Pathology and Genetics o f P a n c re a t i c N e o p l a s m s Omer H. Yilmaz, MD, PhD, Vikram Deshpande, MD* KEYWORDS Pancreatic cancer Molecular pathology Genetic alteration Genetic mutation Neoplasms Molecular markers
his is a state-of-the-art review of the molecular genetics of pancreatic neoplasms. Although understanding of the molecular features underlying pancreatic neoplasms is still in its infancy, a strong emphasis on the relevance of these findings for the practicing surgical pathologist is provided. The application of molecular techniques has yielded a wealth of information that may soon enhance diagnostics, and will also lead to the development of safer, more effective targeted therapies. The pathologist will play a key role in integrating the current pathologic classification system with newly validated molecular markers.
INTRODUCTION Pancreatic cancer is the fourth leading cause of cancer death in both men and women in the United States.1 There have been significant advances in our understanding of the genetics of pancreatic neoplasms in the past 2 decades, as well as an explosion of information over the past 3 years, largely because of the availability of whole genome and exome sequencing technologies. Over the past decade, the role of the pathologist has changed as well, with the widespread availability of endoscopic ultrasound-guided fine-needle aspiration biopsy, a technique that has allowed for sampling of these lesions with relatively little morbidity and mortality. The increased detection of pancreatic cysts in asymptomatic patients has also led to an increase in the number of pancreatic biopsies and surgical interventions. The morbidity and mortality associated with pancreatic resections has fallen in the past 2 decades, with a consequent increase in the number of pancreatic resections.
Unfortunately, in spite of these initial advances in our understanding of the molecular genetics of these entities, there has been little advance in the field of therapeutics. The 5-year survival rate for patients with localized disease after surgical resection is 20% and for those with metastatic disease, the survival rate is only 2%.1 Only about 20% of pancreatic cancers are detected early enough to be surgically resectable. Although individualized targeted therapies are routinely used for patients with lung and breast tumors, personalized medicine has not achieved the same degree of success in the pancreas. Nonetheless, there are several pathways that could be targeted in pancreatic cancer, and consequently there are numerous clinical trials currently under way. This article provides a state-of-the-art review of the molecular genetics of pancreatic neoplasms with a strong emphasis on the relevance of these findings for the practicing surgical pathologist.
PANCREATIC DUCTAL ADENOCARCINOMA CLINICAL FEATURES Ductal adenocarcinoma of the pancreas is the most common malignant neoplasm of the pancreas. Most of these tumors arise in the head of the gland. The presenting symptoms are variable and include jaundice, weight loss, and diabetes mellitus. Triphasic pancreatic-protocol computed tomography is the best initial diagnostic test for pancreatic cancer, and on this modality these tumors appear as a hypodense solid mass.
PATHOLOGY Ductal adenocarcinomas are firm to hard mass lesions that are often poorly defined grossly. Very
Department of Pathology, Massachusetts General Hospital and Harvard Medical School, 55 Fruit Street, Warren 2, Boston, MA 02478, USA * Corresponding author. E-mail address: [email protected]
Surgical Pathology 5 (2012) 941–959 http://dx.doi.org/10.1016/j.path.2012.08.008 1875-9181/12/$ – see front matter Ó 2012 Elsevier Inc. All rights reserved.
Yilmaz & Deshpande occasionally, they are cystic and thus can mimic primary cystic neoplasms of the pancreas. Histologically, most ductal adenocarcinomas are composed of glandlike structures embedded in an abundant desmoplastic stroma. A small proportion of these tumors show large ducts lined by neoplastic cells, the so-called large-duct variant of adenocarcinoma. A proportion of tumors are poorly differentiated with little or no gland formation. In spite of their aggressive biologic behavior, some ductal adenocarcinomas show bland nuclear features, making the distinction of benign lesion from an invasive carcinoma difficult, particularly on small biopsy specimens (Fig. 1). There are several morphologic features that can aid in this distinction (Table 1). The availability of tools to interrogate the transcriptome and proteome, such as expression profiling, have identified a substantial library of products that could aid in distinguishing a well-differentiated pancreatic carcinoma from a benign pancreatic lesion.2 Many of these proteins can be detected immunohistochemically and are thus available to the practicing pathologist (Table 2).3 Some of these markers have been validated on biopsy specimens as well. A study by Levy and coworkers4 evaluated a panel of immunohistochemical markers (S100P, IMP3, and VHL) and reported that 70% of malignant biliary biopsies were S100P1 and IMP31. In contrast, biopsies that were negative for carcinoma were negative for IMP3, and either negative or only focally positive for S100P.5
KEY MOLECULAR EVENTS IN PANCREATIC DUCTAL ADENOCARCINOMA Recent comprehensive sequencing efforts of pancreatic cancers revealed a wide spectrum of mutations, an average of 63 somatic mutations per tumor, although only 4 were identified frequently: 1 oncogene (KRAS) and 3 tumor suppressor genes (CDKN2A, SMAD4, and TP53).6 Mutations in these 4 genes account for the vast majority of alterations in pancreatic ductal adenocarcinoma. The presence of these mutations/deletions can be exploited for the diagnosis of pancreatic carcinoma (Table 3). Other important genetic mutations that occurred in these neoplasms, albeit at a much lower frequency, involved SMARC4A, EPHA3, CDH1, FBXW7, EGFR, and NF1. A subset of pancreatic ductal adenocarcinoma also showed other genetic alterations: overexpression of ERBB2, deletion/ mutations of MAP2K4 and STK11, TGFBR1, and TGFBR2.6 Germline mutations in BRCA2, PALB2, CDKN2A, STK11, PRSS1 genes, and Lynch syndrome, are associated with a substantially increased risk of pancreatic cancer.7 Germline BRCA2 gene mutations account for the highest proportion of known causes of inherited pancreatic cancer. In addition to the genes mutated in pancreatic adenocarcinoma discussed previously, epigenetic (nongenomic) alterations have also been described in pancreatic carcinoma.
Fig. 1. Cellblock preparations from an EUS-guided fine-needle aspiration biopsy of the pancreas. Although in this instance, the features are diagnostic for adenocarcinoma, with lesser amounts of tissue, ancillary tests can often help in establishing the diagnosis of adenocarcinoma (H&E).
Pathology and Genetics of Pancreatic Neoplasms
Table 1 Morphologic features helpful in distinguishing a benign/inflammatory mass from pancreatic ductal adenocarcinoma Chronic Invasive Ductal Pancreatitis Adenocarcinoma Lobular architecture maintained Haphazard distribution of glands Nuclear variation of greater than 4:1 within a gland Perineural invasion Nuclear membrane irregularitiesa a
Best seen on cytologic preparations.
pancreatitis. This makes SMAD4 a powerful tool for distinguishing malignant from benign pancreatic disease. Sequencing for SMAD4 mutations is currently impractical for most clinical molecular laboratories. Fortunately, there is a strong correlation between SMAD4 mutations and loss of immunoreactivity for SMAD4, and thus immunohistochemistry serves as an efficient surrogate marker for the mutation.8,9 Total loss of protein expression in pancreatic ductal adenocarcinoma results from biallelic inactivation. This usually results from mutation in one copy of the gene, followed by either deletion or epigenetic silencing of the second copy. The resultant loss of nuclear reactivity for SMAD4 makes this a robust marker for pancreatic carcinoma (Fig. 2). The adjacent benign pancreatic parenchyma and lymphocytes serve as an internal control. The major caveat, though, is that the intact nuclear reactivity does not exclude a carcinoma. Loss of SMAD4 has been reported to be a marker of aggressive biologic behavior and widespread metastasis.10,11 If validated, cancers that are negative for SMAD4 expression by immunohistochemistry could be prioritized for systemic rather than local (radiation, surgical) treatment.
P53 and p16
These genes can be mutated in pancreatic adenocarcinoma, but there are no significant “hotspots” for mutations and therefore clinical testing is not practical.
SMAD4 is lost in approximately half of all pancreatic ductal adenocarcinomas. The loss of SMAD4 is a late event in pancreatic carcinogenesis. SMAD4 loss is not seen in reactive and inflammatory processes of the pancreas, such as chronic
Table 2 Partial list of molecules overexpressed in most pancreatic ductal adenocarcinomas and may have diagnostic value in distinguishing pancreatic ductal adenocarcinoma from chronic pancreatitis
1 2 3 4 5 6 8 9 10
Mucin 1 S100A6 Mesothelin Mucin 4 Prostate stem cell antigen HSATII (in situ hybridization) SMAD4 IMP3 S100P
MicroRNA Alterations in microRNA expression seem to contribute to cancer development and progression. Overexpression of several microRNAs in pancreatic cancers, including miR-21, miR-34, miR-155, and miR-200, is thought to contribute to neoplastic progression. The altered expression of these microRNAs could be used to aide pathologists in distinguishing pancreatic adenocarcinoma from chronic pancreatitis, although as ancillary tests they are not quite ready for “prime time.”12,13
PRECURSOR LESIONS OF PANCREATIC DUCTAL ADENOCARCINOMA
52 53,54 52 55 9 4 4
The precursor lesion of pancreatic adenocarcinoma is pancreatic intraepithelial neoplasia (PanIN).14 These microscopic lesions typically measure less than 0.5 cm, are uncommon in the young, and the number of these lesions increase with age. Based on the degree of cytologic and architectural atypia, the lesions are classified as PanIN-1, PanIN-2, and PanIN-3.
Yilmaz & Deshpande
Table 3 List of commonly altered genes in pancreatic cancer
% of Pancreatic Cancers Showing Role in Tumorigenesis Alteration
CDKN2A/ TSG p16
Chromosome Location and Function of Protein
Mechanisms of Activation/ Inactivation
Chromosome 12p Activating Encodes membranemutation bound GTPin codon binding protein 12, 13, or 61
KRAS mutation may support a diagnosis of PDAC or IPMN Unlike colon cancer, the mutation does not predict response to chemotherapy Strong diffuse Chromosome 17p Mutation in reactivity in some Maintains G2-M one allele PDACs arrest, regulates and loss of Occasionally, reactive G1-S checkpoint, other allele ducts may be induces apoptosis reactive for P53 Chromosome 18q Homozygous 1. PDAC: loss of Signal transduction deletion or nuclear reactivity Mutation in one supports PDAC allele and loss 2. In metastatic of other allele setting loss of SMAD4 suggests pancreatic primary 3. Prognostic value: SMAD4 loss is associated with poor prognosis and widespread metastasis None Homozygous Chromosome 9p deletion or P16 inhibits progression of cell Mutation in one allele and cycle at G1-S loss of other checkpoint allele or Promoter methylation
Abbreviations: IPMN, intraductal papillary mucinous neoplasm; PDAC, pancreatic ductal adenocarcinoma.
MOLECULAR EVENTS ASSOCIATED WITH PRENEOPLASTIC LESIONS
and SMAD4 are late events in pancreatic carcinogenesis, being typically identified in only PanIN-3.14
Similar to preneoplastic precursors in other organs, PanIN lesions sequentially acquire selected genetic and epigenetic abnormalities. One of the earliest events identified in PanIN lesions are KRAS mutations. Activating KRAS mutations are identified in 45% of PanIN-1 lesions. Telomere shortening is identified in the vast majority of early lesions as well. Mutations in additional genes, such as p16 are identified in PanIN-2, whereas alterations in TP53
VARIANTS OF PANCREATIC ADENOCARCINOMA Variants of pancreatic adenocarcinoma include adenosquamous carcinoma, colloid carcinoma, hepatoid, signet ring, medullary carcinoma, pleomorphic carcinoma, undifferentiated carcinoma, and undifferentiated carcinoma with osteoclasttype giant cells. Most of these variants show
Pathology and Genetics of Pancreatic Neoplasms Fig. 2. An immunohistochemical stain for SMAD4. Note the tumor lacks nuclear expression of this protein. The background non-neoplastic pancreas shows nuclear expression for SMAD4. The presence of this inbuilt control is critical when evaluating this stain. Unequivocal loss of SMAD4 staining is a reliable marker for pancreatic ductal adenocarcinoma (H&E).
a genetic profile similar to conventional ductal adenocarcinomas.15,16
MEDULLARY CARCINOMA The one genetically distinctive carcinoma of the pancreas is medullary carcinoma.17,18 Histologically, this tumor is similar to medullary carcinomas arising in the colon and breast. Morphologically these tumors are characterized by sheets of neoplastic cells with limited gland formation and abundant intratumoral lymphoplasmacytic infiltration. The tumors show poor differentiation and a syncytial growth pattern. These tumors arise in the setting of Lynch syndrome, although sporadic examples are seen as well. Most medullary carcinomas are microsatellite unstable (MSI1). Medullary carcinomas arising in patients with Lynch syndrome show mutations of one of the DNA mismatch repair genes (most commonly MLH1 and MSH2). Interestingly, unlike ductal adenocarcinomas, these neoplasms are wild type for KRAS gene. These neoplasms may also harbor BRAF, ACVR2, and TGFBR2 mutations. In spite of their poorly differentiated nature, the prognosis for patients with this tumor is better than those with ductal adenocarcinoma. Based on the experience with colonic carcinomas with a similar genetic profile (MSI1 tumors), it has been suggested that these pancreatic tumors will not respond to 5-fluorouracil therapy.
Targeted Therapies Pancreatic cancer responds poorly to most chemotherapeutic agents. Targeted therapies have not achieved the same degree of success in the pancreas as in the lung and melanoma. Several targeted agents for pancreatic cancer have been tested in clinical trials, 2 of which are described here.
PARP INHIBITORS Pancreatic cancer cells with defects in the BRCA2PALB2-Fanconi DNA repair pathway are sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. In phase 1 or 2 clinical trials of patients with a germline BRCA2 gene mutation, response rates of about 40% were recorded with olaparib for recurrent breast and ovarian cancer.19 Clinical trials of PARP inhibitors for patients with pancreatic cancer are currently under way.
HEDGEHOG PATHWAY INHIBITORS A number of other trials, such as the hedgehog pathway inhibitor GDC-0449 (Genentech, San Francisco, CA) are also under investigation in a phase 2 clinical trial, in combination with gemcitabine.20,21 Unlike the “blockbuster” drugs for lung carcinoma and melanoma, there have been no such successes in the therapeutic arena for pancreatic carcinoma.
Yilmaz & Deshpande
PANCREATIC ENDOCRINE NEOPLASMS
Pancreatic endocrine neoplasms (PENs) show extensive neuroendocrine differentiation. The peak incidence is between age 30 and 60 years and both the genders are equally affected. A number of hereditary syndromes are associated with an elevated risk of pancreatic endocrine tumor: Multiple Endocrine Neoplasia type I, von Hippel Lindau, and Tuberous sclerosis. PENs may also be associated with hormonerelated syndromes related to the overproduction of a variety of peptide hormones: insulin, glucagon, gastrin, VIP, somatostatin, and ACTH. Most PENs identified in the past decade are in fact nonfunctional.22
Patients with MEN1 and VHL syndrome develop multiple pancreatic endocrine tumors, and these findings suggest that MEN1 and VHL genes are relevant to the pathogenesis of these neoplasms. Indeed, until recently, menin (the product of the MEN1 gene) was the only common mutation identified in sporadic (nonhereditary) neuroendocrine tumors. It was, however, well recognized that pancreatic endocrine neoplasms lacked mutations that are commonly seen in pancreatic ductal adenocarcinoma. A recently published exome-sequencing effort has dramatically changed our understanding of the molecular underpinnings of these neoplasms. The most common mutations in pancreatic neuroendocrine neoplasms involve genes that regulate the epigenome: DAXX, ATRX, and MEN1 (Table 6).25 In contrast, the primary drivers of ductal carcinoma appear to be alterations in the genes themselves (see Table 3). The epigenome is regulated by chemically modifying DNA or histones, which in turn regulate gene expression. This epigenetic modulation enables gene transcription of certain genes while silencing others that are required for tumor development. The derepression of these latter genes by loss of proteins, such as DAXX and ATRX, may play a critical role in driving tumorigenesis. ATRX and DAXX were mutually exclusive (ie, they were never observed in the same tumor).25 Both DAXX and ATRX proteins play a critical role in the incorporation of a histone H3.3 at the ends of chromosomes (telomeres). The alterations in ATRX and DAXX were generally frameshift and nonsense mutations, resulting in complete loss of functional proteins. The loss of these 2 proteins results in genetic aberrations at the telomere, and this alteration drives tumorigenesis.
PATHOLOGY Most neuroendocrine tumors are well-circumscribed lesions. Approximately 20% of these lesions, however, show cystic change, and rarely this neoplasm may present as a unilocular cyst without a solid component, mimicking a cystic pancreatic neoplasm.23 Histologically, these neoplasms are characterized by a variety of growth patterns, including nesting, trabecular, and glandular architecture (Fig. 3A). Occasionally, the neoplasm may lack the typical neuroendocrine architecture, and instead appear as solid sheets. Cytologically, the tumors have a monotonous appearance and will frequently, but not invariably, show coarse socalled “salt-and-pepper” chromatin. PENs are classified based on their proliferating index (Table 4).24 Although grade 1 tumors are associated with the histologic appearance described previously, grade 3 tumors often show features similar to small-cell or large-cell neuroendocrine carcinoma of the lung. That being said, the histologic grade of a PEN is not based on histologic appearance, but instead on the proliferation index: grade 3 tumors are defined by the presence of greater than 20 mitoses per high-power field or a Ki67 labeling index of greater than 20%.
Differential Diagnosis of Pancreatic Neuroendocrine Neoplasm In most instances, the diagnosis of a neuroendocrine neoplasm is fairly obvious on light microscopic evaluation. In some instances, however, a neuroendocrine neoplasm may be indistinguishable from a solid pseudopapillary neoplasm and acinar cell carcinoma. The immunohistochemical profile of these neoplasms is listed in Table 5.
PRACTICAL APPLICATIONS PREDICTIVE FACTORS A subset (15%) of pancreatic neuroendocrine neoplasms have alterations of the mTOR pathway (see Table 6). Clinical trials using mTOR inhibitors in pancreatic endocrine neoplasm have demonstrated some success in treating these tumors (increase in progression-free survival).26 Individuals with activation of the mTOR pathway are potentially more likely to respond to mTOR inhibitors, such as Everolimus, than tumors that do not. Although this development opens an avenue for personalized therapy for these tumors, this
Pathology and Genetics of Pancreatic Neoplasms
Fig. 3. (A) Pancreatic endocrine tumors. This tumor shows the characteristic nuclear monotony. Note the entrapped ducts (arrow) (H&E). (B) An immunohistochemical stain for ATRX. Note the preservation of nuclear reactivity in the endothelial cells (arrow), while the tumor itself is negative for this protein (Immunohistochemistry ATRX).
paradigm has nonetheless not yet been validated in clinical trials.
marker for the genetic alteration of these genes (see Fig. 3B).25 If validated by other studies, this genetic analysis performed at the “microscope” would help in assessing the risk of malignant behavior in pancreatic endocrine tumors.
DAXX and ATRX Gene Mutation A particularly intriguing aspect of these mutations is that individuals with mutations in either ATRX or DAXX show significantly better survival than patients who lack these mutations. Early results suggest that immunohistochemistry could serve as a surrogate
TRADITIONAL PROGNOSTIC PARAMETERS The assessment of risk of aggressive behavior in pancreatic endocrine tumors is far from perfect. The best predictor of outcome is proliferation, as
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Table 4 Grading of neuroendocrine neoplasm Neuroendocrine neoplasm Grade 1 Neuroendocrine neoplasm Grade 2 Neuroendocrine neoplasm Grade 3
<2 mitoses/10 HPFs AND <3% Ki67 index 2–20 mitoses/10 HPFs OR 3%–20% Ki67 index >20 mitoses/10 HPFs OR >20 Ki67 index
Abbreviation: HPFs, high-powered fields.
measured by mitosis and Ki67 labeling index.27 Therapy is determined largely by the grade of the tumor. Somatostatin analogs are the principal agents used for low-grade neoplasms (grade 1 and most grade 2 tumors). These neoplasms are typically slow growing, and it is not uncommon to see prolonged survival even in the face of widespread metastasis to the liver. High-grade (grade 3) tumors are extremely aggressive neoplasms, and are treated similar to their pulmonary counterparts and do not show a response to Somatostatin analogs. Other more traditional factors that predict survival include the presence of liver or lymph node metastasis, perineural and vascular invasion, and tumor necrosis.27 The molecular signature of these neoplasms alluded to previously may provide a novel means of predicting survival in these patients, however.
OTHER PANCREATIC NEOPLASMS A number of pancreatic neoplasms show mutations in the APC pathway, including solid pseudopapillary neoplasms, acinar cell carcinoma, and pancreatoblastoma. Significantly, all 3 neoplasms are composed of sheets or nests of monotonous cells. None of these tumors have been audited by whole genome sequencing and thus it is likely that other genes critical to tumorigenesis remain to be identified. These 3 neoplasms show significant morphologic overlap and one often has to turn to immunohistochemistry to unequivocally diagnose these tumors (see Table 5).
SOLID PSEUDOPAPILLARY NEOPLASM OF THE PANCREAS Solid pseudopapillary neoplasm is a low-grade malignant tumor that occurs predominantly in young women (average age 28 years). Most of these are identified incidentally on imaging, although larger examples of these neoplasms may be symptomatic. Grossly and on imaging these lesions invariably show both solid and cystic components. More than 95% of patients are cured by surgical resection alone. In the rare instance with local spread or metastasis, prolonged survival is not uncommon.
Table 5 Immunohistochemical profile of pancreatic tumor with monotonous tumor appearance Pancreatic Endocrine Tumor
Acinar Cell Carcinoma
Immunohistochemistry Positive in >90% of cases
Chromogranin* Synaptophysin* Keratin
Trypsin* Chymotrypsin Lipase Keratin
Immunohistochemistry Occasionally positive markers (generally weak and focal) Immunohistochemistry Invariably negative Electron microscopy
Chromogranin focal Synaptophysin focal Nuclear beta-catenin reactivity
Solid Pseudopapillary Neoplasm Nuclear beta-catenin reactivity* Loss of E-cadherin reactivity CD 10 Vimentin Synaptophysin Keratin
Chromogranin* Neuroendocrine granules
* Represents key immunohistochemical finding.
Electron-dense zymogen granules varying from 125–1000 nm and irregular fibrillary granules
Numerous mitochondria and secondary lysosomes
Pathology and Genetics of Pancreatic Neoplasms
Table 6 Common mutations in pancreatic endocrine tumors % with Mutations MEN1 (menin) DAXX ATRX PTEN TSC2 PIK3CA
44.1% 25% 17.6% 7.3% 8.8% 1.4%
HISTOPATHOLOGY These neoplasms have a distinctive microscopic appearance created by a combination of solid and pseudopapillary patterns with intervening areas of hemorrhage and cystic degeneration. The tumor cells have a distinctive morphologic appearance: oval nuclei with fine chromatin and longitudinal nuclear grooves (Fig. 4A). The pseudopapillary structures are lined with multiple layers of these cells. The cells lining these pseudopapillae have nuclei that are located away from the stromal cores. The stromal cores themselves are typically either hyalinized or myxoid, the latter appearance being characteristic of a solid pseudopapillary neoplasm.
Molecular Pathology Almost all solid pseudopapillary neoplasms harbor somatic point mutations in exon 3 of CTNNB1, the gene that encodes beta-catenin.28 This mutation enables beta-catenin to escape cytoplasmic degradation, resulting in the intranuclear localization of this protein (see Fig. 4B). This localization activates the transcription of several oncogenes, among them are MYC and cyclin D1. Beta-catenin protein also interacts with E-cadherin, and abnormal localization of the betacatenin is associated with aberrant E-cadherin expression. Thus, most solid pseudopapillary neoplasms show loss of membrane staining. In contrast, the normal pancreatic ducts, islets, and pancreatic endocrine neoplasms all show a strong membranous pattern of reactivity. Interestingly, the pattern of E-cadherin reactivity depends on the antibody used. An antibody directed against the extracellular domain of E-cadherin shows a complete loss of staining in tumor cells, whereas antibodies against the cytoplasmic domain of E-cadherin label the nuclei of the neoplasm.29
beta-catenin protein serves as a robust marker of beta-catenin mutation (see Fig. 4B). In normal cells and in pancreatic endocrine tumors, betacatenin decorates the cytoplasmic membrane of cells.
ACINAR CELL CARCINOMA CLINICAL FEATURES Acinar cell carcinoma is an uncommon neoplasm, representing 1% of all exocrine pancreatic neoplasms. Although equally rare in children, they nonetheless constitute 15% of pancreatic neoplasms in this age group. Males are more frequently affected than females.
PATHOLOGY Acinar cell carcinoma is a highly cellular neoplasm that is generally unaccompanied by stroma. The diagnosis rests on the identification of certain characteristic architectural patterns, among which the formation of acini by the tumor is diagnostically the most useful (Fig. 5). Other patterns commonly encountered include the glandular pattern, the solid pattern, and the intraductal papillary pattern (this being the least common). Cytologically, the cells are monotonous and show large cherry red nucleoli. The cytoplasm varies from amphophilic to eosinophilic, and may be slightly granular. These tumors are mitotically active and, in contrast to most pancreatic neuroendocrine tumors, mitotic figures are easy to find. Although the architectural and cytologic features of these neoplasms are highly characteristic, nonetheless, in some cases they show overlap with pancreatic endocrine neoplasms and solid pseudopapillary neoplasms. Accurate diagnosis of these neoplasms relies significantly on immunohistochemistry.
MOLECULAR FEATURES There is little information available on the genetics of acinar cell carcinoma. Abnormalities in the Wnt signaling pathway has been shown in 25% of cases, and the alterations include activating mutations directly in beta-catenin or indirectly by loss of its negative regulator, the APC tumor suppressor gene.30 Thus, some acinar cell carcinomas will show nuclear reactivity for beta-catenin.
Diagnostically, this genetic profile is a remarkably useful tool, as the intranuclear localization of the
This is an extremely uncommon neoplasm that occurs in children mostly younger than 10 years.31
Yilmaz & Deshpande
Fig. 4. (A) A solid pseudopapillary neoplasm. Some of the nuclear features, particularly nuclear monotony is similar to pancreatic endocrine neoplasm; however, the nuclei in solid pseudopapillary neoplasm are ovalshaped and display fine nuclear chromatin (H&E). The presence of nuclear reactivity for beta- catenin, a reliable marker of beta-catenin mutations, unequivocally distinguishes this lesion from pancreatic endocrine neoplasm (B) (Immunohistochemistry beta-catenin).
Rare examples have, however, been reported in adults. Pancreatoblastoma has also been reported in infants with Beckwith-Wiedemann syndrome. This is a malignant tumor with more than half of these patients showing metastasis either at presentation or following resection.
PATHOLOGY These lesions are typically large (median sized 11 cm) and solid, although uncommon cystic examples have been described. This highly cellular tumor, arranged in islands separated by stromal bands, is composed of monotonous round to oval cells, some but not all
Pathology and Genetics of Pancreatic Neoplasms Fig. 5. Acinar cell carcinoma. The acinar formation (arrow) is typical of this neoplasm (H&E).
of which show unequivocal evidence of acinar cell differentiation, predominantly in the form of wellformed acinar structures. A characteristic feature of pancreatoblastoma is the presence of nests of squamoid morules, although frank keratinization is uncommon (Fig. 6A). Although evidence of endocrine differentiation is invariably identified with immunohistochemistry, unequivocal morphologic features may not be apparent with a hematoxylin and eosin stain.
MOLECULAR PATHOLOGY Most pancreatoblastomas have loss of heterozygosity of chromosome 11p. Alterations in the Wnt signaling pathway have been reported in pancreatoblastoma.32 These mutations result in nuclear accumulation of beta-catenin.33 Unlike solid pseudopapillary neoplasms, however, the nuclear reactivity for beta-catenin is seen predominantly in the squamoid morules (see Fig. 6B).
CYSTIC NEOPLASMS OF THE PANCREAS There has been a dramatic increase in the prevalence of cystic lesions of the pancreas.34 The widespread use of cross-sectional imaging accounts for virtually all these additional cases identified. The 3 common cystic neoplasms of the pancreas are intraductal papillary mucinous neoplasm (IPMN), serous cystadenoma, and mucinous cystadenoma. Additionally, the pancreas
may harbor a host of other non-neoplastic pancreatic cysts such as retention cysts, and pseudocysts. In one study, incidental pancreatic cysts were present in 2.6% of individuals.35 Most of these cystic lesions are detected on cross-sectional imaging in otherwise asymptomatic (at least from the pancreatic perspective) elderly individuals. This poses a significant dilemma for the treating physician. On one hand, these are elderly individuals with significant comorbid conditions in whom surgery is fraught with significant risks. On the other hand, for cysts that harbor invasive carcinoma, surgical intervention at an early stage might represent the best opportunity for a cure. There is an urgent need for a biomarker that could predict malignancy within a pancreatic cyst, thus sparing the large group of individuals with benign cysts (in most cases a benign IPMN) from a potentially debilitating surgical procedure. This section reviews the genetic events in these pancreatic cysts, and demonstrates the utility of these alterations.
INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS IPMNs are cystic epithelial neoplasms of the pancreas composed of mucin-producing cells lining either the main pancreatic duct or its branches.
Yilmaz & Deshpande
Fig.6. Pancreatoblastoma. (A) This particular neoplasm showed numerous squamoid morules (arrow) (H&E). The squamoid morules showed nuclear reactivity for beta-catenin. The adjacent neoplastic cells lack nuclear reactivity for beta-catenin (B) and instead show membranous staining (immunohistochemistry beta-catenin).
CLINICAL AND DEMOGRAPHIC FEATURES
Although IPMNs are found in a broad age range, the vast majority of these lesions are detected in individuals older than 70 years.36 They occur slightly more frequently in males. A significant majority of cases are asymptomatic, often detected during clinical evaluation for another condition.37 Other presenting features include abdominal pain, chronic pancreatitis, and jaundice.
On imaging, IPMNs may either show dilatation of the main pancreatic duct (main duct type IPMN) or isolated dilatation of the peripheral branches of the pancreatic ductal system (branch duct type IPMN). When both sites are involved, the neoplasm is designated as “combined” main and branch duct type IPMN. Grossly, both categories show dilated ducts and cysts filled with mucin (Fig. 7).
Pathology and Genetics of Pancreatic Neoplasms Fig. 7. Intraductal papillary mucinous neoplasm. The large cystic space represents the main pancreatic duct (asterisk). This markedly dilated main pancreatic duct also shows the papillary mass lesion that measured approximately 5 cm in size (arrow). Histologically, this tumor showed evidence of invasion.
HISTOPATHOLOGY IPMNs are lined by mucin-producing epithelium.38 This lining epithelium is typically arranged in a papillary pattern, although occasionally IPMNs may be lined by flat nonpapillary epithelium. The dilated ducts and cysts are lined by varying grades of dysplastic epithelium: mild, moderate, and severe. IPMNs are classified into 4 types based on the differentiation of the lining cells: gastric foveolar, intestinal, pancreatobiliary, and the oncocytic types.39,40 The gastric foveolar type is lined by epithelium that resembles the lining epithelium of the stomach, ie, tall columnar cells with basally oriented nuclei and an apical “cup” of mucin (Fig. 8A). The intestinal type of IPMN closely resembles colonic type tubulovillous adenoma (see Fig. 8B). The pancreatobiliary type of IPMN is lined by cuboidal cells and invariably shows high-grade atypia. The oncocytic type of IPMN shows significantly less mucin than the other variants of IPMN (see Fig. 8C). The neoplastic cells, instead, show abundant granular eosinophilic cytoplasm and thus resemble Hurtle cells; however, architecturally they resemble other IPMNs in that they show complex intraductal papillary proliferation.
INVASIVE CARCINOMA ARISING IN INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS Approximately 30% of resected IPMNs are associated with invasive carcinoma. Histologically, an
invasive carcinoma arising in an IPMN may either resemble conventional ductal adenocarcinomas (the so-called tubular subtype), or is associated with abundant extracellular mucin, the so-called colloid type carcinoma. The colloid type of invasive carcinoma is associated with a significantly better prognosis than the tubular subtype.41
RISK OF MALIGNANCY Intraductal papillary mucinous neoplasms are preneoplastic lesions that may progress to pancreatic ductal adenocarcinoma, although the magnitude of this risk is currently unknown. Although aggressive surgical resection was the accepted paradigm in the past, more recently a significant majority of lesions are deemed at a low risk for progression to cancer and are thus treated conservatively, albeit with close clinical follow-up. The paradigm today is to resect only lesions with a high risk of invasive carcinoma and watchful waiting for the low-risk lesions.42 The risk of malignancy is assessed by cross-sectional imaging. On imaging, the features that suggest malignancy include a dilated main pancreatic duct (main or combined type IPMNs) and a cyst that measures greater than 3 cm with a mural nodule. However, cross-sectional imaging lacks specificity, ie, a substantial number of benign pancreatic cysts show one or more of these risk factors and are thus resected. It should be noted that the vast majority of small (less than 3 cm)
Yilmaz & Deshpande Fig. 8. Examples of IPMN. (A) Gastric foveolar type IPMN (H&E). (B) Intestinal type IPMN (H&E). (C) Oncocytic type IPMN (H&E).
Pathology and Genetics of Pancreatic Neoplasms asymptomatic branch duct IPMNs have a very low malignant potential.
Chemical Analysis of Cyst Fluid Chemical analysis of cyst fluid helps distinguish IPMN from its mimics. A cyst fluid CEA level of greater than 200 ng/mL is highly suggestive of an IPMN.43
CYTOLOGY IN PANCREATIC CYSTS Cytologic assessment of pancreatic cyst fluid has the potential to both distinguish IPMN from its mimics and to assess the risk of malignancy in an IPMN. Unfortunately, cytologic evaluation is associated with numerous pitfalls, and in our experience suffers from both low sensitivity and specificity. A major shortcoming of cytology is the inability to distinguish mucinous epithelium that is derived from the surface of the gastrointestinal tract from neoplastic mucinous epithelium. The other concern is the relatively low sensitivity for malignancy, the negative predictive value being approximately 60%. An attempt to increase the sensitivity for malignancy at the Massachusetts General Hospital has resulted in numerous false-positive diagnoses.44 Thus, cytology is not a panacea for distinguishing benign from malignant pancreatic cysts.
KEY MOLECULAR EVENTS IN INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS The 2 common mutations identified in IPMNs are KRAS and GNAS. Other genetic alterations that have been identified in IPMNs include PIK3CA mutations.45,46
KRAS Eighty-one percent of IPMNs harbored a KRAS mutation, although other studies have detected such mutations in fewer than 50% of cases.46,47 Virtually all these mutations are located on codon 12. Significantly, KRAS mutations are more frequent in gastric foveolar and pancreatobiliarytype IPMNs and are less frequent in intestinaltype IPMNs.
GNAS The GNAS gene is located in chromosome band 20q13. GNAS encodes the G-protein stimulatory subunit (Gsa), a component of heterotrimeric G-protein complexes. GNAS mutations in IPMNs are confined to a single codon, codon 201. GNAS R201C and GNAS R201H mutations were identified in 39% and 32% of the IPMNs, respectively, and 4% of the IPMNs had both mutations.46 The activating mutations in GNAS results in
overproduction of cAMP, which, in turn, overactivates the PKA pathway. GNAS mutations are relatively more common in the intestinal-type IPMNs.
Practical Applications Pancreatic cyst fluid is aspirated under endoscopic ultrasound (EUS) guidance in an attempt to distinguish an IPMN from other benign and non-neoplastic cysts and to assess the risk of malignancy within these cysts. Pancreatic cyst fluid contains both neoplastic cells and neoplastic cell DNA. Analysis of this DNA has the potential to assess the risk of malignancy in these lesions; however, because the cumulative lifetime risk of pancreatic cancer is less than 1%, any test would need to be highly specific so as to not generate a flood of false-positive results. The identification of 2 high-frequency mutations in IPMN, KRAS, and GNAS, identified in 97% of IPMNs, has the potential to make at least one of these critical distinctions (Fig. 9). Because virtually all mutations in the 2 genes are restricted to a single codon, a relatively simple molecular test could distinguish IPMNs from benign mimics. A ligation assay used to assess KRAS and GNAS mutations identified IPMNs with a sensitivity and specificity of 96% and 100%, respectively.46 Furthermore, the assay involved interrogation of just 2 genes and can be performed on as little as 250 mL of fluid, making this an ideal assay for the diagnostic molecular laboratory. Unfortunately, this analysis cannot distinguish benign IPMNs from malignant IPMNs. KRAS and GNAS mutation were identified in both low-grade and high-grade IPMNs, as well as invasive carcinomas arising from IPMNs. Ongoing sequencing efforts in this area may in the future provide biomarkers that would make this distinction.
MUCINOUS CYSTIC NEOPLASMS OF THE PANCREAS CLINICAL FEATURES Mucinous cystic neoplasms are almost exclusively seen in women, most commonly between the ages of 40 and 50 years. These cysts range from unilocular to multilocular and are virtually always identified within the body or tail of the pancreas. Unlike IPMNs, mucinous cystic neoplasms do not communicate with the pancreatic ductal system.
PATHOLOGY Histologically, mucinous cystic neoplasms are lined by mucinous epithelium with atypia that
Yilmaz & Deshpande Fig. 9. Proposed algorithm for the molecular testing of cystic pancreatic lesions.
ranges from mild to severe.48 Most of these cysts, however, show only mild atypia, whereas only a minority are associated with invasive carcinoma. This paradigm of sequential acquisition of cellular atypia is very similar to IPMNs; however, unlike IPMNs, mucinous cystic neoplasms show ovarian-type stroma adjacent to the lining epithelium. In fact, one should not make a diagnosis of a mucinous cystic neoplasm without the presence of ovarian-type stroma. The ovarian-type stroma is identical to the stroma seen in the ovary and shows a similar immunohistochemical profile. Thus, immunohistochemical stains for estrogen and progesterone receptors may be used as evidence to support the diagnosis of mucinous cystic neoplasm.
Practical Applications Mutations in the KRAS gene have been observed in both noninvasive and invasive mucinous cystic neoplasms, albeit at a significantly lower frequency of 33%.46 Alterations in other genes associated with pancreatic ductal adenocarcinomas, such as CDKN2A, SMAD4, and TP53, have been identified in invasive adenocarcinomas arising in mucinous cystic neoplasms. However, alterations in GNAS have not been identified in MCNs.
The paradigm listed in Fig. 9 may assist in distinguishing an MCN from an IPMN and serous cystadenoma, although definitive subclassification may not be possible in most cases.
SEROUS CYSTADENOMA CLINICAL FEATURES Although a relatively uncommon benign neoplasm, its importance lies in that it is often mistaken for an IPMN. The mean age at which this lesion is detected is 60 years. A slight female predominance has often been noted. On imaging and on gross pathology, these lesions typically show a central stellate scar that is surrounded by a multitude of tiny cysts. The oligocystic variant (dominated by cysts measuring larger than 2 cm) and unilocular variant (dominated by a single cyst) lack this appearance and are thus often mistaken for an IPMN.
GENETIC SUSCEPTIBILITY A significant majority of patients with VHL syndrome develop pancreatic serous cystadenoma. This association has led investigators to establish a link between mutations in the VHL
Pathology and Genetics of Pancreatic Neoplasms Fig. 10. Serous cystadenoma. The cysts are lined by a single layer of cells that show distinct cytoplasmic membranes and clear cytoplasm (H&E).
gene and serous cystadenoma. Whereas VHL mutations are ubiquitous in serous cystadenomas arising in VHL syndrome, however, such mutations have been reported in only 22% of sporadic cases of serous cystadenoma.49
KRAS2 mutations. Thus, a biopsy from a serous cystadenoma that inadvertently samples a PanIN lesion would also be positive for KRAS mutation. The aging pancreas is thus virtually a “soup of KRAS mutations.”
Regardless of their gross appearance, these cystic lesions showed a remarkably similar histopathological appearance. The cysts are lined by a single layer of cuboidal epithelium with clear cytoplasm. The nuclei are round and show fine nuclear chromatin and lack nucleoli (Fig. 10). This characteristic cytoplasmic feature owes its presence to abundant intracytoplasmic glycogen, a feature that can be demonstrated using a periodic acidSchiff stain with diastase digestion. Occasionally, papillary projections composed of similar cells are also seen.
The most common genetic alterations in pancreatic ductal adenocarcinoma are KRAS, CDKN2A, SMAD4, and TP53. When confronted with a difficult pancreatic biopsy, the alteration in the corresponding genes, such as SMAD4, may assist the surgical pathologist in confirming the diagnosis of malignancy. The incidence of pancreatic cystic lesions has increased dramatically in the past decade and molecular testing on cyst fluid, specifically KRAS and GNAS, provide tools to distinguish the most common of these cysts, IPMN, from its non-neoplastic mimics. A recent study using exome sequencing identified mutations in RNF43 as a frequent mutation in both intraductal papillary mucinous neoplasms and mucinous cystic neoplasms.56
Practical Application Serous cystadenomas lack mutations seen in pancreatic ductal adenocarcinomas and IPMN. The presence of a KRAS mutation in a fineneedle aspiration/biopsy of the pancreas would virtually exclude the diagnosis of a serous cystadenoma; however, this statement must be accompanied by an important caveat, a qualification that applies to virtually all molecular tests performed on the pancreas that interrogate KRAS. PanIN lesions are ubiquitous in the elderly and invariably harbor
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