Pathology, genetics and precursors of human and experimental pancreatic neoplasms: An update

Pancreatology xxx (2015) 1e13

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Review article

Pathology, genetics and precursors of human and experimental pancreatic neoplasms: An update € ppel b Irene Esposito a, *, 1, Angela Segler b, 1, Katja Steiger b, Günter Klo a b

Institute of Pathology, Heinrich-Heine-University of Düsseldorf, Moorenstr. 5, 40225, Düsseldorf, Germany €t München, Ismaningerstr. 22, 81675, Munich, Germany Institute of Pathology, Technische Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

Over the past decade, there have been substantial improvements in our knowledge of pancreatic neoplasms and their precursor lesions. Extensive genetic analyses, recently using high-throughput molecular techniques and next-generation sequencing methodologies, and the development of sophisticated genetically engineered mouse models closely recapitulating human disease, have improved our understanding of the genetic basis of pancreatic neoplasms. These advances are paving the way for refined, molecular-based classifications of pancreatic neoplasms with the potential to better predict prognosis and, possibly, response to therapy. Another major development resides in the identification of subsets of pancreatic exocrine and endocrine neoplasms which occur in the context of hereditary syndromes and whose genetic basis and tumor development have been at least partially defined. However, despite all molecular progress, correct and careful morphological characterization of tissue specimens both in the context of experimental and routine diagnostic pathology represents the basis for any further genetic investigation or clinical decision. This review focuses on the current and new concepts of classification and on the current models of tumor development, both in the field of exocrine and endocrine neoplasms, and underscores the importance of applying standardized terminology to allow adequate data interpretation and promote scientific exchange in the field of pancreas research. Copyright © 2015, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.

Keywords: Pancreatic cancer Ductal adenocarcinoma Neuroendocrine neoplasm Precursor lesion Mouse model KRAS

Introduction The pancreas is an organ with dual function exerted by an exocrine and endocrine cell compartment. Thus, the neoplasms arising therefrom display different phenotypes in terms of morphology and biology, and are driven by distinct genetic alterations. In order to improve the prognosis and treatment options, especially for those tumors with unfavorable outcome, it is essential to identify precursor lesions and their driving molecular alterations. With this knowledge we will improve our understanding of the tumors' natural history, we may then better select the patients who profit from existing therapeutic approaches and might finally define new therapeutic targets. The development of sophisticated genetically engineered mouse models that closely recapitulate human disease, together with the

* Corresponding author. Tel.: þ49 211 811 8339; fax: þ49 211 811 8353. E-mail address: [email protected] (I. Esposito). 1 Equally contributing first authors.

extensive use of high-throughput molecular techniques and nextgeneration sequencing methodologies have improved our understanding of the genetic basis of pancreatic neoplasms and may prepare our way for clinic-translational approaches. The bottom line for these approaches is the consistent use of established, internationally accepted classification systems and a unanimous nomenclature both in research and routine pathology. The 2010 World Health Organization classification [1] of pancreatic tumors is widely accepted. It separates the tumors in epithelial (including exocrine and neuroendocrine tumors) and non-epithelial neoplasms and subtypes them according to their biological behavior in benign, premalignant and malignant (Table 1). Human neoplasms can be further classified in sporadic, familial and hereditary. A neoplasm occurring in individuals who do not have a germline mutation that confers increased susceptibility to cancer or without a family history of cancer is defined as sporadic. Cancer that occurs in families more often than would be expected by chance and usually at an earlier age compared to the general population is defined as familial. Shared environmental or lifestyle

http://dx.doi.org/10.1016/j.pan.2015.08.007 1424-3903/Copyright © 2015, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.

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I. Esposito et al. / Pancreatology xxx (2015) 1e13 Table 1 a WHO classification of epithelial tumors of the pancreas. Entity

Subtypes

Benign

Acinar cell cystadenoma Serous cystadenoma

Premalignant

Pancreatic intraepithelial neoplasia, grade 3 (PanIN-3) Intraductal papillary mucinous neoplasm Intraductal tubulo-papillary neoplasm Mucinous cystic neoplasm

Malignant

With low- or intermediate-grade dysplasia With high-grade dysplasia With low- or intermediate-grade dysplasia With high-grade dysplasia

Ductal adenocarcinoma

Adenosquamous carcinoma Colloid carcinoma Hepatoid carcinoma Medullary carcinoma Signet ring cell carcinoma Undifferentiated carcinoma Undifferentiated carcinoma with ostoclast-like giant cells

Acinar cell carcinoma Acinar cell cystadenocarcinoma Intraductal papillary mucinous neoplasm with an associated invasive carcinoma Pancreatoblastoma Mixed acinar/ductal/neuroendocrine carcinoma Serous cystadenocarcinoma Solid-pseudopapillary neoplasm Neuroendocrine neoplasms

Neuroendocrine microadeoma Neuroendocrine tumor (NET)

Neuroendocrine carcinoma (NEC) a

Nonfunctional pancreatic NET Functional pancreatic NET - EC cell, serotonin producing - Gastrinoma - Glucagonoma - Insulinoma - Somatostatinoma - VIPoma Small cell NEC Large cell NEC

Slightly modified from Ref. [1].

factors play a role in familial cancer. If a germline mutation predisposes to cancer development, the term hereditary neoplasm is used (for definitions see also www.cancer.gov/dictionary). Among the pancreatic neoplasms the sporadic epithelial nonneuroendocrine tumors are most frequent and account for about 90%. They display either an acinar or ductal cell phenotype. In addition, there are some rare tumors whose phenotypic appearance is not attributable to one of the known cell types. Genetically, the neoplasms with a ductal phenotype usually harbor KRAS mutations (“KRAS-positive”), while those with other phenotypes such as the acinar cell carcinomas, neuroendocrine neoplasms, serous cystic neoplasms and solid pseudopapillary neoplasms have a different genetic profile. In this review, the pancreatic neoplasms and their precursors are classified according to their KRAS-status and their cellular phenotype (see Table 2). In addition, their counterparts in genetically engineered mice are briefly described for comparison. Finally, we present a conceptual approach to the development of pancreatic neoplasms. KRAS-positive neoplasms with ductal phenotype Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor that histologically imitates small and medium-sized pancreatic ducts. PDAC affects usually old and only rarely patients younger than 40 years, occurs slightly more in men than women and is the most common pancreatic neoplasm (85e90%) [2e4]. For the last reason, the term “pancreatic cancer” is often used as synonymous to PDAC. However, since the pancreas gives also rise to

other types of “cancers” distinct from PDAC, the term PDAC should be used in the scientific literature, if this tumor type is discussed. PDACs are solid, firm and poorly defined tumors, measuring about 2.5e3 cm when diagnosed [5] (Fig. 1A). As they predominantly occur in the head (70%) of the pancreas, they often narrow the distal common bile duct and the preampullary pancreatic duct, causing cholestasis on the one hand and fibrosis of the pancreatic parenchyma on the other. The pancreas has no defined capsule [6], and this enables the tumor tissue to easily infiltrate into the retropancreatic space, where it spreads along nerve plexus, it frames and infiltrates the superior mesenteric artery and metastasizes to the peripancreatic, peribiliary, mesenteric and paraaortic lymph nodes. Hematogenous metastases are usually first seen in the liver and only later at other sites [2,3]. Histologically, the typical PDAC shows well-differentiated ductlike structures embedded in a desmoplastic stroma (Fig. 1B). The cancer cells express as main markers the mucin core proteins (MUC1, MUC5AC), the cytokeratins 7, 8, 18, 19 and commonly also 20, carcinoembryonic antigen (CEA) and often p53 and SMAD4 protein [2,3]. The prognosis is determined by histological grade (G), TNM-status and the resection margin infiltration [7,8]. Variants of PDAC according to the WHO classification include adenosquamous carcinoma, colloid carcinoma, hepatoid carcinoma, medullary carcinoma and undifferentiated (anaplastic) carcinoma. Genetically, the typical PDAC harbors mutations in the KRAS gene locus, followed by mutations of CDKN2A/p16, SMAD4 and TP53. Among the PDAC variants, KRAS mutations seem to play a main role in adenosquamous carcinoma and undifferentiated

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Table 2 Sporadic exocrine neoplasms of the pancreas and their precursor lesions. Type

Gender (age range)

KRAS-positive, ductal phenotype PDAC M > F (65e85)

IPMN

Pancreatic intraepithelial neoplasms (PanIN)

Mucinous cystic neoplasms (MCN)

M ¼ F (50e70)

M ¼ F > 40 y

Mostly F (40e60)

Localization

Morphology

Head (60e70%)

1.5e5 cm, solid, invasive Mucin production, Duct formation Intense stromal reaction >1 cm, cystic with papillary projections and thick mucin, multifocality Gastric subtype (branch duct), intestinal, pancreatobiliary, oncocytic (main duct) Microscopic lesions, <0.5 cm, noninvasive Seldom flat, mainly papillary, mucin production 6e10 cm, cystic, unifocal

Head/body

Any

Tail (>90%)

Key facts for diagnosis

Thick walled cysts, mucin production, ovarian-like stroma

KRAS-negative, acinar phenotype Acinar cell carcinoma M >> F (55e65)

Pancreatoblastoma

M ¼ F children

KRAS-negative, uncertain phenotype Serous cystic neoplasm F > M (60e80) Solid-pseudopapillary neoplasms

F>>>M (8e85)

Any

Any

Mostly solid, invasive, softer than PDAC Acinar, solid, trabecular

Solid Acinar structures, squamoid nests

Body-tail

Microcystic, oligocystic, solid

Any

Solid with necrotic-haemorrhagic areas Solid and pseudopapillary, monomorphic cells, PAS þ globuli

anaplastic carcinoma, while in medullary carcinoma and probably also hepatoid carcinoma different molecular pathways may be active (see later). In undifferentiated carcinoma with rhabdoid features it has been recently shown that its morphological phenotype correlates with different genotypes. KRAS amplifications and no alteration of SMARCB1/INI (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1) characterized undifferentiated carcinomas with pronounced pleomorphic appearance, while undifferentiated carcinomas with a more monomorphic appearance had SMARCB1/INI alterations but no KRAS abnormalities [9]. Recent data generated through extensive transcriptome analysis and deep-sequencing methods have suggested that molecular subtypes of PDAC with different clinical behavior and response to therapy exist [10,11]. A first comprehensive exome sequencing of a series of 24 PDAC revealed that each tumor contained an average of 63 mutations [12] and a more recent whole genome sequencing analysis on 100 samples revealed a total of 11,868 somatic structural variants with an average of 119 per individual [9]. Frequent genetic abnormalities, including mutations of KRAS, TP53, CDKN2A and SMAD4 are regarded as “driver mutations”, which seem to be essential for the development of the neoplasms [12]. Subgroup analysis based on variations in chromosomal structure showed that high genomic instability co-segregates with deficiency in DNA damage repair and alteration of DNA-maintenance genes, such as

IHC: MUC1, MUC5AC, CK 7,8,18,19; CEA Common genetic alterations: KRAS, CDKN2A/P16, TP53, SMAD4/DPC4

IHC: MUC1, MUC2, MUC5AC, MUC6, CDX2 Common genetic alterations: KRAS, GNAS, RNF43, CDKN2A/P16, TP53, SMAD4 IHC: MUC1 (mostly in high-grade lesions), MUC5AC Common genetic alterations: KRAS, CDKN2A/P16, GNAS, TP53, SMAD4/DPC4

IHC: CK 7,8,18,19; CEA, MUC5AC, MUC1 in invasive component Common genetic alterations: KRAS, CDKN2A/P16, TP53, SMAD4/DPC4

IHC: CK 7,8,18, 19, Trypsin, chymotrypsin, bcl-10, CEA, AFP (rare) Common genetic alterations: APC-pathway, BRCA2, FAT, TP53 IHC: CK 7, 8, 18, 19, CEA, trypsin, chymotrypsin, synaptophysin, AFP Common genetic alterations: APC-bcatenin pathway, Smad4 IHC: CK 7, 8, 18, 19, Inhibin, MUC6 Common genetic alterations: VHL

IHC: vimentin, CD10, Synaptophysin (focal), bcatenin (nuclear), PR Common genetic alterations: CTNNB1

BRCA1, BRCA2 and PALB2 and good response to platinum chemotherapy [10]. Further defining genotypeephenotype correlations may therefore be extremely valuable in identifying subgroups of patients who might profit from individualized treatment strategies. Precursor changes that give rise to invasive PDAC include preinvasive lesions such as pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm (IPMN), and mucinous cystic neoplasm (MCN). These lesions are stratified by cytological and architectural atypia in low and high-grade dysplasia, which correlates with progressive accumulation of genetic and epigenetic alterations. Given the rarity of IPMNs and MCNs, it is hypothesized that the majority PDACs (>95%) originate from PanINs. PanINs are microscopic (<0.5 cm) flat or papillary, non-invasive duct cell proliferations separated into a group with low-grade (including PanIN 1 and 2) and high-grade dysplasia (i.e. PanIN 3) [13,14]. The histological progression from low-grade to high grade PanIN is mirrored by the accumulation of genetic changes [15e17] Early genetic alterations include activating KRAS mutation and telomerase shortening, detectable in >90% of PanIN1 [18], mutations involved in later stages concern inactivation of the tumor suppressor genes CDKN2A/p16, TP53 and SMAD4 [13]. According to this multistep tumor progression model, immunohistochemical characteristics also vary with the grade of dysplasia. For example,

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Fig. 1. A Gross image of a firm and solid pancreatic ductal adenocarcinoma (PDAC) (arrow) in the head of the pancreas with stenosis of the distal bile duct and dilatation of the main pancreatic duct (arrowhead). B Pancreatic biopsy with well differentiated PDAC showing duct-like structures (arrows) embedded in desmoplastic stroma.

the apomucins MUC1 and MUC5A can be already detected in lowgrade PanIN lesions and show progressively increased expression in high-grade lesions and invasive cancer [19]. Intraductal papillary mucinous neoplasms (IPMNs) are grossly visible epithelial neoplasms (1 cm), growing within the main pancreatic duct or one or more of its branches. The neoplastic proliferations consist of columnar cells displaying a ductal phenotype with mucin production. There are four epithelial subtypes, showing intestinal, gastric, pancreatobiliary or oncocytic differentiation and revealing different features concerning the expression of MUC1, MUC2, MUC5AC and MUC6, frequency, site of occurrence, multifocality, type of associated invasive carcinoma (see below) and prognosis [20,21]. Briefly, IPMN with gastric differentiation, which mostly belong to the branch-duct category, have been shown to bear the best prognosis of the whole group, and pancreatobiliary IPMN the worst, independently from the presence of an associated invasive carcinoma [19]. Recent data derived from studies based on targeted massive parallel sequencing support the concept that different molecular pathways may at least partially explain the differences in biologic behavior (see below, genetic alterations). IPMNs can be further classified according to their degree of dysplasia in low and high-grade lesions [14,22], and according to their association with an invasive carcinoma. Approximately one third of IPMNs is invasive [22], and among these invasive carcinomas there are two distinct types: colloid (mucinous) and tubular (classical ductal) adenocarcinomas. Colloid carcinomas are associated with intestinal-type IPMNs and bear a significantly better prognosis than tubular (ductal) adenocarcinomas that arise from gastric or pancreatobilary-type IPMNs [22]. Genetic alterations in IPMNs are in part similar to those detected in PDAC. The presence and the extent of these genetic alterations are mirrored by the degree of dysplasia and invasiveness of the IPMN. An average of 30e80% of IPMNs harbor a KRAS mutation and the KRAS mutation status correlates with the degree of dysplasia [22,23] and the presence of an associated invasive adenocarcinoma of tubular type [24]. Loss of p16 expression occurs in 100% of

invasive compared with 10% of noninvasive IPMNs [25]. P53 overexpression and loss of SMAD4 are late events associated with highgrade dysplasia and invasive carcinomas [22,23,26]. IPMNs also contain alterations that differ from those involved in PDAC. Approximately 60% of all IPMN types are associated with activating GNAS mutations at codon 201 and 75% with somatic mutations in RNF43 [27]. GNAS mutations occur in 100% of IPMN of the intestinal type [28] and in 83e89% of IPMN-associated colloid carcinomas [24,29]; moreover, they are detected in about 50% gastric and 30% pancreatobiliary IPMN, independently from the degree of dysplasia [24]. In addition, recent data show that gastric type IPMN without GNAS mutations are more frequently associated with an invasive carcinoma than those bearing GNAS mutations [30]. Altogether, these data suggest a dichotomy in the molecular pathogenesis of IPMN, where GNAS mutations are mostly associated with a less aggressive intestinal and epartially- gastric phenotype, whereas KRAS mutations mostly occur in the more aggressive pancreatobiliary IPMN and invasive carcinomas of classical ductal (“tubular”) type. Mutations in BRAF and PIK3CA and promoter hypermethylation of multiple genes (that was associated with an increasing degree of dysplasia) have also been reported in a small fraction of IPMNs [31,32]. The many genetic alterations in IPMNs represent promising targets to improve the sensitivity of screening and diagnostic assays. The examination of pancreatic cystic/juice fluid seems to be a promising diagnostic option because mutations present in the neoplastic cells can be detected in the fluid. Due to 95% mutation rates of either GNAS or KRAS in IPMN, this method could also be used for the differential diagnosis of cystic lesions [33,34]. Mucinous cystic neoplasms (MCNs) are clearly distinct from all other pancreatic neoplasms, but particularly from PanINs and IPMNs. They are not connected to the ducts, mainly located in the pancreatic (body-) tail, and occur almost invariably in women [22,35]. The cells lining the cysts show the mucin producing ductal phenotype, and are supported by progesterone receptor and inhibin-positive ovarianlike stroma [22,36]. As with IPMNs, MCNs can be further classified

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according to their degree of dysplasia in low- and high-grade lesions [14,22]. About 10% of MCNs are associated with an invasive carcinoma, usually with features of classical PDAC. The prognosis for patients with noninvasive or invasive, completely resected, MCNs is favorable [37]. Exceptions are the rare MCNs that primarily present with an invasive undifferentiated carcinoma. MCNs display similar genetic alterations as PDACs. KRAS alterations are common, even in low-grade lesions, while alterations in p16, p53 and SMAD4 have been mainly observed in cases with severe dysplasia and invasive carcinoma [36,38]. Whole-exome sequencing showed that MCNs contain an average of 16 mutations in each tumor. It also identified mutated RNF43, supporting its role as tumor suppressor gene in mucin-producing cystic neoplasms of the pancreas. GNAS mutations were not detected in MCNs and can therefore be used to differentiate between IPMNs and MCNs [27]. KRAS-negative neoplasms with acinar cell phenotype Acinar cell carcinoma (ACC) is a rare neoplasm (<2% of pancreatic malignancies) that can occur anywhere in the pancreas and typically forms a large well-demarcated solid and nodular, and only rarely cystic, mass (Fig. 2A). Men are more frequently affected than women (mean age 55e65 years). The tumor cells produce pancreatic enzymes, notably trypsin and lipase that can be detected by immunohistochemistry (Fig. 2B) [2,39]. Interestingly, some ACCs may contain substantial proportions (>30%) of nonacinar cells showing neuroendocrine (MANEC, mixed adenoneuroendocrine carcinoma) and occasionally ductal differentiation [39e42]. Two recent whole exome-based analyses of pancreatic tumors with acinar differentiation revealed an average of 65 nonsynonymous mutations/tumor respectively, although >30% of the tumors did not show any mutation [43,44]. The genetic alterations in ACC differ from those commonly mutated in PDAC, since alterations in KRAS, TP53, SMAD4, RNF43 and GNAS have only rarely been reported [22,43,45,46]. In ACCs, somatic alterations in the APC/bcatenin pathway (CTNNB1 and APC) have been described in up to 20e25% [47]. Other reported findings are promoter hypermethylation of several tumor suppressor genes and large chromosomal gains and losses [22,45]. In addition, recent studies revealed a microsatellite stable, chromosomal unstable genotype of ACC with defects in genes involved in DNA maintenance (e.g. BRCA2, PALB2, ATM) and high number of large chromosomal changes, potentially involved in the therapy resistance of this tumor type [40,43,44]. Pancreatoblastoma is a very rare tumor, mostly occurring during infancy and showing acinar differentiation in addition to socalled squamoid nests, expressing nuclear b-catenin. To date no precursor lesion of acinar cell type neoplasms has been identified. The so-called acinar cell cystadenoma is unrelated

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to ACC or PB and most probably represents a non-neoplastic cystic change of the acini, with no evidence of malignant transformation in the cases published so far [48,49]. KRAS-negative neoplasms with uncertain phenotype and various genetic changes Solid-pseudopapillary neoplasm (SPN) is a low-grade malignant tumor that typically affects young women. It presents as a solitary, well-circumscribed mass, which often undergoes pseudocystic hemorrhagic degeneration. For this reason, SPNs are usually included among the cystic pancreatic neoplasms. Microscopically, SCNs are composed of monomorphic epithelial cells forming solid and pseudopapillary structures. Genetically, 95% of all SPNs harbor somatic activating mutations in the b-catenin gene (CTNNB1), that lead to nuclear localization of the b-catenin protein, loss of the membranous E-cadherin expression, and downstream activation of a number of transcription factors. Other peculiar immunohistochemical findings are expression of vimentin, CD10, CD56 and (focally) synaptophysin [22,50,51]. Whole-exome sequencing revealed an average of only 3 nonsynonymous mutations per SPN [27]. Serous cystic neoplasms (SCN) are (with very rare exceptions [52]) benign tumors that are subtyped as serous microcystic adenoma or serous oligo- and macrocystic adenomas. The tumors present predominantly in old women. The multicystic tumors are usually large (4e10 cm) and composed of cuboidal cells that express inhibin, NSE and MUC6. Multifocal SCNs occur in the setting of a von Hippel-Lindau (VHL) syndrome, with a VHL germ line mutation combined with a LOH at 3p. Somatic inactivating mutations (point mutations) in the VHL gene occur in up to 50% of sporadic SCN [22,27]. Whole-exome sequencing revealed 10 nonsynonymous somatic mutations per tumor in SCN. These alterations differed from those frequently mutated in other cystic neoplasms of the pancreas, such as IPMNs and MCNs and therefore represent a promising target for diagnostic assays [22,27]. Medullary carcinomas show a solid (“medullary”) histological pattern with pushing borders and a lymphoid stroma reaction. Genetically, some of them are wild-type for the KRAS gene, microsatellite instable and show mutations in one of the DNA repair genes MLH1 and MSH2 [53,54]. Medullary differentiation is associated with better prognosis and is predictor of poor response to certain adjuvant chemotherapies, such as 5-flouoruracile [54e56]. KRAS-negative neoplasms with neuroendocrine phenotype Neuroendocrine neoplasms (NENs) account for 1e2% of all pancreatic tumors. They affect both genders evenly and may occur at a wide range of age (30e60 years). The WHO classification

Fig. 2. A Gross image of an acinar cell carcinoma (ACC) Solid circumscribed tumor filling the head of the pancreas and showing focal hemorrhagic necrosis. B ACC are often composed of acinar and solid structures with a high neoplastic-cell to stroma ratio.

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distinguishes two main groups: the well-differentiated neuroendocrine tumors (PanNETs) and the poorly-differentiated neuroendocrine carcinomas (PanNECs). These subtypes are characterized by their histological differentiation and proliferative activity, assessed by mitotic count or preferably by the Ki-67 index. Accordingly, PanNETs display low (G1, <2%) or moderately enhanced (G2, 3e20%) proliferative activity, and PanNECs are highly proliferating (G3, >20%) neoplasms. Immunostaining for chromogranin A and synaptophysin and the Ki-67 index are essential for the diagnosis and classification [57,58]. PanNENs that show a Ki67 proliferation rate exceeding 20% but retaining a well differentiated histology may be provisionally classified as “PanNET G3”, as these tumors may still respond to the therapy options of the usual PanNETs [59]. PanNETs are rather indolent, slowly growing neoplasms that usually present as solitary, round masses and histologically display several characteristic organoid growth patterns (Fig. 3A and B). In addition, PanNET show expression of peptide hormones and somatostatin receptors and may produce hormonal syndromes. Syndromic (“functioning”) PanNETs include insulinomas, glucagonomas, gastrinomas, VIPomas and others [60]. However, these PanNETs are outnumbered by nonfunctioning tumors, which are not associated with a hormonal syndrome, although many of them may express hormones such as glucagon and PP. Some of these neoplasms are multihormonal and in their metastases they may even produce hormones other than those found in the primary tumor [57,61,62]. Studies in PanNET using transcription factors such as PDX1 (pancreatic and duodenal hoemobox-1), CDX2 (caudal type homeobox 2), NGN3 (neurogenin 3) and ISL1 (islet-1), which are involved in the development and differentiation of pancreatic neuroendocrine cells, revealed expression patterns that correlated with the dominant hormone expression in the tumors [63]. PanNETs can be associated with hereditary tumor syndromes such as MEN1 or VHL. Stage and grade have emerged as the most potent predictive factors in most studies [61,64,65]. When surgical

resection is performed, the prognosis is usually better compared to PDAC [66]. PanNECs, in contrast to PanNETs, are often large tumors with illdefined borders and hemorrhagic-necrotic areas. Histologically, they are separated into a large and small cell subtype, the first subtype showing large solid nests arranged in an organoid pattern (Fig. 3C) the second diffuse irregular sheets of cells (Fig. 3D) [67,68]. Both types are highly proliferative and exhibit intratumoral necroses. They usually express no hormones and thus are not associated with hormonal syndromes. As highly aggressive neoplasms, they often present with extensive metastases at the time of diagnosis [67,68]. Survival ranges from 1 month to a year, despite some initial favorable responses to chemotherapy [69,70]. Whether the small and large cell subtypes of poorly differentiated PanNENs represent different entities, as suggested by their histology, is so far unclear. According to recent studies, their genetic profiles seem to be similar, inasmuch as both subtypes were found to carry TP53 and Rb/p16 mutations in equal frequency and to show similar BCL-1 expression [71]. However, as small and large cell subtypes often differ in their proliferative activity (lower Ki67 index in the large cell subtype) and their association with non-neuroendocrine elements, such as adenocarcinoma components (in case of the large cell subtype), it is conceivable that in the future more detailed genetic studies might reveal some subtle differences between the two subtypes [72]. Pathogenesis. Alterations of the MEN1, VHL, or NF1 genes, which are found as germ line mutations in familial PanNETs associated with MEN1, VHL and NF1 syndromes, play also a role in the development of sporadic NETs (Table 3). Recent whole-exome sequencing of PanNETs revealed three distinct genetic signatures. The largest group of PanNETs, about 45%, harbor somatic mutations of the MEN1 gene and either the DAXX or ATRX gene [67], genes that are involved in the maintenance of the telomere structure, without using the telomerase system (alternative lengthening of

Fig. 3. A Gross image of a pancreatic neuroendocrine tumor (PanNET) showing a well-demarcated, solitary, yellow-whitish surface (arrows). B PanNET with trabecular cell arrangement. C Pancreatic nenroendocrine carcinoma (PanNEC) of large cell type: Poorly differentiated neoplasm with distinct cell pleomorphism and small necrotic foci but a still retained organoid growth pattern. D PanNEC, small cell type, characterized by sheets of small blue irregular cells.

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telomeres, ALT) [67,73]. The second signature, which is related to HIF-alpha and VHL alterations, is found in approximately 25% of the PanNETs [74]. Finally, the third signature, detected in about 15% of pancreatic NETs, involves alterations in the mTOR cell signaling pathway, which is connected to various tyrosine kinase receptors and plays an important role as possible target for therapy [20,67,68]. Noteworthy is that the common mutations identified in PDACs (e.g. TP53, KRAS, CDKN2A, SMAD4) are not found with any significant frequency in PanNETs [69]. In poorly differentiated PanNENs, two possibilities have to be considered. First, poorly differentiated PanNENs may derive from well-differentiated PanNENs through a process of dedifferentiation. Second, the poorly differentiated GEP-NENs are de novo neoplasms that have their own tumor stem cell distinct from that giving rise to well differentiated NENs. In support of the latter assumption is the observation that well differentiated PanNENs, which in the setting of liver metastases show increased proliferative activity to values consistent with a grade 3 (Ki67 > 20%), usually a feature of the poorly differentiated NENs, retain the typical well differentiated histological pattern with ISL1-positivity and no expression of p53 and do not convert histologically into poorly differentiated NENs [75]. Further evidence for a clear distinction between well and poorly differentiated PanNENs comes from recent immunohistochemical and genetic findings [71,76], showing in well differentiated PanNENs the consistent expression of the transcription factor ISL-1 and the common mutation of the genes MEN1, ATRX and DAXX [76], findings which are absent in poorly differentiated PanNENs. Neuroendocrine precursor lesions. A hyperplasia-neoplasia sequence has been proposed to explain for the origin of NET in MEN1-patients [77]. The activation of the germ line MEN1 mutation is the effect of the loss of the second allele at the MEN1 gene locus on 11q3, followed by the development of microadenomas (<0.5 cm) from hyperplastic lesions, mainly affecting the glucagon-producing cells [78]. Recently, it has been shown that alterations in ATRX and DAXX are late events in the tumorigenesis of pancreatic well differentiated NENs in MEN1 patients [79]. A similar hyperplasia-neoplasia sequence as in MEN1 is also observed in the recently discovered glucagon cell adenomatosis, which is, in half of the cases, associated with a germ mutation in the glucagon receptor gene [80]. Another disease that might also follow a hyperplasiaeneoplasia sequence is insulinomatosis, causing recurrent hyperinsulinemic hypoglycemia [81]. Poorly differentiated PanNENs, which are on the other end of the differentiation spectrum, are not associated with MEN1 or any other hereditary syndrome, and are not preceded by any precursor changes involving protodifferentiated NE cells. It seems therefore

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likely that poorly differentiated NEN originate from an early neuroendocrine precursor cell tackled by mutational hits, which affect genes of general importance for cell life and involved in cell cycle control, such as TP53, Rb and CDKN2A/p16. Familial/hereditary pancreatic neoplasms PDAC are reported in approximately 5e10% of patients with a family history of pancreatic cancer [82]. The terms hereditary and familial pancreatic cancer have been frequently interchangeably used in the literature, sometimes causing confusion among their meaning and definition. While the term “hereditary pancreatic cancer” refers to pancreatic cancers arising in patients with recognized genetic syndromes (Table 4), “familial pancreatic cancer” (FPC) defines patients with PDAC having at least two firstdegree relatives with confirmed PDAC and who do not fulfill the criteria of other inherited tumor syndromes. Studies have suggested that FPC is mostly autosomal inherited with a heterogeneous phenotype [83] but the disease-causing mutation has not been identified so far. Pancreatic carcinomas arising in the context of FPC or another hereditary syndrome are usually conventional ductal adenocarcinomas and are indistinguishable from sporadic PDAC. However, a careful examination of the surrounding noncancerous parenchyma may support the suspicion that a specific PDAC arises in the context of a genetic syndrome. It has been shown that familial and hereditary PDAC is associated with the appearance of multiple precursor lesions, including PanINs with low-grade or rarely high-dysplasia, and IPMNs (Fig. 4), which in some cases are so numerous that they can even be appreciated grossly [56,84]. Most of the IPMNs are small side-branch lesions of gastric type [83] and show high-grade dysplasia in up to one-fourth of the cases [85]. These precursor lesions are often associated with so-called lobulocentric atrophy of the pancreatic parenchyma, characterized by lobular fibrosis with loss of acinar tissue and slight inflammation. These findings may provide a basis for screening of patients at risk, because the patchy heterogeneity of the pancreatic parenchyma may be detected by endoscopic ultrasound [84,86,87]. Observations in the pancreas of individuals with a family history of PDAC and findings coming from genetically engineered mouse model (GEMM) studies have spurred the idea that, in addition to the PanIN/IPMN pathway, there may be an alternative pathway to the development of PDAC. PDACs may not only develop from ductal progenitor cells via PanINs or IPMNs, but also from acinar-ductalmetaplasia (ADM) lesions [88e90] that convert into ductal structures referred to as tubular complexes [91]. Tubular complexes show a loss of expression of acinar markers, such as chymotrypsin,

Table 3 Sporadic neuroendocrine neoplasms of the pancreas. Type

Gender (age range)

Localization

Morphology

Pancreatic neuroendocrine tumor (PanNET)

M ¼ F (30e80)

Any

Well-demarcated, solid, usually small (2e3 cm)

Organoid growth patterns (trabecular, acinar, gyriform, insular)

Pancreatic Neuroendocrine Carcinoma (PanNEC)

M > F (>40)

Head

Key facts for diagnosis

IHC: CK 8, 18, 19, Synaptophysin, Chromogranin, ISL-1, various hormones Common genetic alterations: MEN1, VHL, DAXX, ATRX, mTOR signaling pathway

Ill-defined borders, solid, large (>4 cm)

Small and large cell subtypes

IHC: CK 8, 18, 19, Synaptophysin, Chromogranin, p53 Common genetic alterations: TP53, Rb, CDKN2A/p16, bcl-1

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Table 4 Hereditary PDAC syndromes. Syndrome name

Gene(s)

PDAC risk, –fold

Other tumor types

Histology

Familial atypical multiple mole melanoma (FAMMM) Hereditary breast and ovarian cancer

CDKN2A/p16

9 to 47

Melanoma

Ductal (tubular) adenocarcinoma

3.5e10

Breast, ovary, prostate

Ductal (tubular) adenocarcinoma

Ataxia-teleangiectasia Li-Fraumeni

BRCA2, FANC-C, FANC-G, PALB2 ATM TP53

unknown unknown

Ductal (tubular) adenocarcinoma Ductal (tubular) adenocarcinoma

Peutz-Jeghers

STK11/LKB1

132

Familial adenomatous polyposis (FAP)

APC

4

Hereditary nonpolyposis colorectal cancer syndrome (HNPCC)

Mismatch repair genes (MSH2 and MLH1) PRSS1/SPINK1 Unknown

increased

e Sarcomas, lymphomas, carcinomas in numerous organs Gastrointestinal tract, breast, gynecologic, testis and lung Thyroid, gastrointestinal, ampullary Colon, endometrium, ureter, liver

Hereditary pancreatitis Familial pancreatic cancer

50e80 9e32

and express ductal markers such as keratin 19 [92e94]. Atypical flat lesions (AFL) are recently described microscopic (<0.5 cm) mostly non-mucinous tubular structures [88,95] arising in areas of lobulocentric atrophy in association with ADM and often bearing KRAS mutations [96]. AFLs are therefore discussed as another precursor lesion of PDAC and seem to represent the link between ADM and ductal carcinogenesis. Presently, AFLs have been found in human FPC, but their existence in sporadic PDAC remains to be shown. Pancreatic carcinomas arising in patients with HNPCC may have a distinct “medullary” histological appearance and display microsatellite instability, as described above [54,56]. PanNETs can be found in association with five inherited syndromes: multiple endocrine neoplasia type 1 (MEN1), von HippelLindau syndrome (VHL), neurofibromatosis type 1 (NF1), the tuberous sclerosis complex (TSC) and glucagon cell adenomatosis (GCA) [1]. PanNETs are most frequent in patients with MEN1 and GCA, less frequent in VHL, and very rare in NF1 and TSC. The main characteristics of NETs arising in the context of a genetic syndrome are shown in Table 5. NET associated with VHL seems to develop via

Fig. 4. PDAC precursor lesions. Low power view of pancreatic tissue from an individual with a history of familial pancreatic cancer. The picture shows multiple lesions: Duct with PanIN 3 lesion (PanIN 3), IPMN (IPMN) and lobulocentric fibrosis (LF) with tubular complexes.

e e

Ductal (tubular) adenocarcinoma

Ductal (tubular) adenocarcinoma, pancreatoblastoma Medullary carcinoma

Ductal (tubular) adenocarcinoma Ductal (tubular) adenocarcinoma

a hyperplasia-dysplasia sequence, as described for MEN1 [97e99], but this has yet not been shown in NF1 and TSC. Other pancreatic neoplasms occurring in the setting of hereditary syndromes. Pancreatoblastomas have been reported in association with FAP and Beckwith-Wiedemann syndrome. Multiple SCN, sometimes in combination with PanNETs, are associated with von-Hippel-Lindau (VHL) syndrome. These SCNs have a germline mutation of the VHL gene coupled with somatic inactivation of the second allele on 3p. Comparative pathology in animal models of pancreatic neoplasms PanIN ePDAC. First approaches to mimic human PDAC in animal models focused on chemical induction of PDAC. These include tumors induced by N-nitrosobis(2-oxopropyl)amine (BOP) in syrian hamsters [100] and by 9,10-dimethyl-1,2-benzanthracene (DMBA) in rat and mice and exhibit close histological and genetic similarities to their human counterparts (recently reviewed by Murtaugh [101]). The first GEMM that successfully recapitulated the human disease was introduced by Hingorani et al., in 2003 [102]. This model, with a pancreas specific expression of a KrasG12D oncogene from early embryonic development on (KC model), shows the full spectrum of PanIN lesions, ADM, AFL [88] and (late) PDAC. Morphologically, the criteria defined for GEMMs of PDAC by Hruban et al., in 2006 [103] follows the human classification, underscoring the close similarities of the lesions at the morphologic level. On the molecular level, some limitations of GEMM have been overcome in the last years (for example by using inducible models [104]), but some still remain (such as expression of mutant allels within the whole organ/cell type and not only in a single cell). Mouse PanIN (mPanIN), as their human counterparts, are noninvasive lesions graded according to their degree of dysplasia. The resulting invasive ductal adenocarcinomas are composed of welldeveloped glandular and duct-like structures expressing cytokeratin 19 and mucins [103], and are also associated with a fibroinflammatory response. Undifferentiated carcinoma (anaplastic or sarcomatoid) have also been described in some mouse models of pancreatic cancer (see review by Mazur and Siveke in 2012) [105]. Intra- and interindividual heterogeneity of tumor differentiation and growth patterns can be observed in mouse models of PDAC. Based on the KC model, tremendous new insights into PDAC initiation and progression have been achieved to date. The progression

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Table 5 Hereditary pancreatic neuroendocrine tumor syndromes. Syndrome

Gene

Frequency of NEN

Characteristics

Other tumors

MEN1

Menin

80e100%

Von-Hippel Lindau

VHL

10e20%

Microadenomas, insulinoma, gastrinoma (duodenum) Non-functioning, clear cell morphology

Type 1 neurofibromatosis Tuberous sclerosis

NF1

1e10%

Pituitary/parathyroidadenomas, adrenocortical tumors, thymic NEN, stomach NEN Pheochromocytoma, renal cell carcinoma, serous cystadenoma, haemangioblastoma, others Neurofibroma, pheochromocytoma, brain tumors, GIST

TSC2, TSC1

Rare

Somatostatinoma (often duodenal periampullary) Non-functioning and functioning

of PanIN lesions to PDAC is dramatically accelerated in the KC model, when additional mutations in loci encoding tumor suppressors known to be involved in human PDAC (e.g. p16/Cdkn2a, Tp53, and Brca2) are added (see review by Murtaugh 2014 [101]). Currently, the most commonly used mouse model in pancreatic cancer research is the Pdx1-Cre;LSL-KrasG12D;LSL-Tp53R172H/þ model, the “KPC model” displaying tumors with marked molecular heterogeneity and chromosomal instability, also common features in human PDAC [106]. An overview of the most popular GEMM and their relevance for the human disease is provided in Table 6. IPMN. Cystic papillary neoplasms in mouse models have been defined by Hruban et al., in 2006 [103] as cystic structures (>1 mm) with a papillary, noninvasive epithelial proliferation showing varying degrees of cellular atypia. Cystic papillary neoplasms arising in the larger ducts may resemble human IPMNs and can be subclassified similarly to human IPMN subtypes by their mucin expression pattern [107]. As genetic and morphological relationships have been described in human IPMN subtypes [26], subclassification of IPMN-like lesions in GEMM could provide new insights into the genetic background of different epithelial morphologies. IPMN-like lesions have been observed after additional deletion of Smad4 in the KC mouse model [38,108,109]. Cystic papillary lesions in mice with concomitant expression of TGFa and KrasG12D [110] and in a KC- model carrying floxed allels of Brg1 [107] reveal key characteristics of human IPMN of the pancreatobiliary type. Deletion of (Tif)-1g in the KC model led to the development of cystic tumors resembling human IPMN without further subclassification [111]. MCN. Cystic neoplasms with a mucinous epithelium and a dense ovarian-type stroma with wavy nuclei expressing estrogen- and progesterone receptors, as defined by Hruban et al. [103], have been described in mice after ablation of Notch2 or Smad4 in KC mice [112,113]. Progression from MCN to PDAC occurred in the Ptf1aCre/ þ ;LSL-KrasG12D;Notch2lox/lox model developed by Mazur et al. [112].

Angiomyolipoma, brain tumors, angiofibromas

Acinar cell neoplasms. Acinar cell carcinoma in mouse models are solid to cystic invasive epithelial neoplasms with acinar differentiation that can be demonstrated by immunostaining for exocrine enzymes such as chymotrypsin and amylase [103]. The relevance of APC mutations for the development of these neoplasms was confirmed by Oghamian et al. [114] in the ApcMin/ þ ;Tp53/ model. Acinar cell carcinoma in transgenic mice were also induced by expression of several different genes under the acinar cell specific elastase promoter [103]. In some of these models also a mixed acinar-ductal phenotype occurred, proving the morphological plasticity of pancreatic acinar cells. Targeting KrasG12D expression to the Mist1 locus leads to the development of acinar cell carcinoma and cystic papillary neoplasms with acinar differentiation [115]. Serous cystic neoplasms. Neoplasms consisting of a single layer of uniform low cuboidal cells, separated by a region of stromal cells, thus closely resembling human serous cystic neoplasms, have been described by Bardeesy et al. using MT-TGFa Ink4a/Arf and MT-TGFa Tp53 models [116], -mutations that seem not be relevant in the human tumors. Lesions resembling serous cystic neoplasms were also observed in a model with Keratin5 (K5) driven Cox2 expression [117]. Vhl inactivation, as one of the most frequent genetic events in human serous cystic neoplasms, has not yet been successfully developed in GEMMs. Solid-pseudopapillary neoplasms. Tumors with necroses and pseudopapillae consisting of small, polygonal cells that were induced in a mouse model with stabilization of b-catenin (Ptf1aCre;ß-catact), demonstrated the previously detected mutations in bcatenin as the proximate cause of human solid pseudopapillary neoplasm [118]. Neuroendocrine neoplasms. GEMMs of PanNETs have been rarely described. The most widely used models are the RIP-Tag model originally described by Hanahan et al. (1985) [119] and the Glucagon-SV40 model of Rindi et al. (1991) [120]. Although

Table 6 Most widely used GEMM of PDAC and their relevance for human disease. Genotype

Mouse pancreatic phenotype

Relevance in human PDAC

Ref.

Pdx1-Cre;KrasG12D Ptf1aþ/Cre; KrasG12D Ptf1aþ/Cre; KrasG12V Pdx1-Cre;KrasG12D; p53R172H/þ Pdx1-Cre;KrasG12D; p53lox/lox

PanIN/late PDAC; G12D appears to be slightly faster transforming Metastatic PDAC Accelerated PDAC development with locally infiltrating phenotype Metastatic PDAC Undifferentiated PDAC IPMN-derived PDAC MCN-derived PDAC Slightly accelerated PDAC development Accelerated PDAC development

Most prevalent KRAS mutations in human PDAC; G12D with tendency towards lower survival

[101,102,127,128]

TP53 alterations are usually associated with aggressive features and chemoresistance; mutated protein exerts gain of function activities; nonsense mutations leading to a p53-“null” phenotype need further investigation

[106,129e133]

Loss of P16 expression is associated with poorer survival Loss of SMAD4 expression is an independent poor prognostic factor in PDAC and a late event in human IPMN and MCN Models for familial PDAC

[108,133] [22,108,109,113,133]

Ela-tTa-Cre;KrasG12V; p53þ/ Pdx1-Cre;KrasG12D; p16/ Pdx1-Cre;KrasG12D; Smad4fl/fl Ptf1aþ/Cre;KrasG12D; Smad4fl/fl Pdx1-Cre;KrasG12D; Brca2þ/ Pdx1-Cre;KrasG12D; p53R270H/þ; Brca2þ/

[134]

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References

Fig. 5. Development of pancreatic cancer from progenitor cells and precursor lesions. Both established pathways and those still under investigation are shown.

artificially created, these models closely recapitulate the human precursor e neoplasia sequence with progression from hyper- and dysplastic islets to neuroendocrine tumors. Other models of PanNET mostly involve ablation of Men1 resulting in the development of insulinomas [121]. Models based on other genes recently identified as being mutated in a significant number of pancreatic neuroendocrine tumors, as DAXX/ATRX and mTOR pathway genes [76], have not yet been developed. Familial/hereditary neoplasms. GEMMs are the ideal tool to confirm the relevance of human germline mutations for development of pancreatic neoplasms. As described above, hetero- or homozygous mutations in Cdkn2a, Brca2 or Tp53 dramatically accelerate PDAC development in mice, validating them as familial susceptibility genes. Mutations in the Prss1 gene in mice are related to an increased susceptibility to pancreatic injury [122] as a risk factor of PDAC development. Morton et al. [123] described a model with an additional Lkb1 floxed allele in a KC model resulting in increased numbers of PanIN and development of PDAC tumors with reduced latency. Recently, GEM models developed by Flandez et al. [124] and von Figura et al [125] could confirm the potential role of Nr5a2 as a risk allele for PDAC. Regarding PanNENs, mice with a heterozygous Men1 display several features of the MEN1 syndrome including the development of insulinomas and parathyroid adenomas [126], and, as described above, pancreas-specific Men1 ablation also results in the development of insulinomas [121]. To date, no GEMMs are available with inactivation of Vhl successfully modeling pancreatic pathology in VHL syndrome. Conclusions Recent progress in defining the detailed pathological features of pancreatic tumors and their precursor lesions in correlation with underlying molecular changes and experimental models has enormously improved our knowledge about tumor development in the pancreas. These improvements may help in defining biomarkers of early disease, predicting prognosis, detecting targets for medical treatment and acquire new insights into tumorigenesis. Fig. 5 summarizes our conceptual approach on the origin of pancreatic neoplasms and their progression to invasive tumors. Acknowledgments This work was supported by the COST action BM1204 “EU Pancreas: An integrated European platform for pancreas cancer research: from basic science to clinical and public health interventions for a rare disease” and was written on behalf of the members of working group 1 (eupancreas.com).

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