Molecular Genetics of Pancreatobiliary Neoplasms

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Chapter 34

Molecular Genetics of Pancreatobiliary Neoplasms

Laura D. Wood

Ralph H. Hruban

Introduction

Decades of research have established that cancer is a genetic disease caused by accumulation of somatic mutations in oncogenes and tumor suppressor genes. Some patients inherit gene mutations that predispose them to cancer, whereas others acquire all alterations somatically. However, in both inherited and sporadic cancers, tumors acquire numerous somatic alterations throughout their development—some are driver alterations that play crucial roles in tumorigenesis, whereas others are passenger alterations with no functional consequences. Knowledge of somatic mutations has greatly advanced understanding of basic tumor biology and has revolutionized the practice of oncology, with mutation-specific diagnostic and treatment strategies now available for multiple tumor types.

Pancreatobiliary neoplasms are among the best genetically characterized tumors. Studies of their molecular features have shown that morphologically defined entities also represent molecularly distinct neoplasms: genetics mirrors morphology. With this newly acquired understanding of the molecular biology of pancreatobiliary neoplasms, the field is primed to move into an era of genomic medicine. In the coming decades, knowledge of the molecular underpinnings of a tumor will be crucial for individual patient care, and pathologists will continue to play a pivotal role in the translation of genetic findings into clinical tests. Therefore, a sound understanding of the molecular genetics of pancreatobiliary neoplasms will be required to care effectively for patients in the era of genomic medicine.

Pancreatic Neoplasms

Ductal Adenocarcinoma

Germline Alterations

Approximately 10% of pancreatic ductal adenocarcinomas (PDAs) have a familial basis, and several genetic syndromes lead to an increased risk of PDA (Table 34.1). However, the genetic basis for most cases of familial PDA remains unknown.1 Still, the influence of inherited genetics is clear: a family history of PDA significantly increases an individual’s risk for the disease.2

Germline mutations in genes in the Fanconi anemia pathway, which encode proteins involved in repair of DNA cross-linking damage, have been strongly associated with familial PDA.1 Germline mutations in BRCA2, a crucial component of this pathway, result in increased risk of breast, ovarian, pancreatic, and other cancers.3,4 Importantly, some patients with BRCA2-related familial PDA have no personal or family history of breast or ovarian cancer, and the low penetrance of some germline BRCA2 mutations necessitates a high index of suspicion for this genetic alteration in patients with familial PDA.4,5 In addition to germline BRCA2 mutations, germline alterations in other genes in the Fanconi pathway also play important roles in familial pancreatic cancer. The gene PALB2 (also known as FANCN) encodes a protein that interacts with the BRCA2 protein. Germline mutations in PALB2 account for a subset (~3%) of patients with familial PDA.6 Germline mutations in other Fanconi pathway genes, such as FANCC and FANCG, have been reported in young patients with PDA, but their specific role in familial PDA remains to be firmly established.710 In addition, although the importance of FANCA in familial PDA has been investigated, germline mutations in this gene do not significantly contribute to familial PDA.11 Finally, germline mutations in BRCA1 may slightly increase the risk of PDA in breast cancer kindreds, but mutations in this gene only minimally contribute to familial PDA in the absence of breast or ovarian cancer.12,13

In addition to syndromes associated with germline mutations in Fanconi pathway genes, increased risk of PDA is also a key feature in several other inherited syndromes (see Table 34.1). Germline mutations in p16/CDKN2A cause familial atypical mole melanoma syndrome (FAMMM); patients with this syndrome have an increased risk of melanoma (with multiple nevi and atypical nevi) and PDA.1418 Germline mutations in STK11/LKB1 are associated with Peutz-Jeghers syndrome (PJS), a syndrome associated with gastrointestinal hamartomas and pigmented macules on the lips and buccal mucosa, as well as cancer predisposition, including an increased risk of PDA.19 PDAs in these patients show somatic loss of the wild-type STK11/LKB1 allele, indicating the importance of biallelic inactivation of the gene for carcinoma development in these patients.20 Patients with hereditary pancreatitis are also at markedly increased risk for PDA.21,22 Hereditary pancreatitis, caused by germline mutations in PRSS1 and SPINK1, is characterized by young age at onset and continuous or relapsing chronic pancreatic inflammatory disease.2325 In contrast to patients with germline mutations in tumor suppressor genes, the increased risk of cancer in these patients appears to be the result of repeated bouts of inflammation and repair, and the increased cancer risk is limited to the pancreas.

In other inherited syndromes, the increased risk of PDA is less well established. Hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome) is caused by germline mutations in hMSH2, hMLH1, hPMS1, hPMS2 or hMSH6 (formerly GTBP), which lead to defects in DNA mismatch repair (and thus microsatellite instability), as well as markedly increased risk of carcinomas of the colon and other sites. There appears to be a slight, but real, increased risk of PDA in patients with HNPCC.1,2628 In addition, microsatellite instability caused by somatic mutations in one of these mismatch repair genes occurs in sporadic PDA and has been associated with a characteristic “medullary” morphology.27

Germline mutations in the ATM gene, which encodes a protein with roles in DNA damage response as well as cell cycle regulation, also rarely occur in familial PDA kindreds.29 Whereas biallelic germline mutations in ATM cause ataxia-telangiectasia, a syndrome characterized by cerebellar ataxia, sensitivity to ionizing radiation, and increased frequency of multiple malignancies, germline heterozygous ATM mutations have been reported in approximately 2% of patients with familial PDA.29 Germline mutations in APC are the cause of familial adenomatous polyposis (FAP), a syndrome with a marked increase in adenomatous colorectal polyps as well as colorectal adenocarcinoma. Although an increased risk of PDA has been reported in patients with FAP, some of this risk probably reflects the increased risk of duodenal carcinomas, which can invade the pancreas and mimic primary PDA.1,30

Somatic Alterations

In sporadic PDA, somatic alterations clearly play a crucial role in tumorigenesis (Table 34.2). Whole exome sequencing of PDA revealed that each carcinoma contains an average of 48 nonsynonymous somatic mutations.31 Aside from known driver genes (discussed later), the individual genes altered in each carcinoma are markedly heterogeneous, but there are 12 core cellular pathways that are altered in the vast majority of carcinomas.31 These pathways, including KRAS signaling, transforming growth factor-β (TGF-β) signaling, DNA damage control, and cell adhesion, represent key cellular processes that are dysregulated in the development of PDA.31

Table 34.2

Somatic Mutation Prevalence of Commonly Altered Genes in Pancreatobiliary Neoplasms

Neoplasm Gene Chromosome Alteration Prevalence Mechanisms of Alteration
PDA KRAS 12 95% Missense mutation
p16/CDKN2A 9 95% Intragenic mutation with LOH, homozygous deletion, promoter methylation
TP53 17 75% Intragenic mutation with LOH
SMAD4/DPC4 18 55% Intragenic mutation with LOH, homozygous deletion
IPMN KRAS 12 80% Missense mutation
RNF43 17 75% Intragenic mutation with LOH
GNAS 20 60% Missense mutation
p16/CDKN2A 9 Only in HGD/invasive carcinoma Intragenic mutation with LOH, homozygous deletion, promoter methylation
TP53 17 Only in HGD/invasive carcinoma Intragenic mutation with LOH
SMAD4/DPC4 18 Only in HGD/invasive carcinoma Intragenic mutation with LOH, homozygous deletion
PIK3CA 3 10% Missense mutation
MCN KRAS 12 80% Missense mutation
RNF43 17 40% Intragenic mutation with LOH
p16/CDKN2A 9 Only in HGD/invasive carcinoma Missense mutation with LOH, homozygous deletion, promoter methylation
TP53 17 Only in HGD/invasive carcinoma Intragenic mutation with LOH
SMAD4/DPC4 18 Only in HGD/invasive carcinoma Intragenic mutation with LOH, homozygous deletion
SCA VHL 3 50% Intragenic mutation with LOH
PanNET MEN1 11 45% Intragenic mutation with LOH
DAXX/ATRX 6/X 45% Intragenic mutation with LOH
mTOR pathway Multiple 15% Multiple
VHL 3 25% Promoter methylation, intragenic mutation
SPN CTNNB1 3 95% Missense mutation
ACC CTNNB1 3 5% Missense mutation
APC 5 15% Intragenic mutation with LOH
PB CTNNB1 3 55% Missense mutation
APC 5 10% Intragenic mutation with LOH
Unknown 11 85% Loss of heterozygosity
CCA KRAS 12 Variable Missense mutation
p16/CDKN2A 9 85% Intragenic mutation with LOH, homozygous deletion, promoter methylation
TP53 17 50% Intragenic mutation with LOH
SMAD4/DPC4 18 50% Intragenic mutation with LOH, homozygous deletion
PIK3CA 3 Variable Missense mutation
IDH1/IDH2 2/15 20% Missense mutation
BAP1 3 20% Intragenic mutation with LOH
ARID1A 1 15% Intragenic mutation with LOH
PBRM1 3 15% Intragenic mutation with LOH
GBC KRAS 12 Variable Missense mutation
BRAF 7 30% Missense mutation
p16/CDKN2A 9 75% Intragenic mutation with LOH, homozygous deletion, promoter methylation
TP53 17 60% Intragenic mutation with LOH
SMAD4/DPC4 18 20% Intragenic mutation with LOH, homozygous deletion
PIK3CA 3 10% Missense mutation
CTNNB1 3 10% Missense mutation
KEAP1 19 30% Intragenic mutation with LOH

image

ACC, acinar cell carcinoma; CCA, cholangiocarcinoma; GBC, gallbladder carcinoma; HGD, high-grade dysplasia; IPMN, intraductal papillary mucinous neoplasm; LOH, loss of heterozygosity; MCN, mucinous cystic neoplasm; mTOR, mammalian target of rapamycin; PanNET, well-differentiated pancreatic neuroendocrine tumor; PB: pancreatoblastoma; PDA, pancreatic ductal adenocarcinoma; SCA, serous cystadenoma; SPN, solid-pseudopapillary neoplasm.

Numerous studies have shown that KRAS is the most frequently altered oncogene in PDA (somatic KRAS mutations are present in >90% of cancers), clearly indicating that this gene is a driver of tumorigenesis in the pancreas.3136 KRAS is a small guanosine triphosphatase (GTPase) that mediates downstream signaling from growth factor receptors. Somatic mutations in KRAS cluster in a few specific hotspots (most commonly in codon 12), confirming the role of KRAS as an oncogene.32,33,37 Mutations have been reported in other oncogenes in the same cell-signaling pathway (including BRAF) in rare KRAS wild-type carcinomas.38,39

Several frequently altered tumor suppressor genes have also been identified in PDA, including p16/CDKN2A, TP53, and SMAD4/DPC4, and these genes also represent drivers in pancreatic tumorigenesis. p16/CDKN2A is the most frequently altered tumor suppressor gene in ductal adenocarcinoma, with loss of p16 protein function identified in more than 90% of carcinomas.31,40,41 Multiple genetic and epigenetic mechanisms underlie the loss of p16 protein expression, including intragenic mutation coupled with loss of the second allele, homozygous deletion of both copies of the gene, and promoter methylation.31,42,43 Loss of p16 results in cell cycle dysregulation, because this protein normally blocks cell cycle progression by preventing inactivation of the retinoblastoma tumor suppressor protein Rb, another important cell cycle regulator.37,44

TP53, which encodes a protein with a pivotal role in the cellular stress response, is another key tumor suppressor gene in PDA. Somatic mutations have been reported in approximately 75% of cases.31,35,37,45,46 These somatic mutations almost always occur through intragenic mutation followed by loss of the wild-type allele.31

Somatic inactivation of SMAD4/DPC4 also occurs frequently in PDA. Homozygous deletion or intragenic mutation followed by loss of the wild-type allele occurs in approximately 55% of carcinomas.31,41,4749 The Smad4 (Dpc4) protein mediates cellular signaling downstream of the TGF-β receptor. Less frequently, somatic mutations occur in other components of the same signaling pathway, including the TGF-β receptors, TGFBR2 and TGFBR1 (ALK-5).37,50

In addition to these known driver genes, somatic mutations are present at a low prevalence in numerous other genes in PDA.31 However, the role of these genes as drivers or passengers is more difficult to establish when only a few mutations are identified. Infrequent mutations in genes known to be altered in other tumor types are likely to play a functional role in pancreatic neoplasia. For example, ARID1A, which encodes a chromatin remodeling protein, is rarely mutated in PDA (with somatic mutations in approximately 8% of carcinomas) but is frequently mutated in ovarian clear cell carcinomas (somatic mutations in >50% of carcinomas). Although the mutations in the pancreas are infrequent, the gene’s importance in ovarian cancer supports a role for mutations in ARID1A in pancreatic neoplasia.51 Similarly, the histone methyltransferase KMT2C (MLL3) is mutated in only a small percentage of PDA, but somatic mutations in this gene have been reported in several other tumor types, supporting a role for this gene in tumorigenesis in multiple organs.31,5254

In addition to small somatic mutations, large chromosomal gains and losses and complex karyotypic abnormalities also occur frequently in ductal adenocarcinoma. Some alterations target known driver genes, whereas many others (such as the frequently altered chromosome 6p) identify regions that may contain specific loci with important roles in pancreatic tumorigenesis. Further characterization and validation of specific target genes is necessary.55 Additionally, PDAs also contain alterations in microRNAs, small, noncoding RNAs that regulate gene expression. Differential expression of multiple microRNAs has been reported in PDA compared with nonneoplastic pancreatic tissue.5658

Although pancreatic intraepithelial neoplasia (PanIN) have been recognized histologically for years, data on the genetic alterations in PanIN lesions firmly established them as noninvasive precursors to PDA in 2001.59 As PanINs acquire increasing cytologic and architectural abnormality with increasing grade, they also sequentially acquire genetic alterations—the same alterations that occur in invasive PDA. Some molecular changes occur early and are present in low-grade PanINs, whereas others are limited to severely dysplastic and invasive lesions. KRAS mutation and loss of p16 expression are early events that are present in low-grade PanINs.6065 When extremely sensitive techniques are used, KRAS mutations are identified in more than 90% of low-grade PanINs, suggesting that KRAS mutations may represent a key initiating step in pancreatic neoplasia.66 In contrast, loss of Smad4 and TP53 mutation are late events, occurring only in high-grade PanIN and invasive PDA.63,67 For both SMAD4/DPC4 and TP53, allelic loss may occur earlier than somatic mutation, with a subset of intermediate-grade PanINs showing loss of one allele.68

Alterations other than those in known driver genes also occur in PanINs. Telomere shortening is one of the most common early events in pancreatic tumorigenesis, with shortening detected in approximately 90% of low-grade PanINs.69 Early telomere shortening may make the cells susceptible to chromosomal fusion and anaphase bridges, which may produce some of the chromosomal abnormalities observed in invasive PDA.

In addition to expanding knowledge of precursors to PDA, detailed study of somatic mutations in primary tumors and metastases has deepened understanding of the process of clonal evolution within tumors, enabling estimation of evolutionary time in tumors.70 These studies suggest a time window of approximately 15 years between tumor initiation and the acquisition of metastatic ability, providing a broad time window for early detection and subsequent clinical intervention.70

Implications for Pathology

Knowledge of the genetic underpinnings of familial PDA has direct clinical implications for pathologists in several ways. First, the pathologist is the first to have the opportunity to recognize subtypes of pancreatic cancer (e.g., “medullary” carcinoma) that are associated with specific familial syndromes. Second, some familial syndromes have specific treatment implications, which highlights the importance of recognizing them clinically at the time of diagnosis. For example, PDAs in patients with germline alterations in the Fanconi anemia pathway (BRCA2, PALB2) are exquisitely sensitive to drugs that target their specific DNA repair defect, such as mitomycin C and inhibitors of the enzyme poly(ADP-ribose) polymerase (PARP).1,71 Carcinomas in patients with HNPCC also possess specific recommendations for treatment: tumors with microsatellite instability are resistant to fluorouracil-based chemotherapy.72 Finally, clinical recognition of familial PDA syndromes lead to increased screening of at-risk patients. Knowledge of these syndromes is crucial in the interpretation of specimens from these patients.73

Somatic alterations in sporadic PDA also affect pathology practice. Immunohistochemistry can be used to demonstrate alterations of protein expression that are indicative of characteristic gene mutations. For example, TP53 mutations result in strong diffuse TP53 nuclear labeling by immunohistochemistry, providing a histologic surrogate for the genetic alteration and a potential technique for identifying neoplastic pancreatic cells (Fig. 34.1).41,45,74 In addition, loss of Smad4 protein expression by immunohistochemistry correlates with genetic alterations in the SMAD4/DPC4 gene (Fig. 34.2).75,76 This technique can be used to distinguish PDA (loss of Smad4) from nonneoplastic pancreatic diseases (retention of Smad4) and may suggest that a metastatic adenocarcinoma is of pancreatobiliary origin.75,76 Importantly, this technique can also be used to evaluate prognosis in ductal adenocarcinomas, because mutations in SMAD4/DPC4 have been associated with a worse prognosis.77

Molecular studies interrogating genetic alterations also demonstrate clinical utility. For example, molecular analyses of KRAS, TP53, and SMAD4/DPC4 can supplement morphologic diagnosis in cytology specimens to increase the sensitivity of fine-needle aspiration.78 In addition, microRNA levels in fine-needle aspiration specimens show diagnostic promise as indicators of ductal adenocarcinoma or mucinous pancreatic cysts. It has been suggested that microRNA profiles can be used to prognosticate in some cases.56,79,80 Finally, with the continuous development of targeted therapies, molecular studies will likely serve a critical role in determining eligibility for therapy.

Variants of Ductal Adenocarcinoma

There are many uncommon morphologic variants of PDA. Although some share molecular features of ductal adenocarcinoma, others contain unique genetic alterations, and these entities have clinical implications.

Compared with PDA, colloid carcinoma (mucinous noncystic carcinoma) has a better prognosis, a lower prevalence of KRAS mutations (approximately 30%) and TP53 mutations (approximately 20%), and a high prevalence of somatic mutations in GNAS, an oncogene that is frequently altered in intraductal papillary mucinous neoplasms (IPMNs) and their associated carcinomas.81 Medullary carcinoma, another variant with a better prognosis than ductal adenocarcinoma, has a high prevalence of microsatellite instability and lacks somatic mutations in KRAS, although oncogenic BRAF mutations have been reported.27,39,82

Undifferentiated carcinoma is an aggressive neoplasm with a poor prognosis; in addition to frequent KRAS mutations, these carcinomas exhibit frequent loss of E-cadherin protein expression, which provides a possible explanation for the carcinoma’s discohesive morphology (Fig. 34.3).8385 Undifferentiated carcinoma with osteoclast-like giant cells, another aggressive carcinoma with poor prognosis, contains two distinct cell populations. Although the neoplastic mononuclear cells contain frequent KRAS mutations and TP53 overexpression, the osteoclast-like giant cells are non-neoplastic and only contain mutant KRAS from phagocytized neoplastic mononuclear cell DNA.8690 Adenosquamous carcinoma has molecular features similar to ductal adenocarcinoma, showing frequent alterations in KRAS, p16/CDKN2A, SMAD4/DPC4, and TP53, but it is an aggressive neoplasm with poor prognosis.91 Finally, hepatoid carcinoma is an exceedingly rare variant, with too few cases to comprehensively determine clinical outcome or characterize molecular alterations.92

Careful integration of molecular findings with histopathology has helped explain some of the morphologic variants of PDA and is forming the basis for a new classification of pancreatic neoplasia, one that integrates molecular genetics together with tumor histopathology.

Intraductal Papillary Mucinous Neoplasm

Germline Alterations

IPMNs are large, noninvasive, mucin-producing precursor lesions that arise in the larger pancreatic ducts. IPMNs occur rarely in inherited cancer predisposition syndromes (see Table 34.1). For example, they have been reported in patients with PJS, caused by germline mutations in the STK11/LKB1 gene on chromosome 19p.93 IPMNs in patients with PJS frequently undergo loss of heterozygosity (LOH) at the STK11/LKB1 gene locus, suggesting a second somatic hit to the remaining wild-type allele. In addition, IPMNs have been reported in patients with HNPCC (Lynch syndrome) and FAP, suggesting that IPMNs may be uncommon extracolonic manifestations of these syndromes.94,95 Germline alterations in BRCA2 have also been reported in patients with IPMN and a family history of PDA.96

Somatic Alterations

Recent whole exome sequencing of noninvasive sporadic IPMNs identified an average of 26 nonsynonymous mutations per IPMN (approximately half as many as in invasive ductal adenocarcinoma).97 Many of the genes mutated in IPMNs are the same ones commonly mutated in PDAs (see Table 34.2). Somatic point mutations in KRAS occur frequently in IPMNs (30% to 70% of cases), with increasing mutation prevalence in neoplasms with higher grades of dysplasia or invasive adenocarcinoma.98104 When extremely sensitive techniques are used, KRAS mutations are identified in 80% of IPMNs.81 Somatic mutations in TP53 have also been reported in IPMNs with high-grade dysplasia or invasive carcinoma, and TP53 overexpression similarly occurs in the most dysplastic areas.98,103105 Smad4 expression is retained in most noninvasive IPMNs but is lost in approximately one third of IPMN-associated invasive carcinomas.106,107 Therefore, the frequency of Smad4 loss is lower in IPMN-associated invasive carcinomas than in invasive ductal adenocarcinomas not arising in association with an IPMN. Loss of p16 protein expression occurs in IPMNs both with and without associated invasive carcinomas, but the frequency of p16 loss is much higher in IPMNs with associated carcinoma (as much as 100% of IPMN-associated invasive carcinomas, compared with 10% of noninvasive IPMNs).107 Promoter hypermethylation of the p16/CDKN2A gene is a frequent mechanism of expression loss; it occurs in more than 50% of all IPMNs, including those with and without associated carcinoma.108

In addition to alterations in genes frequently altered in PDA, IPMNs also contain mutations in genes that are unique among pancreatic neoplasms (see Table 34.2). Somatic mutations in GNAS have been reported in approximately 60% of IPMNs.81,109 The protein encoded by GNAS plays a crucial role in numerous signaling pathways by coupling transmembrane receptors to their downstream signaling proteins.110 The GNAS mutations in IPMNs all occur in a previously described oncogenic hotspot (codon 201), providing strong evidence for their functional importance in IPMNs. Mutations in GNAS are most prevalent in intestinal-type IPMNs but occur in most histologic subtypes, with the possible exception of oncocytic IPMNs.81 In IPMNs with associated invasive carcinoma, GNAS mutations are identified in both noninvasive and invasive components, providing further genetic evidence that IPMNs give rise to invasive carcinoma.81

The gene RNF43 is also frequently altered in IPMNs; approximately 75% of IPMNs contain somatic mutations in RNF43, which encodes an E3 ubiquitin ligase.97 The majority of these alterations are nonsense substitution mutations that lead to the insertion of stop codons and thus to loss of function of the encoded protein. This mutation pattern, along with frequent LOH at the RNF43 locus on chromosome 17q, provides strong evidence that RNF43 is a tumor suppressor gene in IPMNs. Approximately 10% of intestinal-type IPMNs contain somatic mutations in PIK3CA, the catalytic component of a crucial cell signaling pathway known to be an oncogene in several other tumor types, and some somatic mutations in IPMNs occur at previously described oncogenic hotspots in PIK3CA.101,102,104 Approximately 5% of sporadic IPMNs contain mutations in STK11/LKB1 (the locus of PJS), and rare mutations in EGFR (epidermal growth factor receptor) and ERBB2 (also called Her2/Neu) have been reported in IPMNs, although the functional significance of these latter mutations is not yet established.93,103

Genetic mechanisms other than somatic mutation also likely play a role in the development of IPMNs. Large chromosomal alterations have been identified in IPMNs with all grades of dysplasia, but these alterations are more frequent in neoplasms with intermediate- or high-grade dysplasia compared with those with low-grade dysplasia.111,112 Promoter hypermethylation occurs in several genes in IPMNs, and hypermethylation is more prevalent and more extensive in IPMNs with associated invasive adenocarcinoma than in those without adenocarcinoma.108 MicroRNAs may also play a role in IPMN pathogenesis, because significantly higher expression of miR-21, miR-221, and miR-17-3p has been reported in IPMN cyst fluid compared with fluid from nonmucinous cysts.80

Implications for Pathology

These genetic data provide strong evidence for the role of IPMNs as a precursor to PDA, a role strongly supported by clinical data as well. The knowledge of frequent somatic mutations in IPMNs can also be used in the development of diagnostic assays. As in PDAs, neoplastic cells with mutations in TP53 can be identified with immunohistochemical stains. In addition, mutations from neoplastic cells can be detected in aspirated cyst fluid, indicating that molecular analyses of cyst fluid may be useful as a preoperative diagnostic tool in pancreatic cysts.81,113 Importantly, more than 95% of IPMNs contain a somatic mutation in either KRAS or GNAS, suggesting that a molecular assay involving these two genes may be highly sensitive for the identification of IPMNs.81 In addition, the specificity of RNF43 mutations for mucin-producing cystic lesions suggests that assays for these mutations may hold future diagnostic promise.97 Finally, identification of microRNAs shows promise in preoperative diagnosis of pancreatic cysts, because several microRNAs are differentially expressed in mucin-producing versus non–mucin-producing cysts.80

Mucinous Cystic Neoplasm

Germline Alterations

There is no known genetic predisposition or association with particular genetic syndromes for mucinous cystic neoplasm (MCN).

Somatic Alterations

Frequent somatic mutations occur in MCNs. Recent whole exome sequencing of noninvasive MCNs revealed an average of 16 nonsynonymous mutations per MCN, fewer than in IPMN or invasive ductal adenocarcinoma.97 Similar to IPMNs, MCNs contain frequent somatic alterations in genes that are commonly mutated in PDA (see Table 34.2). Somatic mutations in KRAS oncogenic hotspots occur frequently in MCNs. Mutation prevalence correlates with degree of dysplasia (30% KRAS mutation prevalence in MCNs with low-grade dysplasia versus 80% KRAS mutation prevalence in MCNs with high-grade dysplasia or associated invasive carcinoma).100,114116 Somatic mutation of TP53, as well as TP53 protein overexpression, are limited to MCNs with high-grade dysplasia.97,115,117,118 Loss of Smad4 protein expression, indicating somatic mutation in the SMAD4/DPC4 gene, has been reported primarily in invasive carcinomas associated with MCNs. Loss of Smad4 expression occurs in only 14% of MCN-associated carcinomas, a far lower prevalence rate than in invasive carcinomas not associated with MCNs.117,119 The p16/CKDN2A gene is also infrequently altered in MCNs; Somatic mutation has been reported in a neoplasm with high-grade dysplasia, and promoter hypermethylation occurs in approximately 15% of MCNs.116,118 In addition, aneuploidy has been reported in MCNs and is associated with poor prognosis.120

These findings suggest a stepwise genetic progression in MCNs, from low-grade dysplasia to invasive carcinoma, in which KRAS mutation is an early event and loss of Smad4 is a late event. This concept is supported by the finding in a mouse model of pancreatic neoplasia that oncogenic KRAS mutation and haploinsufficiency of SMAD4/DPC4 led to the development of MCNs, associated with the occurrence of additional somatic alterations in TP53, p16/CDKN2A, and SMAD4/DPC4.121

In addition to mutations in genes frequently altered in PDA, MCNs also share alterations in genes unique to mucin-producing cyst-forming neoplasms of the pancreas (see Table 34.2). Similar to IPMNs, MCNs contain frequent alterations in RNF43; approximately 40% of MCNs harbor somatic mutations in RNF43, and these mutations are enriched for nonsense substitutions.97

Molecular studies have also provided insight into the biphasic nature of MCNs. Gene expression studies suggest different expression profiles in the epithelial and stromal components. Activation of the Notch pathway (JAG1 and HES1) occurs in the epithelial component and activation of estrogen metabolism (STAR and ESR) occurs in the stromal component.122 The genetic or epigenetic bases for these differences remain unclear. In exceedingly rare mixed malignant neoplasms with both epithelial and high-grade “sarcomatous” components, LOH studies suggest a monoclonal origin for the two components with subsequent genetic and morphologic divergence.123

Implications for Pathology

Immunohistochemical staining for TP53 and Smad4 facilitates identification of high-grade neoplastic cells in MCNs, although the prevalence rate of alterations in these genes is far lower in MCNs than in PDAs. As in IPMNs, identification of mutations in DNA from cyst fluid (including mutations KRAS and RNF43) represents a promising preoperative diagnostic test to determine the likelihood of a premalignant mucin-producing cyst.

Serous Cystadenoma

Germline Alterations

Serous cystadenomas (SCAs) occur in approximately 90% of patients with von Hippel-Lindau (VHL) syndrome, an autosomal dominant disorder that is characterized by the development of clear cell neoplasms in multiple organs (see Table 34.1).92,124 VHL is caused by germline mutations in the VHL gene on chromosome 3p, which encodes a regulator of the hypoxia-inducible factor 1 (HIF1α) pathway.92,125,126 SCAs that occur in this syndrome are often combined serous-neuroendocrine neoplasms.92

Somatic Alterations

A recent whole exome sequencing study of sporadic SCAs identified approximately 10 nonsynonymous somatic alterations per tumor, approximately half the number of alterations in IPMN and far fewer than in invasive PDA.97 Importantly, SCAs lack somatic alterations in genes frequently mutated in invasive PDA, such as KRAS and TP53 (see Table 34.2). Sporadic SCAs frequently contain somatic mutations of the VHL

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