Pancreatic intraepithelial neoplasia and pancreatic tumorigenesis: of mice and men.
The poor prognosis of pancreatic cancer is mainly because the disease is almost always detected in a late stage, when advanced tumor growth exists and curative resection is no longer possible. Surgery is the only option for cure, but even when surgical resection is intended as a measure for complete removal of the malignancy, most patients will develop recurrence of the disease and will die due to metastatic growth. (3-5)
Early detection, at a stage when there is still an option to cure, is at present the only way to improve the dismal outlook of patients diagnosed with pancreatic cancer. Recent advances in our understanding of pancreatic carcinogenesis may indeed translate to therapeutic options that will benefit pancreatic cancer patients. It is now well established that invasive pancreatic cancer develops through stepwise tumor progression and it is therefore preceded by preinvasive stages amenable to curative treatment. The precursor lesions of invasive pancreatic cancer are known as pancreatic intraepithelial neoplasia (PanIN) and the consecutive stages are morphologically well defined. (6) Pancreatic intraepithelial neoplasias are not the only precursors of invasive pancreatic cancer: Intraductal papillary mucinous neoplasms and mucinous cystic neoplasms, for example, are also preinvasive stages of carcinoma, (7) but PanINs are the most common precursor of conventional ductal adenocarcinomas of the pancreas, and they are the subject of this review. Pancreatic tumor progression is not only morphologically relatively well defined but many of the molecular genetic alterations leading to pancreatic cancer are also known. Consecutive stages of tumor progression are accompanied by cumulative genetic disarray in which specific genetic alterations are the major players. (8) These specific genetic abnormalities are potential markers that enable early diagnosis and may become used to target intervention. Finally, recent genetically engineered mouse models of pancreatic cancer recapitulate the human disease quite accurately and these transgenic mouse models provide potentially interesting tools for translational research purposes. (9)
[FIGURE 1 OMITTED]
PANCREATIC INTRAEPITHELIAL NEOPLASIA
It is now recognized that in the pancreas, ductal adenocarcinoma develops through a stepwise tumor progression model analogous to the adenoma-carcinoma sequence in the colorectum, in which consecutive preinvasive stages are relatively well defined and morphologically distinctive (Figure 1). These preinvasive lesions are designated PanINs and they are classified based on the degree of architectural and cytonuclear abnormalities in PanIN-1A or PanIN-1B, and PanIN-2 or PanIN- (3.6,7,10) Pancreatic intraepithelial neoplasia lesions are found in the smaller pancreatic ducts (<5-mm diameter). In PanIN lesions the normal cuboidal flat epithelial lining of the ducts is replaced by columnar mucinous cells, either flat or papillary, with various degrees of dysplasia. Typically, PanIN-1A lesions contain a lining of flat mucinous epithelium and PanIN1B lesions are covered by a papillary mucinous epithelium, in both cases with minimal cytonuclear atypia. In PanIN-2 lesions there is more cytonuclear atypia, pseudostratification, and loss of polarity consistent with low-grade dysplasia, whereas the features in PanIN-3 are those of high-grade dysplasia or carcinoma in situ with a more complex papillary architecture, nuclear hyperchromatism and pleomorphism, crowding, mitotic figures, and sometimes necrosis; the lesion is, however, still confined within the basement membrane and there is no invasive growth of tumor cells. (6)
Pancreatic intraepithelial neoplasias are proliferative lesions that are considered precursors of pancreatic ductal adenocarcinoma and are therefore neoplasms in the strict sense. The evidence that PanINs are indeed neoplastic lesions is derived from various sources and observations. Autopsy studies showed an increase of the prevalence of PanIN with age corresponding with the increase of pancreatic cancer rates in the older age groups. (11) In resection specimens with pancreatic ductal adenocarcinoma, PanINs are typically present in the area surrounding the tumor. PanIN-1 is found in 75%, PanIN-2 in 65%, and PanIN-3 in 50% of cases. (12) Well-documented cases in which high-grade PanINs subsequently developed into invasive carcinoma have also been described. (13)
Proteins that show an increased expression in ductal pancreatic carcinoma are also overexpressed in PanIN lesions. Cyclin D1 overexpression is associated with a poor prognosis in pancreatic cancer.14 Nuclear cyclin D1 overexpression is not present in the normal pancreatic ducts or in PanIN-1 lesions. It is, however, seen in 29% of the PanIN-2 lesions and in 57% of the PanIN-3 lesions. (8) Cyclooxygenase 2 is a rate-limiting enzyme in the prostaglandin pathway and its overexpression is implicated in tumor cell growth, invasion, angiogenesis, and prognosis. 15 Cyclooxygenase 2 is upregulated in pancreatic cancer and overexpression is increasingly encountered in consecutive grades of PanIN. (16) Cyclooxygenase 2 overexpression in PanINs is particularly interesting because it is a potential target for chemotherapy by means of selective cyclooxygenase 2 inhibitors. (17)
The strongest evidence that PanINs are precursors of pancreatic ductal carcinoma and truly neoplastic lesions comes, however, from molecular genetic studies in which the specific genetic alterations commonly encountered in pancreatic cancer were also found in PanINs, and, more importantly, these mutations were shown to accumulate during progression through the various stages of PanIN lesions.
GENETIC ALTERATIONS IN PANINS
Cancer is a disease of the genes. An interplay between activated oncogenes and loss of function alterations in tumor suppressor genes leads to uncontrolled and autonomous tumor cell growth. Genetic instability makes the genome vulnerable to mutations in tumor suppressor genes and oncogenes and this instability increases during tumor progression. Genes responsible for the maintenance of the DNA integrity, such as DNA repair genes, can also be involved and their loss of function contributes to genetic alterations and instability of the genome. This genetic model for tumor growth is best known for colorectal tumorigenesis, (18) but it holds true for pancreatic tumorigenesis as well, and in this regard pancreatic cancer is one of the better understood solid tumors. (19)
The most common activating point mutation occurs in the KRAS2 oncogene and more than 90% of the pancreatic cancers contain this mutation (20) (Figure 2, A). The mutation in pancreatic carcinomas always takes place in codon 12, and it is therefore easily detectable with simple molecular testing. (21,22) The KRAS2 gene encodes a guanosine-5'-triphosphate binding protein with intrinsic guanosine-5'-triphosphatase activity. The guanosine-5'-triphosphatase activity is disrupted by the mutation, and as a result the protein remains constitutively active. (21,22) The protein regulates cell cycle progression via the mitogen-activated protein kinase and AKT pathways. (23) KRAS2 mutations belong to the earliest genetic alterations observed in pancreatic tumorigenesis. KRAS2 mutations are found in 10% to 30% of PanIN-1, 45% of PanIN-2, and 85% of PanIN-3 lesions. (24) Pancreatic intraepithelial neoplasia lesions may harbor the same mutation as the adjacent carcinoma, but this is not necessarily the case. (25) The high frequency of KRAS2 mutations suggests that they can be considered an initiating event, and the genetically engineered animal models for pancreatic cancer support this notion: KRAS2 mutation is a sine qua non for the development of ductal neoplasia in the mouse and the zebrafish. (26,27) Because in the human pancreas mutations always occur in the same codon, they can be detected in small samples. (28) These mutations are therefore a potentially interesting target for early diagnosis. (21,22,29) Nevertheless, false positivity is a concern because the mutations occur in the earliest lesions and the natural history of these lesions may not justify resection. (30) In high-risk patients and in combination with other high-resolution imaging methodology to visualize early lesions, KRAS2 mutational analysis in cytology or pancreatic juice samples may have a role in diagnostic evaluation.31 As a target for therapeutic intervention, the RAS signaling pathway has been disappointing so far. (32)
[FIGURE 2 OMITTED]
In contrast to the dominantly acting oncogenes, tumor suppressor genes are recessive and, in general, both the paternal and the maternal copy need to be mutated to disrupt their function. Tumor suppressor genes inhibit tumor cell growth and disruption of their function contributes to tumorigenesis. The most important tumor suppressor genes that have a role in pancreatic tumorigenesis are p16/INK4A, TP53, and DPC4/SMAD4.
Virtually all pancreatic carcinomas harbor mutations in the p16/INK4A gene located on chromosome 9p. Homozygous deletion, intragenic mutation in combination with loss of the remaining allele, or hypermethylation of the promoter region constitute the various mechanisms of silencing of this gene in pancreatic carcinogenesis. (33,34) The gene encodes a cell cycle checkpoint protein that binds to Cdk4 and Cdk6 and in this fashion binding to cyclin D1 is prevented resulting in cell cycle arrest. (35) When there is loss of function of p16, inappropriate phosphorylation of Rb (retinoblastoma) leads to progress of the cell cycle and increased cellular proliferation. (36) The p16/Rb pathway is inactivated in 95% of the invasive pancreatic cancers, and absence of nuclear protein expression of p16, as assessed by immunohistochemistry, is found in 30%, 55%, and 70% of the invasive pancreatic cancer-associated PanIN-1, PanIN-2, and PanIN-3 lesions, respectively, indicating that loss of function is a relatively early event in pancreatic tumorigenesis. (37) This is also demonstrated by the fact that patients with a germline mutation in this gene, and particularly those with a p16-Leiden deletion, carry an increased risk for invasive pancreatic cancer (38,39) (Figure 2, B through D). In such patients early detection of precursor lesions through surveillance could be considered and may lead to a decision of prophylactic surgery. (38) Homozygous deletions of p16 may encompass adjacent genes. MTAP is such a gene and it is located 100 kilobases telomeric to the p16/INK4A gene. (40) Therefore, this gene is regularly lost with deletions of p16. This may have therapeutic implications as the MTAP protein product plays a role in the biosynthesis of adenosine, and chemotherapeutic agents have been developed to specifically target the MTAP loss in pancreatic neoplasms. (40)
The TP53 tumor suppressor gene, located on chromosomal arm 17p, is the most frequently inactivated tumor suppressor gene in human solid neoplasms, and it is also inactivated in 55% to 75% of the invasive pancreatic cancers. The protein product acts as a transcription factor that binds to DNA and when mutated, this binding is disrupted. (41) The protein has a number of critically important functions, most notably as a checkpoint protein that enables repair of DNA damage during the cell cycle arrest or as a protein that induces apoptosis when the DNA damage is irreparable. (42) In the pancreas, loss of functional p53 typically occurs through an intragenic mutation in combination with loss of the remaining wild-type allele. (43) TP53 mutations generally lead to stabilization of the protein product and positive nuclear staining with immunohistochemistry. 44 Positive immunostaining is therefore an accurate surrogate marker for mutations. (44) Positive p53 staining is typically seen in PanIN-3 lesions, that is at the transition from in situ carcinoma to invasive pancreatic cancer8 (Figure 3, A and B). TP53 mutations are a relatively late event in pancreatic tumorigenesis. Positive p53 immunostaining may nevertheless be of value in the early detection of neoplasia by means of brush cytology in high-risk patients. (45)
[FIGURE 3 OMITTED]
Another tumor suppressor gene that is frequently inactivated in invasive pancreatic cancer is DPC4 or SMAD4 located on chromosomal arm 18q. DPC stands for deleted in invasive pancreatic cancer and indeed this genetic alteration is quite specific for pancreas carcinomas, although it can occasionally be encountered in colon, breast, ovarian, and bile duct cancers as well. (46,47) The available antibodies against the DPC4/SMAD4 protein are highly suitable for immunohistochemistry and expression accurately mirrors the gene status. (48) Absence of nuclear expression in immunohistochemical stainings can therefore be of diagnostic value. (49) Loss of function is generally caused by a homozygous deletion or an intragenic mutation associated with loss of the second allele in invasive pancreatic cancer and 55% of the pancreatic carcinomas harbor such alterations. (47) The DPC4/SMAD4 protein is an intermediate in the transforming growth factor [beta] signaling pathway. On transforming growth factor [beta]/activin receptor activation, SMAD2 and SMAD3 proteins are phosphorylated and heterodimerize with the SMAD4 protein. (50) Thereafter the complex translocates to the nucleus where SMAD4 acts as a transcription factor and transactivates target genes involved in growth inhibition and apoptosis. (50) Like TP53, mutations in DPC4/SMAD4 occur relatively late during pancreatic tumorigenesis and are only seen in PanIN-3 lesions (30%-40%) and invasive cancers (49) (Figure 3, C and D).
Loss of function of genes that play a role in DNA repair and in maintaining the integrity of the genome also contribute to pancreatic carcinogenesis. This includes among others the Fanconi anemia family of genes. Fanconi anemia is a hereditary cancer susceptibility syndrome and affected family members usually die at a young age due to hematologic malignancies. (51) Those who survive develop solid tumors at an older age. The genes of the Fanconi anemia pathway involved in pancreatic tumorigenesis include the FANCC and FANCG genes and most notably the BRCA2 gene. (52) The BRCA2 gene product plays a role in DNA repair of so-called DNA-interstrand cross-links through homologous recombination. (53) Because these DNA-interstrand cross-links are deliberately introduced by certain chemotherapeutic agents, such as cis-platinum and mitomycin, it is likely that pancreatic neoplasms with disruption of the Fanconi anemia pathway may be more susceptible to such chemotherapeutic agents. (53) The percentage of invasive pancreatic cancers with BRCA2 mutations is less than 10%. (54) BRCA2 germline mutation is accompanied by increased risk for invasive pancreatic cancer and a founder mutation exists among the Ashkenazi Jewish population. (55) In patients with invasive pancreatic cancer known to carry a BRCA2 germline mutation, loss of the second allele was found in a PanIN-3 lesion but not in the low-grade lesions, indicating that complete loss of function of BRCA2 is a late event comparable with the timing of loss of function of TP53 and SMAD4/DPC4. (56)
Mismatch repair genes encode proteins that form complexes that act as repair systems for replication errors occurring during DNA replication, particularly in DNA nucleotide repeat tracks. These errors, if not repaired, introduce so-called microsatellite instability into the genome and predispose to tumorigenesis. (57) Defects in the mismatch repair genes are rare in pancreatic tumorigenesis and less than 5% of invasive pancreatic cancers contain such mutations. When defects in the mismatch repair system occur, they are accompanied by microsatellite instability and medullary type carcinomas. (58) Similar to colorectal cancer, these tumors have a better prognosis than conventional ductal adenocarcinomas of the pancreas, and KRAS2 mutations are not found. (57,58)
It is thought that the earliest event leading to genomic instability in pancreatic carcinogenesis involves a telomeric crisis initiated by erosion of the telomeres. (59) Telomeres are present at the end of the chromosomes and consist of specific DNA sequence repeats bound to proteins. These caps on the chromosomal ends protect against fusion of the chromosomes. Extreme shortening of the telomeres leads to the formation of anaphase bridges during mitosis and consequently to numerical and structural instability of the genome. (60) Telomere erosion occurs during the early stages of pancreatic tumorigenesis and is seen in more than 90% of the lowest grade PanIN lesions. (59) It is believed that the chromosomal instability due to this telomeric crisis may be the underlying cause of further genetic disarray, which develops during tumor progression in most invasive pancreatic cancers. (59,60)
GENETICALLY ENGINEERED MOUSE MODELS
Genetically engineered mouse models for invasive pancreatic cancer make use of the transduction of genetically manipulated DNA into the developing animal and it is necessary to refer briefly to pancreatic embryology to provide a framework for the understanding of the findings from transgenic mouse models. The pancreas is formed by the dorsal and ventral pancreatic buds from foregut endoderm. After fusion of the buds, the proximal duct of the ventral bud and the distal duct of the dorsal bud fuse to form the main pancreatic duct in humans. Formation of the dorsal and ventral bud, subsequent branching morphogenesis, and cytodifferentiation in specialized endocrine and exocrine cells is governed by a transcriptional machinery in which activation and repression of specific transcription factors dictate an orderly organogenesis.61 The dorsal bud develops through inhibition of Hedgehog signaling in a specific region of the endoderm, whereas the default development of the ventral bud is toward pancreas and here the induction of Hedgehog signaling limits the extent of the ventral bud formation. (62,63) Early morphogenesis of the pancreas is governed by 3 transcription factors: Pdx1, HlxB9, and p48. (64) Homozygous deletion of Pdx1 leads to pancreatic agenesis. (64) Loss of HlxB9 expression leads to agenesis of the dorsal but not the ventral bud. (65) The pancreatic epithelium contains all the precursor cells of the mature organ and differentiates along islet cell, acinar cell, and ductal cell lineages giving rise to the mature organ. Cells committed to the exocrine cell lineage lose Pdx1 expression but maintain p48 expression. Islet cell precursors maintain Pdx1 expression, lose p48 expression, and acquire neurogenin 3 expression. (66,67) An important role in the development and maturation of the pancreas is played by Notch. Notch signaling inhibits p48, and loss of Notch signaling permits differentiation into acinar cells. (68,69) Notch expression serves to maintain a pool of undifferentiated cells in the mature organ. (69) Also, various components of the notch signaling pathway, which are extinguished in the mature organ, are aberrantly reactivated in PanIN lesions and invasive pancreatic cancer. (70) In vitro Notch induction induces acinar to ductal metaplasia, whereas [gamma] secretase inhibitors (inhibiting Notch) have the opposite effect. (70) Similarly, components of the Hedgehog signaling pathway are upregulated during pancreatic tumorigenesis, whereas no expression occurs in the mature normal pancreas. (71) Cyclopamine, an inhibitor of Hedgehog, reduced tumor mass of xenografted invasive pancreatic cancers in nude mice. (71) These numerous observations support the notion that pancreatic tumorigenesis appears to be associated with reactivation of embryonic pathways that play a role in developmental morphogenesis, similar to (tumorigenesis in) other compartments of the digestive tract. (72)
Various genetically engineered mouse models of invasive pancreatic cancer have been developed and the pathology of most of them has been critically evaluated, with special emphasis on the similarities and differences with respect to human disease. (73)
Mutant KRAS endogenously expressed in the developing murine pancreas is a prerequisite for the development of ductal type adenocarcinomas, (26) and only these mouse models developed bona fide mouse pancreatic intraepithelial neoplasia (mPanIN) lesions ranging from low to high grade. Hingorani et al (26) developed a model in which expression of an oncogenic [KRAS.sup.G12D] allele is activated by a Pdx1-Cre transgene. The Pdx1-Cre, KrasG12D mice develop mPanIN of various degrees and some develop ductal type invasive cancers as well, thereby faithfully recapitulating the natural history of the human disease (Figure 1). Not only do these mPanIN lesions resemble the human lesions microscopically, but they also overexpress proteins such as Notch, Hedgehog, and cyclooxygenase 2, similar to the human situation. (26) When crossed with a biallelic INK4A/ Arf deletion or an oncogenic [Trp53.sup.R172H] allele, these mice develop more aggressive tumors with metastasis, short latency, and full penetrance. (74,75) Importantly, sole abrogation of INK4A/Arf or TP53, without mutant KRAS, does not lead to pancreatic carcinogenesis. (74,75)
The development of the genetically engineered mouse models of ductal type invasive pancreatic cancer has enhanced our understanding of the disease considerably. The mouse models have confirmed the importance of mutantly activated KRAS in ductal pancreatic adenocarcinogenesis. The fact that the carcinomas are also accompanied by low- and high-grade mPanINs in these mouse models, microscopically resembling their human counterparts, is yet another argument in favor of their role as the preceding noninvasive precursor stages of ductal pancreatic adenocarcinoma and supports the legitimacy of the postulated human tumor progression model.
Interestingly, despite the previous bona fide mouse models of human pancreatic carcinogenesis, the earliest lesion and initiating event, that is the cell of origin of invasive pancreatic cancer, remains to be elucidated. Given their ultimate ductal phenotype and because the precursor lesions also exhibit features of ductal differentiation, one would perhaps assume that the ductal epithelial cells give rise to the carcinomas. Surprisingly, targeting of oncogenic KRAS into the mature ductal epithelium does not induce neoplastic growth in the murine pancreas. (76) Growth of mPanINs and ductal adenocarcinomas can be produced in the adult pancreas of mice by conditional expression of mutant KRAS in the acinar/centroacinar compartment, but only if accompanied by ongoing injury and chronic inflammation. (76) These findings implicated the acinar/centroacinar compartment as the site of origin of ductal pancreatic adenocarcinoma and they underscored the importance of chronic pancreatitis as a risk factor for cancer, a long-standing well-known fact from epidemiologic studies. (77) These observations also led to a critical reappraisal of the morphologic findings in specimens in which chronic inflammation surrounds the pancreatic carcinoma, with particular attention to foci of so-called acinar to ductal metaplasia. (73) This metaplasia is a common finding in a variety of genetically engineered mouse models and is characterized by tubular structures that have features of acinar as well as ductular differentiation. (77) It is also one of the first abnormalities seen after ligation of the pancreatic duct in other animal models. (78) In the human pancreas, acinar to ductal differentiation is particularly seen in the setting of chronic pancreatitis and these lesions can be associated with PanINs. (79) From the previous mouse model in which there is induction of chronic injury, it appears that the chronic inflammation resulting in acinar to ductal metaplasia causes otherwise quiescent cells to adapt a progenitor-like cell type, susceptible to oncogenic transformation by mutant KRAS activation; this could be due to dedifferentiation of mature acinar cells, expansion of a centroacinar progenitor cells, or both. (77) As mentioned earlier, in vitro systems show a similar phenomenon after Notch activation, and the observed reactivation of Notch pathway components in PanIN lesions is of particular interest in this context. (70) In an attempt to integrate all these data into pathologic findings in human disease, a recent study evaluated resection specimens harboring acinar to ductal metaplasia accompanied by PanIN lesions and performed KRAS2 mutational analysis. Mutations were not found in acinar cells or isolated foci of acinar to ductal metaplasia, that is cells distant from the PanIN lesions. Of the metaplastic foci associated with a PanIN lesion, one-quarter harbored a KRAS2 mutation, and this mutation was identical to the one in the accompanying PanIN lesion. In the remaining PanIN lesions with a KRAS2 mutation, the associated acinar to ductal metaplasia did not contain this KRAS2 mutation. (80) Under the assumption that KRAS2 mutation is the earliest genetic event in pancreatic carcinogenesis, the findings do not seem to support the conclusion drawn from the mice studies that dedifferentiated acinar cells or centroacinar progenitor cells are the putative cell of origin for human ductal pancreatic adenocarcinoma.
Ductal pancreatic adenocarcinoma is the most common cancer of the pancreas and a lethal disease; early detection, in a stage when surgical resection is still an option for cure, is currently the only hope for a better outcome. The noninvasive precursor stages of ductal pancreatic adenocarcinoma, called PanINs, are well-defined and potential targets for early diagnosis or treatment. Genetically engineered mouse models for ductal pancreatic carcinoma are an important adjunct to the armamentarium of translational research. These models faithfully recapitulate the natural course and biology of the human disease. In both mice and men, pancreatic cancer appears to be initiated by oncogenic KRAS2. Inactivation of various tumor suppressor genes then accelerates tumor progression in the mouse and the human exocrine pancreas, including TP53, P16/INK4A, and SMAD4 or transforming growth factor [beta] receptor 2. Interestingly, recent evidence suggests that the cell origin of human pancreatic carcinogenesis may differ from that in the mouse.
We thank Marjon Clement, BSc, for her assistance with the preparation of the manuscript.
Accepted for publication October 21, 2008.
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Niki A. Ottenhof, BA; Anya N. A. Milne, MD, PhD; Folkert H. M. Morsink, BSc; Paul Drillenburg, MD, PhD; Fiebo J. W. ten Kate, MD, PhD; Anirban Maitra, MBBS; G. Johan Offerhaus, MD, MPH, PhD
From the Department of Pathology, University Medical Center, Utrecht, the Netherlands (Ms Ottenhof, Drs Milne, ten Kate, and Offerhaus, and Mr Morsink); the Department of Pathology, Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands (Dr Drillenburg); and the Department of Pathology, The Sol Goldman Pancreatic Research Center, The Johns Hopkins Medical Institution, Baltimore, Maryland (Dr Maitra).
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: G. Johan Offerhaus MD, MPH, PhD, Department of Pathology, University Medical Center, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands (e-mail: email@example.com).
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|Author:||Ottenhof, Niki A.; Milne, Anya N.A.; Morsink, Folkert H.M.; Drillenburg, Paul; ten Kate, Fiebo J.W.;|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Mar 1, 2009|
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