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.^98–104 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.^98,103–105 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

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).^{100,114–116} 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.

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 gene, which has been reported in as many as 50% of SCAs.^97,127,128 LOH at the VHL locus on chromosome 3p also occurs in a large proportion of sporadic SCAs.^116,127 In addition to frequent loss of chromosome 3p, allelic loss of chromosome 10q has been reported in 50% of SCAs, and loss of several other chromosomal regions occurs less frequently.¹²⁸ No target genes for these latter losses have been identified. Aberrant methylation at a few loci has been reported in only a small proportion of SCAs.¹¹⁶

Implications for Pathology

The frequency of SCAs in patients with VHL syndrome requires a high index of suspicion when examining pancreata from these patients. Conversely, the finding of multiple SCAs or a combined serous-neuroendocrine neoplasm should raise the suspicion that the patient has VHL. In addition, VHL mutations have been identified in DNA from pancreatic cyst fluid.⁹⁷ The specificity of these mutations for SCAs suggest that molecular assays to identify VHL mutations in cyst fluid may provide a useful preoperative diagnostic test, separating cysts with a risk of malignant progression (IPMNs and MCNs) from benign cysts (SCAs).

Somatic Alterations

In sporadic PanNETs, frequent somatic mutations occur in multiple genes (see Table 34.2). Sequencing of all protein coding genes in 10 PanNETs revealed an average of 16 nonsynonymous somatic mutations per PanNET, far fewer than in invasive PDA.¹³⁵ Somatic mutations in MEN1 occur in 45% of sporadic PanNETs.^135–140 LOH at the MEN1 locus also frequently occurs in PanNETs. Loss occurs in 30% to 70% of sporadic PanNETs, including some without somatic MEN1 mutations.^137–140 Somatic mutations in MEN1 cause defects in MEN1 protein function, with loss of expression or aberrant localization.¹⁴¹ Additional mechanisms also likely cause MEN1 protein dysfunction, because some neoplasms without MEN1 mutation show aberrant MEN1 protein localization.¹⁴¹

Somatic mutations in the ATRX and DAXX genes are also common in sporadic PanNETs.¹³⁵ These somatic mutations, which occur in approximately 45% of sporadic PanNETs, inactivate the encoded proteins that function in a chromatic remodeling complex.¹³⁵ The proteins encoded by ATRX and DAXX are components of a complex involved in telomere maintenance. Inactivating mutations in PanNETs are associated with the telomerase-independent telomere maintenance mechanism termed “alternative lengthening of telomeres (ALT).”¹⁴² Inactivating mutations in ATRX and DAXX are associated with acquisition of the ALT phenotype, indicating that the telomeres of PanNETs are fundamentally different from the telomeres of PDA, which exhibit telomere shortening and reactivation of telomerase.^69,142,143 Moreover, mutations in ATRX and DAXX (as well as the ALT phenotype) occur late in the development of PanNETs: they occur only in large tumors (>3 cm) and are absent from microadenomas.¹⁴⁴

The cell signaling pathway of mammalian target of rapamycin (mTOR) is also altered in some PanNETs. Mutations in components of this pathway (including PIK3CA, PTEN, and TSC2) occur in approximately 15% of sporadic PanNETs.¹³⁵ In addition to somatic mutations, LOH at the TSC2 locus on chromosome 16p occurs in approximately 30% of sporadic PanNETs.¹⁴⁵

Although rare alterations in some genes have been reported, PanNETs lack frequent alterations in genes commonly mutated in PDA, including KRAS, TP53, SMAD4/DPC4, and p16/CDKN2A.^135,146 Somatic mutations in KRAS and BRAF have not been identified in PanNETs.^135,146,147 Mutations in TP53 are rare in PanNETs, occurring in approximately 3% of neoplasms, a far lower prevalence rate than in PDA.¹³⁵ However, dysregulation of the TP53 signaling pathway may still be important in PanNETs, because copy number alterations of TP53 regulators (e.g., MDM2) have also been reported in PanNETs.¹⁴⁸ No mutations were identified in SMAD4/DPC4 in a recent whole exome sequencing study of PanNETs.^135,149 Neither homozygous deletion nor somatic mutation of p16/CDKN2A has been identified in PanNETs, but promoter hypermethylation of that locus occurs in approximately 50% of gastrinomas.¹⁵⁰

Several large chromosomal gains and losses (including some recurrent alterations) have been reported in PanNETs, but the target genes for most alterations remain to be identified.^{145,151–153} Promoter hypermethylation and deletion of the VHL locus on chromosome 3p occurs in approximately 35% of sporadic PanNETs,¹⁵⁴ but no other target genes for aberrant methylation in PanNETs have been identified.

Implications for Pathology

Knowledge of the genetic syndromes predisposing to PanNETs (including MEN1 and VHL) allows recognition of these neoplasms in syndromic patients. In particular, pathologists need to carefully examine the duodenum in patients with MEN1, because small duodenal neuroendocrine tumors (gastrinomas) in these patients can metastasize to a peripancreatic lymph node and mimic a primary pancreatic neoplasm.¹⁵⁵ In addition, some somatic alterations have prognostic implications—mutations in MEN1, ATRX, and DAXX have been associated with improved prognosis.¹³⁵ Immunohistochemistry can be used to show loss of ATRX or DAXX protein expression in mutant cases (Fig. 34.4). In contrast, the presence of multiple chromosomal abnormalities confers a worse prognosis in sporadic PanNETs regardless of the specific loci altered, and patients with these neoplasms are more likely to be seen with metastatic disease.^151–153

Finally, some somatic mutations in PanNETs may influence therapy. Alterations in the mTOR pathway can have profound clinical significance, because drugs targeting the mTOR pathway have already been developed for clinical use and may be specifically efficacious in patients with somatic alterations in this cellular pathway.¹⁵⁶

Somatic Alterations

Recent whole exome sequencing of SPNs revealed very few somatic alterations—only the β-catenin gene (CTNNB1) was mutated in more than one SPN (see Table 34.2).¹³⁵ These studies revealed an average of only three nonsynonymous somatic mutations per SPN, the lowest number for any solid neoplasm sequenced to date. Several of the SPNs studied contained only one or two somatic mutations, but every SPN contained a CTNNB1 mutation. These studies showed that SPNs lack alterations in genes commonly altered in PDA (e.g., KRAS, TP53, SMAD4/DPC4) or in other pancreatic cysts (e.g., GNAS, VHL, RNF43).¹³⁵

SPNs are characterized by frequent activating somatic mutations of CTNNB1, which occur in approximately 95% of SPNs, leading to the abnormal nuclear accumulation of β-catenin protein in almost 100% of cases.^158–161 Importantly, the other common pancreatic neoplasms (including invasive PDA and PanNET) lack somatic mutations in CTNNB1.¹⁶² The protein product of this gene has diverse functions in normal cells, regulating cell adhesion through its interaction with E-cadherin and serving as an important target of Wnt signaling.^37,163 (β-catenin is normally targeted for degradation by APC and GSK-3β, but when degradation of β-catenin is inhibited by Wnt signaling, the protein translocates to the nucleus, leading to transcription of target genes.) Somatic mutations in CTNNB1 frequently affect key phosphorylation sites, preventing degradation of the β-catenin protein and leading to nuclear translocation of β-catenin and transcriptional activation.^158,159

Somatic mutations in CTNNB1 in SPNs lead to overexpression of cyclin D1, a key cell cycle regulator and important downstream target of β-catenin.^158,159 In addition, expression of the E-cadherin protein is also dramatically altered in SPNs—expression of the extracellular domain of E-cadherin is lost in these neoplasms, whereas the intracellular component of the E-cadherin protein is abnormally expressed in the nucleus of neoplastic cells.^{161,164–166} Based on gene expression analyses, SPNs exhibit activation of both the β-catenin pathway (with overexpression of AXIN2, TBX3, SP5, and NOTUM) and the Notch signaling pathway (with the overexpression of HEY1, HEY2, and NOTCH2).¹⁶⁷

The role of large chromosomal alterations in SPNs remains controversial, with conflicting reports in the literature. Although there are many case reports of translocations and complex chromosomal abnormalities in single patients, no recurrent abnormalities have been identified.^168,169 Similarly, some studies have shown recurrent chromosomal gains and losses in small case series, but others have failed to identify any chromosomal alterations.^160,170,171 Therefore, although the importance of activating somatic mutations in CTNNB1 is well established in SPN, the role of other alterations remains unclear.

Implications for Pathology

The discovery of a frequent genetic alteration in SPNs (i.e., CTNNB1 mutation) has led to the development of a diagnostic test (β-catenin immunohistochemistry) that, when combined with routine histopathology and other immunostains, helps to distinguish this neoplasm from other solid cellular neoplasms of the pancreas (Fig. 34.5). In the appropriate setting, immunohistochemical labeling for β-catenin to identify aberrant nuclear labeling represents a key step in the diagnostic algorithm for SPN. In addition, abnormalities in E-cadherin expression may explain this lesion’s discohesive morphology.

Somatic Alterations

Recurrent somatic alterations of only a few genes have been identified in ACCs (see Table 34.2). Somatic mutations in the APC/β-catenin pathway occur in 20% to 25% of ACCs; both activating mutations in CTNNB1 and inactivating mutations in APC have been reported.¹⁷³ Importantly, ACCs lack frequent alterations in genes commonly mutated in PDA. Although rare mutations of KRAS and TP53, as well as rare loss of Smad4 protein expression, have been reported, most studies have identified no alterations in these genes in ACC.^{146,173–177} A subset of ACCs exhibit microsatellite instability.¹⁷³

Large chromosomal alterations occur frequently in ACCs. Numerous regions of gain and loss have been reported, including several regions that are altered in multiple ACCs.^{55,173,177,178} Allelic loss of chromosome 11p is one of the most frequent alterations, occurring in approximately 50% of ACCs.¹⁷³ Each ACC commonly harbors numerous large chromosomal alterations.^55,176,178 Although specific target genes for these alterations have not been identified, the regions altered in ACC differ from those frequently altered in PDA.¹⁷⁸ In addition, promoter methylation of several tumor suppressor genes has been identified in a small number of ACCs, although the functional consequences of these epigenetic alterations are unknown.¹⁷⁷

Implications for Pathology

Because of frequent alterations in the APC/β-catenin pathway, immunohistochemical labeling for β-catenin shows the characteristic nuclear accumulation in a subset of ACCs.¹⁷³ However, immunohistochemistry for β-catenin must be interpreted in the context of other immunohistochemical, morphologic, and clinical data to avoid confusion with other neoplasms with aberrant β-catenin staining, such as SPN.

Somatic Alterations

Sporadic pancreatoblastomas show frequent allelic loss at chromosome 11p, occurring in 86% of cases in one study (see Table 34.2).^181,182 Loss of 11p has also been reported in other embryonal neoplasms, such as hepatoblastoma¹⁸³ and Wilms tumor,¹⁸⁴ suggesting the possibility of a common genetic pathway in embryonal tumors. In addition, most pancreatoblastomas have somatic alterations in the APC/β-catenin pathway, including activating mutations in CTNNB1 and inactivating mutations in APC.¹⁸¹ These alterations lead to abnormal nuclear accumulation of β-catenin as well as overexpression of cyclin D1, both of which occur most frequently in the squamoid nests of pancreatoblastoma.¹⁸⁵ Pancreatoblastomas lack frequent alterations in genes commonly mutated in PDA: although rare loss of Smad4 expression has been reported, KRAS mutations and alterations of TP53 expression have not been identified in pancreatoblastoma.¹⁸¹ In addition, there have been several case reports of complex karyotypes with several large regions of loss and gain, and trisomy 8 has been reported.^186–189 However, target genes for these large chromosomal alterations have not been identified.

Implications for Pathology

Because of the associated germline predisposition to pancreatoblastoma, this tumor should be suspected in patients with Beckwith-Wiedemann syndrome. In addition, because of genetic alterations in the APC/β-catenin pathway, many pancreatoblastomas show abnormal nuclear accumulation of β-catenin that can be detected by immunohistochemistry.¹⁸¹ Although nuclear β-catenin staining may be useful diagnostically, if other histopathologic features are not considered, it can also create confusion in the differential diagnosis with SPN and other neoplasms with aberrant β-catenin labeling.

Somatic Alterations

Sporadic gallbladder carcinomas contain somatic alterations in many of the driver genes that are altered in PDA (see Table 34.2). The prevalence rate of somatic mutations in KRAS is lower in gallbladder carcinoma than in other pancreatobiliary carcinomas, but the precise rate varies widely among studies (from 0% to 30%).^{203,230–233} Oncogenic mutations in KRAS also occur in carcinomas from patients with pancreatobiliary maljunction, who are at increased risk for development of biliary cancer. Interestingly, carcinomas from these patients have a higher prevalence rate of KRAS mutation, showing mutations in 50% to 60% of carcinomas.¹⁹⁶ Similar oncogenic KRAS mutations also occur in the precursors to gallbladder carcinoma, including 25% of adenomas.^228,234 Some experts argue that the difference in KRAS mutation prevalence rate between adenomas and carcinomas provides evidence for the model of multiple tumorigenic pathways in the gallbladder—a more common pathway in which carcinoma arises from flat dysplasia and a less common pathway in which carcinoma arises from an adenoma (which on its own has low malignant potential). In addition to KRAS mutations, somatic mutations at a previously described oncogenic hotspot in BRAF also occur frequently in gallbladder carcinoma (>30% of carcinomas in one study).²³⁵

Somatic mutations in tumor suppressor genes known to be drivers in other pancreatobiliary carcinomas also occur in gallbladder carcinoma. p16/CDKN2A alterations are common, occurring in 60% to 75% of gallbladder carcinomas, by mechanisms including point mutation, chromosomal loss, and promoter methylation.^199,233 These somatic p16/CKDN2A alterations are associated with a poor prognosis.¹⁹⁹ Loss of expression of RB1 (pRb), a protein that functions in the same cell cycle control pathway as p16, occurs infrequently in gallbladder carcinomas, with loss in only 4% of carcinomas.²³³ Alterations in TP53 are common, with abnormal p53 protein expression present in approximately 60% of carcinomas.^201,233 Moreover, whole exome sequencing studies of gallbladder carcinoma identified TP53 as the most frequently altered gene, with somatic mutations in almost half of the tumors sequenced.¹⁹¹ LOH at the TP53 locus also frequently occurs in gallbladder carcinomas.²³⁶ Both TP53 loss and mutation have been identified in dysplastic gallbladder mucosa as well as histologically normal mucosa adjacent to carcinomas, and different mutations have been reported in carcinomas and adjacent non-neoplastic mucosa, suggesting the possibility of a field effect in this tumor type.²³⁶ TP53 mutations occur both in sporadic carcinomas and in those associated with pancreatobiliary malfunction,¹⁹⁶ and serum antibodies to the TP53 protein have been reported in a subset of patients with gallbladder carcinoma.²³⁷ Loss of Smad4 expression also occurs in gallbladder carcinomas, although less frequently than in other tumor types; fewer than 20% of gallbladder carcinomas show loss of this protein.^201,233

Mutations in genes not frequently altered in other pancreatobiliary neoplasms have also been reported in gallbladder carcinoma. Somatic mutations in CTNNB1 occur in fewer than 10% of gallbladder carcinomas.^{232,238–240} Gallbladder adenomas exhibit a much higher CTNNB1 mutation prevalence rate (approximately 60%), providing further evidence for the model of separate genetic pathways underlying adenomas and carcinomas of the gallbladder.^238–240 Activating somatic mutations in the oncogene PIK3CA occur in approximately 10% of gallbladder carcinomas.^192,203 Rare mutations in the EGFR tyrosine kinase domain have been reported.²⁰⁶ Somatic mutations in KEAP1, a regulator of the cellular response to oxidative stress, have been reported in several gallbladder carcinomas, with a mutation prevalence rate of approximately 30%.²⁴¹

Microsatellite instability has been reported in sporadic gallbladder carcinomas, but reports of its prevalence are conflicting (range, 3% to 35%).²⁴² Microsatellite instability frequently occurs in gallbladder carcinomas in patients with pancreatobiliary maljunction (>70%).¹⁹⁶ Some studies point to regional variation in microsatellite instability: In a comparison between carcinomas from Japan and Hungary, microsatellite instability was much more frequent in the Japanese cases (42% versus 7%).²⁴³

In addition to somatic point mutations, large chromosomal alterations and epigenetic abnormalities frequently occur in gallbladder carcinoma. Large gains and losses have been reported in several genomic regions, but the target genes for most of these alterations remain to be identified.^214,231 A few amplifications with known target genes have been identified: amplifications with overexpression of ERBB2 and of EGFR each occur in approximately 10% of gallbladder carcinomas.²¹⁷ In addition, a small number of gallbladder carcinomas (<5%) exhibit amplification of the MYC oncogene.²⁴⁴ Aberrant methylation has also been reported in multiple genes, including p16/CDKN2A and APC.²⁴⁵ Aberrant methylation seems to be a progressive process throughout gallbladder tumorigenesis, with increasingly aberrant methylation in dysplasia and carcinoma.²⁴⁶

Small cell carcinoma of the gallbladder represents a distinct clinical, histologic, and molecular entity. These neoplasms, which have histologic features similar to those of small cell carcinomas in other organs, have a dismal prognosis. They frequently occur as one component of a mixed neoplasm, often combined with more conventional adenocarcinoma.²⁴⁷ In contrast to conventional gallbladder adenocarcinoma, all small cell carcinomas of the gallbladder assayed showed inactivation of the p16/pRb pathway, with 67% of tumors showing loss of RB1 and the remaining 33% showing loss of p16.²³³ These neoplasms also showed very high prevalence rate of TP53 alterations, with abnormal TP53 protein expression in 83%.²³³ However, KRAS mutation and loss of Smad4 have not been identified in small cell carcinomas of the gallbladder, which indicates significant differences in the molecular features of this tumor type compared with conventional gallbladder adenocarcinoma.

Implications for Pathology

As with other types of pancreatobiliary neoplasms, immunohistochemical assays for TP53, Smad4, and β-catenin have clinical utility in gallbladder carcinoma, reliably identifying tumors with gene mutations. Immunolabeling for β-catenin may help differentiate carcinomas that occur through a separate tumorigenic pathway, because mutations in CTNNB1 are much more common in gallbladder adenomas. However, the precise clinical relevance of this distinction remains to be determined.^238–240 As with cholangiocarcinoma, identification of patients with alterations in ERBB2 or EGFR may help identify those who are likely to benefit from specific targeted therapies. In addition, methylation patterns of some genes have prognostic significance. There is an association of methylated DLC1 with poor prognosis and an association of methylated MGMT with improved prognosis.²⁴⁶ Finally, recognition of small cell carcinoma of the gallbladder as a morphologically and genetically distinct entity will facilitate the identification of a subset of patients with gallbladder carcinoma, which carries a far worse prognosis.