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

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.^31–36 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.^{31,41,47–49} 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.^31,52–54

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.^56–58

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.^60–65 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).^83–85 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.^86–90 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.

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

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

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