50 Despite these difficulties for the genetic studies, at least three genes are now known to be frequently altered, forming the basis for a genetic model for this cancer. Moreover, it is hoped that with increases in our knowledge about the underlying genetic alterations in pancreatic carcinoma, better approaches to diagnosis, prognostication, prevention, and treatment may emerge. This work was supported by NIH grant CA62924 and by Deutschen Krebshilfe. Dr. Kern is a McDonnell Foundation Scholar.
From the Departments of Pathology (SAH, SEK) and Oncology (SEK), The Johns Hopkins Medical Institutions, Baltimore, Maryland
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CLONAL SELECTION AND TUMORIGENESIS
One of the fundamental theorems of cancer biology is that of clonal selection. 41 This theory explains cancer as a consequence of acquired (somatic) mutations, which provide an individual cell and its progeny (a "clone" of cells) with a selective growth advantage over the surrounding cells. The mutations lie within genes involved in growth control, and mutant forms of these genes allow cells to grow in what would otherwise be limiting conditions. Clones with different mutational spectra co-exist and therefore compete. Those combinations best able to survive in the poor nutritional environment of a tumor, best able to produce progeny, or best able to invade surrounding tissue predominate. Thus the competition of the different clones at any given stage of tumor progression leads to the selection of a predominant clone for that stage (Fig. 1). The presence of a predominant clone allows the determination of the specific genetic changes harbored by it; these may identify the mutations that were critical for the production of the neoplasm. TUMOR PROGRESSION MODEL OF PANCREATIC NEOPLASMS
Macroscopic and microscopic studies of various human neoplasms demonstrate that most epithelial malignancies evolve through several stages of precursor lesions. Such a morphologic progression is seen in cervical carcinoma, ductal mammary carcinoma, colorectal carcinoma, head and neck carcinoma, and lung carcinoma and is strongly suggested in ductal adenocarcinoma of the pancreas. Owing to their extraordinary accessibility, colorectal neoplasms (carcinomas and their precursor lesions, comprising adenomas of various stages) became a productive model for the genetic study of tumor development. It is now thought that the emergence of colorectal cancer depends upon a series of genetic changes. These changes appear to occur in a preferred order, genetically defining certain stages within the progression of a colonic adenoma toward its full manifestation as a metastatic carcinomaY,56 Within a pancreas harboring a ductal adenocarcinoma, it is not unusual to find "hyperplastic" ductal lesions.?, 61 These can have either normal flat mucinous epithelium or a papillary architecture. Some le-
Figure 1. Clonal selection model for tumor progression. Sequential steps in the clonal evolution of a neoplasm are depicted schematically. Each level of evolution is a consequence of acquired (somatic) mutations, which provide one individual cell and its progeny with a selective growth advantage over the surrounding cells (neighboring clones and subclones are represented by circles of various shading). Thus, the competition among the different clones at any given stage of tumor progression will lead to the selection of a predominant clone for that stage, and eventually may lead to the evolution of a clone with malignant behavior.
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Figure 1. See legend on opposite page
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sions harbor classic signs of atypia, such as nuclear pleomorphism and hyperchromatism. A tumor progression model for the pancreas, analogous to that of epithelial neoplasms in other sites, would characterize these duct lesions as a form of pancreatic intraepithelial neoplasm (PIN). PIN occurs in persons with or without associated pancreatic disease, and each lesion carries a low risk of progression to an invasive lesion. One might then hypothesize, based on this model, that PIN and pancreatic ductal adenocarcinoma should share some clonal genetic changes, and the more histologically advanced types of PIN should be associated with a gain of certain mutations. One would hope that such an understanding would allow the detection of patients at especially high risk for cancer, before the emergence of invasive tumor.
ONCOGENES INVOLVED IN PANCREATIC ADENOCARCINOMA
Oncogenes, the activated mutated forms of normal cellular "protooncogenes," were the first gene class identified to be involved in tumorigenesis. Most oncogenes code for proteins that are thought to be major players in the control of cell growth. 2,57 One member of the ras oncogene family, K-ras, was found to be mutated in pancreatic adenocarcinomas at a frequency unprecedented among other human tumors. Various studies confirmed a mutation rate of up to 85%.16,23,26,44,47 The mutations are generally confined to codon 12 and rarely to codon 13. Mutations in the other family members, Nras and H-ras, are uncommon. The duct lesions (PIN) of the pancreas often harbor K-ras mutations. Papillary and atypical forms have the highest frequency (well Over 50%), whereas flat mucinous lesions have a much lower rate?, 61 These K-ras mutations occur with similar frequency whether they are associated with chronic pancreatitis or pancreatic cancer. The nearly universal presence of K-ras mutations in the carcinomas, combined with the high frequency of K-ras mutations in those lesions that could be viewed as precursors of the carcinoma, implies that the development of a K-ras mutation is a key rate-limiting step early in the neoplastic progression of pancreatic cancer. For technical reasons, the restriction of K-ras mutations to only codons 12 and 13 allows the development of dedicated sensitive tests for these mutations. It has been shown, for example, that K-ras mutations can be detected in stool specimen of patients with either chronic pancreatitis or pancreatic carcinoma? One limitation of this assay is that it involves sophisticated molecular techniques that are not readily applicable to clinical use. But more importantly, the very identification of K-ras mutations produces a clinical dilemma. It is likely that clinically trivial pancreatic duct lesions and small colorectal adenomas could produce a positive assay result. A considerable waste of diagnostic effort would result from the indiscriminate application of such sensitive assays. We
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still know too little about pancreatic adenocarcinoma and its precursor lesions to propose an algorithm for their rational use. Another interesting facet of ras concerns its known biochemical features. Ras encodes a guanine nucleotide-binding protein, and its function in signal transduction is rather well studied. 4. 38 One necessary step in the ras pathway involves the farnesylation of the ras protein via the farnesyl transferase enzyme. Without farnesylation, the ra:s protein cannot associate with the plasma membrane of the cell, a prerequisite for its cell-transforming activity. Recently, several farnesyl transferase inhibitors have been engineered/8. 25. 33 and studies of their effects on neoplastic cells are ongoing. The development of ras farnesyl transferase inhibitors in the form of a drug may offer some hope of a rational therapeutic option for pancreatic carcinoma, as well as other ras mutation-positive tumors. TUMOR-SUPPRESSOR GENES IN PANCREATIC ADENOCARCINOMA
The tumor-suppressor genes contribute to tumorigenesis through their loss of function, in contrast to the oncogenes, which exhibit a gain of function caused by an activating mutation. 58 Many tumor-suppressor genes can be described by a simple model: In each cell, most genes are represented by both a maternal and a paternal copy. When only one copy of a tumor-suppressor gene harbors an inactivating mutation, the remaining wild-type copy may be adequate to maintain the tumor suppressor function. 32 Therefore, mutations in tumor-suppressor genes generally involve both copies, and mutations in these genes are termed recessive. The second inactivating mutation is most often the loss of a chromosomal region containing this gene, also called allelic loss or loss of heterozygosity (LOH). Of the known tumor-suppressor genes, p53 and MTS1 have been found to be involved in pancreatic carcinogenesis. p53, MTS1, AND CELL CYCLE CONTROL
p53, the 1993 molecule of the year9 because of its importance in many types of human cancer, is located on the short (p) arm of chromosome 17,21 An allelotype (a genome-wide survey for allelic loss) in pancreatic tumors showed a high frequency of LOH for chromosome 17p, involving the area of the p53 gene. 50 Sequence analyses of the p53 gene found that 50% to 70% of pancreatic carcinomas had mutations of p53.26. 44. 47 A mutation in one copy of the p53 gene was always accompanied by the loss of the wild-type allele, as expected for a classic tumorsuppressor gene. p53 is a nuclear phosphoprotein with the ability to bind to specific sequences of DNA and to activate gene transcriptionY· 3!. 42 Small changes in the p53 amino acid sequence can prevent its binding to these DNA regulatory sites. 3!
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MTSl/CDK4I, a tumor-suppressor gene recently cloned and located on chromosome 9p,27,49 was found to have mutations in 38% of pancreatic carcinoma, again always with loss of the wild-type allele,6 The genomic analysis of MTSI in pancreatic carcinoma, as well as in other tumor types,6, 27, 40 revealed an unusual number of cases that inactivated the MTSI gene through another mechanism-the loss of both alleles. Homozygous deletions, as this second form of genomic change is termed, are thought to be the result of two steps-the loss of a larger chromosomal region and the separate loss of a smaller region that targets a tumor-suppressor gene. The total frequency of mutations and of homozygous deletion involving the MTSI gene in pancreatic carcinoma is nearly 80%, effectively rivaling the frequency of p53 mutations described in this tumor.6 MTSI and p53 have an interesting feature in common. They both contribute inhibitory signals to the control of the cell division cycle (Fig. 2)12,45,49,59; that is, they form part of the control system for movement of a cell through the cell cycle, acting to determine which cells replicate their DNA and undergo mitosis. The alternating phases of DNA replication and mitosis strongly depend on the interaction of three classes of proteins-the cyclin-dependent kinases (CDKs), the cyclins, and the CDK inhibitors. 46 The importance of this was underscored when it was found that wild-type but not mutant p53 can induce the transcription of a 21-kD protein (WAFl), a CDK inhibitor. 12,19,60 WAFI in tum can bind to cyclin-CDK complexes, inhibiting cells from progression into S phase (Fig. 2).19,60 The 16-kD product of the MTSI gene (p16) is itself a CDK inhibitor.49
p53-WAF1 Pathway Activation of Transcription
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Figure 2. Schematic model for the action of p53 and Mts1 in cell cycle control. The mutational inactivation of p53 and Mts1 in most pancreatic carcinoma removes these two key control mechanisms, presumably allowing the inappropriate entry of cells into the phases of DNA synthesis and subsequent cell division. Other suggested pathways, such as the ability of p53 to mediate apoptosis (programmed cell death), are also likely to be affected by such mutations.
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It binds CDK4, which is responsible for the phosphorylation of the Rb
protein. Because the phosphorylation of Rb allows the release of E2F, a stimulus for DNA replication, the inhibition of this pathway by p16 normally should control the frequency of cell division (Fig. 2). Thus, a common pathway through which p53 and MTSI regulate cell growth and mediate tumor-suppressive action seems to be their ability to control the cell division process. Such controls might, for example, allow cells the ability to repair damaged DNA or to turn on a program that leads these cells to undergo apoptosis.1O,28, 62 It is obvious that any mutation in these genes which loosens this set of "cell cycle brakes" may potentially support neoplastic growth. 29 OTHER GENES
Studies of allelic loss revealed a high frequency of loss (88%) of chromosome 18q (Fig. 3, unpublished observation), the site of the DDC (deleted in colorectal carcinoma) gene. Other studies suggested a low expression of the gene in pancreatic cancer. 20, 5! Owing to the incredible length and complexity of the gene (29 exons spanning 1.4 megabases),8 sequence analysis has not been possible to date in this form of carcinoma. Allelic loss studies also identified a moderate rate of loss of 13q (Fig. 3, unpublished observations), the site of the RBI (retinoblastoma) gene. Yet expression studies failed to confirm an abnormality of Rb. 50 It is possible that another tumor suppressor gene lies on chromosome 13q and is involved in pancreatic carcinoma. Chromosome 5, the site of the APC (adenomatous polyposis coli) gene, is uncommonly lost in pancreatic carcinoma (Fig. 3), and APC mutations have not been found in a well-described series from the United States50 or in a set of pancreatic carcinoma celllines. 52 A report of APC mutations in a small series from Japan22 has not been confirmed. The "mutator phenotype" describes a series of genetic changes seen at high rate in some tumors, especially those of hereditary nonpolyposis colorectal cancer (Lynch syndrome).!' 37 The increased risk for colon cancer in these families is attributable to mutations in several DNA repair genes (hMSH2, hMLHl, hPMSl, and hPMS2).5, 14, 34, 39, 43 The proteins from these genes cooperate to perform a repair function for DNA mismatch mutations. The lack of such a repair function in a cell dramatically increases the rate of mutation. By chance, some of these mutations should occasionally affect genes involved in neoplastic transformation. Another feature of this mismatch repair defect is that it produces numerous anomalies at simple base pair repeats, which provide a sensitive assay system for this defect. Detailed studies of three separate series in the United States found only the rare occurrence of a mismatch repair defect (Ref. 6 and 50, and unpublished observation). A smaller series from Japan reported finding the mutator phenotype at a high rate,17 but this has not been confirmed.
Figure 3. Frequency of allelic loss on selected chromosomal arms found in a series of 18 pancreatic adenocarcinomas. Allele-specific polymorphic markers were used in a PCRbased assay to evaluate for the individual sites of loss (unpublished data). Losses of 9p and 17p target the MTS1 and p53 genes, respectively. The suspected tumor-suppressor genes at the other sites of frequent loss have not been identified to date. Black bar = p arm; shaded bar = q arm.
ALLELIC LOSS AND A GENERAL MEASURE OF GENETIC DAMAGE Initially, surveys for genetic damage in tumor cells were done with karyotyping, but the rapid development of DNA probes has replaced karyotyping for certain kinds of studies. 55 It is now possible to do a genome-wide analysis of hundreds of sites, with an approximate resolution of 1 to 5 Mb and covering all chromosomes, through a highresolution allelotype. This allows one to map each chromosomal region individually for sites of allelic deletions in a particular tumor. The chromosomal regions showing frequent allelic loss are presumably those most likely to harbor tumor-suppressor genes. Sites with a high frequency of loss in pancreatic carcinoma include chromosomes 9, 17, and 18, mentioned above (Fig. 3). Allelic loss on chromosome 22q, including the site of the NF2 (neurofibromatosis type II) gene, was found to be present in 55% of cases (unpublished observation). Upon study, none of seven pancreatic tumors having 22q loss contained a terminating mutation of NF2, the consequence of the type of NF2 gene alteration frequently found in the syndrome (unpublished observation).24, 48, 54 Therefore, NF2 is not a likely candidate tumor suppressor gene for pancreatic carcinoma. Another potential use of an allelotype is in the calculation of the
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fractional allelic loss (FAL) value, providing a crude parameter for the estimate of the total genomic damage in a tumor. FAL is defined as the number of chromosomal arms having allelic loss, divided by the total number of chromosomal arms.3D An allelotype done with a small number of patients was able to compare the FAL value with the survival data. This study found a trend toward better survival when the FAL was below a certain threshold so and suggested that the aggressive behavior of pancreatic carcinoma may be related to the loss of multiple tumor suppressor genes. Recent data suggest that the average FAL value for pancreatic adenocarcinoma may be near 40%, the highest rate of LOH yet reported for human cancer (unpublished observation).
POTENTIAL CLINICAL APPLICATION
Insights into the molecular genetics of pancreatic carcinogenesis are beginning to form a genetic model for pancreatic cancer and its precursors, and it is reasonable to ask whether there will be a clinical utility for such an understanding. Despite progress on the molecular genetic level, the potential usefulness for clinical application is still to be defined for risk assessment, early diagnosis, prognosis, and therapeutics.
Risk Assessment
Among the risk factors attributed to pancreatic cancer are tobacco smoking and, possibly, pre-existing chronic pancreatitis. 15, 35 Families are known that have an increased frequency of pancreatic cancer, but specific genetic linkage, such as that found in familial forms of colorectal and breast carcinoma, has yet not been demonstrated. 36 These considerations currently make the identification of patients at risk difficult. It is therefore hoped that a genetic understanding of the disease, together with epidemiologic data, might eventually shape some distinctive risk groups. It will be of special interest whether molecular profiles of carcinomas and the precursor duct lesions may allow us to directly address the question of a potential link between chronic pancreatitis and pancreatic carcinoma.
Early Diagnosis
A strategy to prevent pancreatic carcinoma would be effected by the availability of a sensitive screening procedure, identifying those patients with incipient pancreatic neoplasms, at a stage when a curative treatment regimen is successful. A molecular genetic detection method might provide such a high sensitivity.7,s3 For example, a positive result
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in a stool ras mutation assay might direct clinical attention to a subset of patients? As of now, neither the sensitivity nor the specificity of such a test can be assured. Such tests await a better definition of the mutational spectra of the intraductal and invasive pancreatic neoplasms.
Prognosis It would be of great general interest to elucidate those factors in pancreatic carcinoma that account for its aggressive clinical behavior. For example, some sites of allelic loss, as identified in the allelotype of pancreatic cancer, might distinguish among patient groups with varying prognosis. An understanding of specific genetic factors involved in the prognosis of this form of cancer might provide clues useful in other malignancies as well.
Therapy
The most likely hope for curative treatment would come from earlier diagnosis. Further, better prognostication might help to discriminate patients who need more aggressive therapy. Eventually, however, it is hoped that unresectable or residual disease might be amenable to improved therapeutic strategies. Knowledge of the genetic mutations of a neoplasm might determine the biochemical alterations of the cells which might be targeted by specific drugs. Such is the promise held out by ras farnesyl transferase inhibitors, for example.
SUMMARY
Molecular genetic studies of pancreatic ductal adenocarcinoma have revealed the common co-existence of K-ras, p53, and MTSI mutations. The finding of K-ras mutations in epithelial lesions of ducts suggests them as a precursor intraepithelial neoplasm. The clinical importance of this line of work can only be anticipated at present, and a fuller understanding of genetic alterations in these neoplasms is necessary.
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6l. Yanagisawa A, Ohtake K, Ohashi K, et al: Frequent c-Ki-ras oncogene activation in mucous cell hyperplasias of pancreas suffering from chronic inflammation. Cancer Res 53:953, 1993 62. Yonish-Rouach E, Resnitzky D, Lotem J, et al: Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352:345, 1991 Address reprint requests to Scott E. Kern, MD 628 Ross Research Building The Johns Hopkins University School of Medicine 720 Rutland Avenue Baltimore, MD 21205-2196