diagnostic immunohistochemistry || immunohistology of the pancreas, biliary tract, and liver

15

541

• Pancreas 541

• ExtrahepaticBiliaryTract(GallbladderandExtrahepaticBileDucts) 559

• Ampulla 563

• Liver 565

• Summary 576

PANCREASThe pancreas is one of the most versatile organs in the types of neoplasia it generates. This is partly because it is almost unique in harboring two functionally entirely distinct components, exocrine and endocrine, in an oth-erwise intimate mixture, and tumors arising from these two components have vastly different pathologic and biologic characteristics. Therefore pancreatic neoplasms are classified on the basis of their cellular lineage (i.e., which component of the organ they recapitulate: acinar, ductal, endocrine, or others); however, cross differentia-tion also does occur. For example, acinar carcinomas often contain numerous endocrine cells or a separate endocrine component. Additionally, the pancreas is one of a few organs, along with liver and kidney, that has an organ-specific “blastic” tumor of its own, pancre-atoblastoma, which is characterized by differentiation along all components of this organ. Thus immunohis-tochemistry (IHC) plays a crucial role in delineating the differentiation of neoplasms that arise in this organ and is an invaluable adjunct in the often-challenging differ-ential diagnosis. It has been an important tool in unrav-eling the mechanisms of tumorigenesis as well.

In this chapter the cellular lineage markers and the application of IHC in the diagnosis and management of specific tumor types are reviewed.

Here, the authors feel obliged to make a caution-ary statement. It is the strong bias of these authors that IHC is an extremely powerful tool, but only if it is used

cautiously and in combination with morphology. It is the authors’ opinion (and experience) that there is no magic IHC marker that makes the diagnosis by itself. Exceptions always occur, and unfortunately, they tend to merge when IHC is necessary the most. This should not come as a surprise. Basic morphology and IHC are two different facets of the same phenotypic process in pathologic conditions. Where one is “unusual” or out-side of the general realm, the other tends to be so as well.1-3

Biology of the Antigens and AntibodiesEPITHELIALMARKERS

As expected, so-called “pan-epithelial” markers such as CAM 5.2, CK8, and CK18 are expressed in the pan-creatic acini and ducts, as well as in the extrahepatic and peri-ampullary ducts4; however, certain subsets show differential expression patterns. Acinar cells gen-erally do not label with AE1/AE3 or CK7 (Fig. 15.1) and CK19, whereas ductal cells are strongly positive for these markers. Both acini and ducts are typically nega-tive for CK20, which is positive in the intestinal mucosa adjacent to ampulla. In general, expression of CKs, even the wide-spectrum CKs (CAM 5.2, AE1/AE3, CK8, and CK18), is typically less, if not absent, in islet cells com-pared with other elements.

GLANDULARANDDUCTALMARKERS

Mucin-RelatedGlycoproteinsandOncoproteins

In the pancreas and ampulla, the glandular/ductal system is characterized and distinguished by mucin production. With the exception of centroacinar cells/intercalated ducts in the pancreas and the serous cystadenomas that presumably recapitulate this earliest component of the ductal system, virtually all glandular elements

ImmunohistologyofthePancreas,BiliaryTract,andLiverOlca Basturk • Alton B. Farris III • N. Volkan Adsay

IMMUNOHISTOLOGY OF THE PANCREAS, BILIARY TRACT, AND LIVER542

and their neoplasms exhibit some degree and type of mucin formation. Among the mucin-related glycopro-teins and oncoproteins that are commonly expressed in the glandular/ductal neoplasms of this region, the most widely are MUCs, CA19-9, carcinoembryonic anti-gen (CEA), B72.3 (TAG-72), and ductal-of-pancreas-2 (DUPAN-2).5-10 Most of these are well described else-where in this book. The ones more pertinent to pancreas are discussed next.

mucins Mucins are high-molecular-weight glyco-proteins, which are produced by various epithelial cells. They are categorized into membrane-associated mucins (MUC1, MUC3 MUC4, MUC12, MUC16, and MUC17), gel-forming mucins (MUC2, MUC5AC, MUC5B, and MUC6), and soluble mucin (MUC7).11,12

MUC1, pan-epithelial membrane mucin or the “mammary-type” mucin, is constitutively expressed in the cell apices of the centroacinar cells, intercalated ducts, intralobular ducts, and focally in the interlobu-lar ducts, but not in the main pancreatic ducts, acini, or islets. It is thought to have an inhibitory role in cell-cell and cell-stroma interactions and in cytotoxic immunity.13 MUC1 also appears to function as a sig-nal transducer, closely interacting with the epidermal growth factor receptor (EGFR) family and participat-ing in the progression of carcinogenesis.13 It is expressed in almost all examples of pancreatobiliary type adeno-carcinomas (invasive ductal adenocarcinomas of the pancreas, cholangiocarcinomas of the bile duct, and a subset of ampullary adenocarcinomas that presumably arise from peri-ampullary ductules). The expression is predominantly confined to the luminal membrane in the duct-forming areas, whereas it is also intracytoplasmic in the poorly differentiated areas. Therefore MUC1 is considered a marker of aggressiveness.14,15

MUC2, also known as “intestinal-type” secretory mucin, “goblet-type” mucin, or gel-forming mucin, is not constitutively expressed in the pancreas or ampullary ductules with the exception of the scattered goblet cells, where it functions as a protective barrier. It is a product

of the MUC2 gene, which is known to have tumor sup-pressor properties, and as such considered to be respon-sible for the more indolent behavior of the tumors.14,15 Carcinomas with prominent intestinal differentiation, namely villous/intestinal type pancreatic intraductal papillary mucinous neoplasms (IPMNs), colloid car-cinomas that often arise in association with IPMNs, and intestinal type adenocarcinomas arising from the ampulla/duodenum, all typically show diffuse expres-sion of MUC2 and CDX2. CDX2 is a transcription fac-tor responsible for intestinal programming and is also an important upstream regulator of MUC2. Although dif-fuse expression of MUC2 is mostly confined to tumors with intestinal differentiation, CDX2 can be expressed to some degree in pancreatobiliary type tumors as well.

Like MUC1, MUC4 is a membrane-associated mucin; however, it is not expressed in normal pancreatic tissue. It is less well studied, but preliminary evidence suggests that, like MUC1, MUC4 may be a marker of ductal ade-nocarcinoma and a sign of aggressiveness.16

“Gastric-type” mucins, especially those marking gas-tric surface-epithelial (foveolar) mucin such as MUC5AC, are fairly ubiquitous in the gastrointestinal tract includ-ing the ampulla, wherever there are gastric-like glands, as well as the tumors arising from these sites, presum-ably due to the close embryologic foregut association with pancreatobiliary tissue. Normal pancreatic ducts, however, do not express this marker.12 The expression of MUC6, gastric pyloric glandular mucin or pyloric-type mucin, is somewhat more restricted. In addition to decorating Brunner’s glands, intercalated ducts of the pancreas, and the pyloric-like glands that occur in the walls of some preinvasive neoplasia such as IPMNs and mucinous cystic neoplasms (MCNs), MUC6 is also expressed extensively in some subsets of intraductal neo-plasia that show oncocytic phenotype (intraductal onco-cytic papillary neoplasms)17 or those with nondescript morphology (“intraductal tubular carcinomas”).18

ACINAR(ENZYMATIC)MARKERS

It has long been known that trypsin, one of the best-characterized serine proteinases, is produced as a zymo-gen (trypsinogen) in the acinar cells of the pancreas. It is secreted into the duodenum, activated into the mature form of trypsin by enterokinase, and functions as an essential food-digestive enzyme. It also catalyzes the cleavage of the other pancreatic proenzymes (e.g., chymotrypsinogen, prophospholipase, procarboxypep-tidase, proelastase) to their active forms.4,19 To date, four trypsin (or trypsinogen) genes—trypsin 1, 2, 3, and 4—have been characterized in humans and three of them—trypsins 1, 2, and 3—have been demonstrated as the zymogens in human pancreatic juice. Immuno-histochemical methods demonstrate trypsin 1 in acinar cells along with pancreatic enzymes such as chymotryp-sin, lipase, amylase, and elastase. Ductal and endocrine cells are negative for these enzymes. More importantly, although studies have shown that trypsins or trypsin-like enzymes are produced by other human cancer cells such as stomach, ovary, lung, and colon, immunohisto-chemical identification of pancreatic enzyme production

FIGURE 15.1 Pancreatic ductal cells are strongly positive for CK7, whereas acinar cells generally do not label with this marker.

543PANCREAS

is helpful in confirming the diagnosis of pancreatic acinar cell carcinoma (ACC).19 Although this requires confirmation, it has been reported that the tumor-derived trypsin is likely to contribute to tumor invasion and metastasis by degrading extracellular matrix pro-teins and by activating the latent forms of matrix metal-loproteinases (MMPs).19

ENDOCRINEMARKERS

The immunohistochemical demonstration of the spe-cific endocrine cell peptides allows the classification of the pancreatic endocrine neoplasms (PENs). How-ever, it is not always possible to demonstrate these in PENs. Therefore it is of diagnostic importance to use broad-spectrum endocrine cell markers for the general identification of the endocrine nature of islet cells and PENs. These protein markers, localized in the secretory granules in the cytosol or in the cellular membrane, are present in most (rarely in all) normal and neoplastic endocrine cells. The markers most commonly used in routine histopathology have been the secretory granule proteins chromogranin and synaptophysin and the cyto-solic enzyme neuron-specific enolase (NSE).20 Of these, chromogranin is the most specific but its sensitivity is about 80% to 90% (Fig. 15.2).

Chromogranins are a family of glycoproteins consist-ing of types A, B, and C. Among these, chromogranin A in particular has attracted great interest. Different chro-mogranin A antibodies are commercially available, and the staining results with these antibodies may vary. Also, the intensity of chromogranin varies with the amount of neurosecretory granules in the cytoplasm, which tend to be more abundant in the perivascular zones in normal islets.20

Synaptophysin, also called protein p38, is a glyco-protein initially found in small vesicle membranes of neurons and of chromaffin cells in the adrenal medulla. It has been routinely used as a broad-spectrum marker in normal and neoplastic neuroendocrine cells including those of the pancreas; however, strong synaptophysin immunoreactivity has been well-documented in tumors without any endocrine differentiation including solid-pseudopapillary neoplasm (SPN) of the pancreas, which is one of the most important differential diagnoses of endocrine neoplasia in this organ.20

NSE is a cytosolic isoenzyme of the glycolytic enzyme enolase, which catalyzes the conversion of 2- phosphoglycerate to phosphoenolpyruvate. It has been considered a marker for neuroendocrine cells and tumors including PENs. Its staining intensity is unrelated to the content of secretory granules and their peptide storage. Therefore NSE can immunostain even degranu-lated tumor cells. It may also be demonstrated in some non-neuroendocrine tumors. Thus the use of NSE as an endocrine marker requires careful evaluation, and it is important not to rely on NSE staining results alone but always to use them in combination with other markers of endocrine differentiation.20

Cell membrane–associated proteins such as neuro-nal cell adhesion molecule (NCAM, CD56) and leu-7 (CD57) have raised interest as neuroendocrine cell

markers, but they lack specificity because they are also expressed in non-neuroendocrine cells and tumors.20

The authors’ own experience and analysis of the lit-erature also indicate a high incidence of “unexpected” or “unexplained” positivity reported with various antibodies in the islets.21 For most of the antibodies, the staining pattern is faint and does not show any preferential staining pattern of the islet hormones, suggesting that it is a cross-reaction with a cytosolic component.21

ADHESIONMOLECULESANDOTHERMARKERS

E-Cadherin

Cadherins are transmembrane glycoproteins that are prime mediators of cell-cell adhesion via calcium-dependent interactions. Different members of the fam-ily are found in different locations (e.g., E-cadherin is found in epithelial tissue, N-cadherin is found in muscle and adult neural tissues, P-cadherin is found in the placenta).22

E-cadherin plays a key role in the maintenance of epithelial integrity and polarity function.23,24 Normal E-cadherin immunoexpression is localized to the cell membrane with a crisp staining pattern. Decrease in membrane staining compared with normal or complete absence of staining (with the antibody raised against the extracellular domain of E-cadherin) and/or nuclear staining (with the antibody recognizing the cytoplasmic domain of E-cadherin), as seen in a myriad of invasive cancers including solid-pseudopapillary neoplasms (SPNs) of the pancreas, is regarded as abnormal.23,25 Therefore E-cadherin staining is of diagnostic use in the immunohistochemical workup of SPNs23 because all cases will show either absence of membrane staining or nuclear positivity depending on the antibody that is employed.24 The exact mechanism by which E-cadherin enters the nucleus is not known, but it is considered that it is closely related to several partner molecules such as β-catenin.23 The cytoplasmic domain of E- cadherin interacts with the catenin molecules that mediate its binding to the actin cytoskeleton.24 Cytoplasmic dotlike

FIGURE 15.2 Immunohistochemistry for chromogranin shows strong positive staining in islets of Langerhans, more prominent in the peripheral cells. Ducts and acini are negative.


staining has also been described with the antibody to full-length E-cadherin.23

β-Catenin

β-catenin plays a key role in the Wnt signaling path-way as a transcriptional activator. Its normal immuno-expression shows distinct membrane decoration of the pancreatic acini and the ducts. Activating mutations in β-catenin gene (CTNNB1) result in (1) dysregulation and redistribution of β-catenin protein leading to char-acteristic strong cytoplasmic and nuclear immunoreac-tivity, which is seen in 100% of SPNs and in 50% to 80% of pancreatoblastomas, usually in the squamoid corpuscles and (2) overexpression of its target gene, cyclin D1, which is variably reported in 74% to 100% of SPNs24 and in most pancreatoblastomas.

Exocrine NeoplasmsDUCTALADENOCARCINOMA

Ductal adenocarcinoma (DA) is the most common tumor of the pancreas (>85% of pancreatic tumors).26 It often forms a solid mass, which can be closely mimicked by pancreatitis, and therefore most of the cases require FNA or core biopsy for diagnosis. Unfortunately, the tumor is usually scirrhous. It has abundant stromal component and low tumor cell yield, which makes the diagnosis of DA one of the most challenging in surgical pathology. Moreover, DA also has an insidious growth pattern. Along with ovarian cancer, it is the most com-mon cause of “intra-abdominal carcinomatosis” and is one of the most common sources of carcinomas of unknown primary. Therefore it is important to know its immunolabeling pattern.3 The immunohistogram in Table 15.1 is a summary for ductal adenocarcinomas.

Ductal adenocarcinomas express CKs and EMA. The keratins expressed consistently are CK7, CK8, CK18, CK19 (as normal ductal cells), CK13,9,27-31 and in a lesser percentage CK4, CK10, CK17, and CK20.9,27-50 Although CK7 is expressed relatively dif-fusely and strongly in the vast majority of the cases (Fig. 15.3A), CK20 expression is less common, detected

in about one third of the cases and is usually focal (Fig 15.3B).31,34,35,37,39,42-44,48,51,52 This pattern of immunolabeling can be diagnostically useful because most acinar and endocrine neoplasms of the pancreas do not express CK7 and most colorectal cancers express CK20 but not CK7.4 Areas of squamous differentiation in DA are also appropriately CK5/6 and p63 positive (Fig. 15.4).45,53,54

Ductal adenocarcinomas express several mucin-related glycoproteins including MUC1 (Fig. 15.5), which is reported to be associated with a poorer prognosis,55-58

TABLE 15.1 ImmunohistogramofPancreaticDuctalAdenocarcinomawithSelectedAntibodies

100%90%80%70%60%50%40%30%20%10%0%

Mes

othe

linEM

A

CAM5.

2CK19

AE1/AE3

CK18CK7

S-100

A4pC

EACK8

MUC1

CA19.9

MUC5A

CB72

.3

PSCA

Loss

of D

PC4

CDX2CK20

MUC2

A

B

FIGURE 15.3 CK7 is expressed diffusely and strongly in the vast majority of ductal adenocarcinoma cases (A); however, CK20 ex-pression is less common and is usually focal (B).

545PANCREAS

MUC3, MUC4, MUC5AC, and in a lesser percentage MUC636,55,56,59-65 but not MUC2. MUC2 is virtually nonexistent in DAs unless there is focal mucinous dif-ferentiation or there are metaplastic goblet cells. The intestinal differentiation marker CDX2 is also positive in only about 30% of DAs and its labeling is typically fainter and variegated compared with colonic adenocar-cinomas.66-69

Oncoproteins that are widely expressed in DAs include CA19-9, CEA, B72.3 (TAG-72), ductal-of-pancreas-2 (DUPAN-2), and CA125.5-10,70-74 The expression of CEA (Fig. 15.6), B72.3, and CA125 may be useful in distinguishing DA or PanIN-3 from reac-tive glands because non-neoplastic glands are often negative or only focally positive for these markers.5-10 However, caution needs to be exercised because over-laps are too common. Moreover, even the lower-grade PanINs (PanIN-1A) can express these markers. Recently another glycoprotein, CEACAM1 (a member of the CEA family), was reported to be positive in DA and not in normal pancreas or chronic pancreatitis, so serum levels of CEACAM1 might serve as a useful indicator for the presence of pancreatic cancer.75

DAs are typically negative for pancreatic enzymes such as trypsin, chymotrypsin, and lipase70,76 unless there is a mixed acinar component, which is uncom-mon. They also fail to label with endocrine markers; however, in 30% of DAs there are scattered, possibly non-neoplastic, endocrine cells in close association with the neoplastic cells, which can be highlighted with immunostains for chromogranin A, synaptophysin, and NSE.23-26,77-79 The latter two markers can occasionally show more diffuse expression, which should not be regarded as evidence of “neuroendocrine differentia-tion” if the tumor is an otherwise conventional adeno-carcinoma.

The desmoplastic stroma associated with DAs expresses a variety of inflammatory and stromal mark-ers. The inflammatory cells are mostly T cells (CD3 positive).80 Scattered B cells (CD20 positive) and mac-rophages (MAC387 and KP1 positive) are also usually present.80 The spindle cells express alpha-smooth muscle actin, smooth muscle myosin heavy chain, and collagen IV, markers of myofibroblastic differentiation.81 They are also reported to be positive for heat shock protein 47 and fibronectin, as well as for proteins associated with tissue remolding such as urokinase-type plasminogen

FIGURE 15.4 Among invasive carcinomas of the pancreas, p63 expression is detected only in areas with squamous differentiation.

FIGURE 15.5 MUC1 is expressed in all pancreatobiliary-type ad-enocarcinomas (invasive ductal adenocarcinoma of the pancreas is shown here). The expression is predominantly confined to the lumi-nal membrane in the duct-forming areas, whereas it is also intracy-toplasmic in the poorly differentiated areas.

FIGURE 15.6 Carcinoembryonic antigen labeling in ductal ad-enocarcinoma, with areas of well-defined tubule formation show-ing more luminal surface labeling. Non-neoplastic glands are often negative or only focally positive for this marker. However, it should be kept in mind that overlaps occur.


activator, the matrix metalloproteinases, and the tissue inhibitors of metalloproteinases.82-85

GenomicApplicationsofImmunohistochemistry

It has been recognized that DA, like other malignant processes, is a genetic disease produced by progressive mutations in cancer-related genes.

tumor suppressor genes Numerous studies have shown that inactivation of TP53 gene occurs in 50% to 80% of cases.86-96 As is well known, however, immunolabel-ing for the p53 protein is not entirely specific for a TP53 gene mutation.4

DPC4 (Deleted in Pancreatic Carcinoma locus 4) or SMAD4 (Mothers Against Decapentaplegic homolog 4) gene is inactivated in approximately 55% of the DAs but practically never in benign conditions.87,97-100 Immu-nohistochemical labeling for the SMAD4/DPC4 gene product has been shown to mirror SMAD4/DPC4 gene status.99 Therefore the immunohistochemical absence of its protein product in the ductal epithelium of a biopsy specimen is strongly suggestive of carcinoma,92,101 as long as the in-built controls are labeling properly. Also, inactivation of DPC4 gene is relatively uncommon in nonpancreatobiliary carcinomas. Thus loss of DPC4 may also serve as a marker of a pancreatic DA in small fine-needle aspirate samples and in metastatic sites.97,102 This is particularly useful in the ovary in distinguish-ing metastatic pancreatic adenocarcinoma from primary ovarian tumors.

P16/CDKN2A is inactivated in approximately 95% of DAs (75% to 80% genetic inactivation plus 15% silencing by hypermethylation)87-90,93,103-105; however, because of the fact that a small minority of benign duc-tal epithelial cells also labels for its protein product (p16), loss of p16 is not as strongly suggestive of carci-noma.91-93,106

oncogenes The oncogene most frequently activated in pancreatic cancer is the KRAS oncogene,107 which is discussed below, but a number of other oncogenes can be activated in DA including HER2/neu (overexpressed in ≈ 70% of the cases)108-111 and AKT2 (amplified in 10% to 20% of cases).112,113

novel tumor markers Gene expression analyses of DA have identified a large number of genes that are differ-entially overexpressed in DA compared with normal pancreatic tissue.114-117 Among them, mesothelin is expressed in close to 100% of DAs,62,118-120 sea urchin fascin in 95%,83,120 a number of S-100 protein subtypes in 93%,121-123 14-3-3 sigma in 90%,115 and prostate stem cell antigen (PSCA) in 60%.124 The concurrent use of KOC and S-100A4 protein has been found in some studies to improve the diagnostic sensitivity of biliary brushings cytology and demonstrates similar specificity as cytology alone in the diagnosis of pancreatobiliary malignancy.125 In addition, secreted or membranous proteins expressed in pancreatic cancer such as meso-thelin or PSCA, which are shed into pancreatic secre-tions or blood, are under scrutiny as potential future markers for primary or recurrent disease.126

BeyondImmunohistochemistry:AnatomicMolecularDiagnosticApplications

DNA ploidy analyses have yielded aneuploid patterns in about half of the tumors, the incidence being higher with the poorly differentiated forms.127-130 Also cytoge-netic analyses of large series have revealed recurrent pat-terns of alterations in specific chromosomes131-133 such as the most frequent whole chromosomal gains being chromosomes 20 and 7 and the most frequent whole chromosomal loss being chromosome 18.131,132

A number of studies have identified that STK11/LKB1 Peutz-Jeghers gene, a tumor suppressor gene, is inactivated in a minority (5%) of DAs.134-139 This is clin-ically important because patients with the Peutz-Jeghers syndrome have a greater than 130-fold increased risk of developing pancreatic cancer.140 STK11/LKB1 Peutz-Jeghers gene is also commonly altered in intraductal papillary mucinous neoplasms (see later).

Among all human cancers, DAs have the highest frequency of KRAS alterations, the oncogene being constitutively activated in approximately 90% of DAs.87-90,107,136-139,141-145 However, it is important to point out that KRAS mutations are seen even in earliest forms of neoplastic transformation (namely PanIN-1A); they are a common incidental finding in pancreas, as well as in patients with chronic pancreatitis lacking invasive carcinoma90,97; thus they are by no means specific for “cancer.” Other oncogenes that are found to be activated in pancreatic cancer include AIB1, BRAF, c-MYC, and c-MYB.112,113,146-149

Microsatellite instability (MSI) is a rare event in DAs and such cancers appear to have a distinct morphology called “medullary150-153 (see later) and improved survival rate relative to those with conventional DAs.150-153 The MLH1 gene is often inactivated in MSI-high pancreatic carcinomas (characterized with loss of expression of hMLH1 at immunohistochemical level) by either muta-tion or hypermethylation.150,152

Ductal Adenocarcinoma

▪ Ductal adenocarcinomas (DAs) are consistently positive for CK7 diffusely and strongly, whereas CK20 is often either very focal or absent.

▪ Several mucin-related glycoproteins (MUC-1, MUC3, MUC4, and MUC5AC) and oncoproteins (CA19-9, CEA, B72.3, DUPAN-2, and CA125) are also typically positive in DAs to varying degrees. None of these is entirely specific for this tumor type.

▪ Scattered, possibly non-neoplastic endocrine cells in DAs can be demonstrated with immunostains for chromo-granin A, synaptophysin, and NSE.

▪ Loss of DPC4 staining in the pancreatic ductal epithe-lium is suggestive of carcinoma provided that this loss is confirmed by the presence of in-built controls. DPC4 may also prove to be a helpful marker for differentiating pan-creatic adenocarcinoma from other carcinomas in small fine-needle aspirate samples and in metastatic sites.

K E Y D I A G N O S T I C P O I N T S

547PANCREAS

OTHERDUCTALCARCINOMAS

UndifferentiatedCarcinoma

In some DAs the hallmarks of ductal differentiation may be lacking. Such cases are classified as “undifferenti-ated carcinoma.”155 In some, epithelial- to-mesenchymal transition can be so complete that the tumor may resem-ble sarcomas (i.e., sarcomatoid carcinoma)155 and only after adjunct studies such as immunohistochemistry can the ductal nature of the tumor be elucidated.155

Immunohistochemically, CKs are expressed in the well-formed epithelial component; however, the sarcomatoid component might be negative or focal/weak positive, hindering the differential diagnosis with sarco-mas.28,156,157 It should be kept in mind that sarcomas

are exceedingly uncommon in the pancreas, and any sarcomatoid neoplasm ought to be regarded as suspect carcinoma rather than sarcoma. The keratin with the most diffuse and strongest staining has been reported to be the monoclonal so-called “pan-cytokeratins” (AE1/AE3 or OSCAR)158; however, these are also the kera-tins that are more prone to be expressed in true sarco-mas. CAM 5.2, CK7, CK8, CK18, and CK19 are also positive in a variegated and less intense pattern.28,156,157 As is typical of most undifferentiated carcinomas, the neoplastic cells diffusely and strongly stain with vimen-tin.28,157,159,160 They may also express MUC1, CA19-9, CEA, and DUPAN-2.156,161 EMA and B72.3 are nega-tive in the majority of cases.157 Immunolabeling for chromogranin, synaptophysin, and NSE is also nega-tive.4,162 In undifferentiated carcinomas with heterolo-gous stromal elements, there may be immunoreactivity consistent with the line of mesenchymal differentiation (e.g., myoglobin or myogenin in striated muscle, S-100 protein in chondroid elements).4

Recently, it has been demonstrated that noncohesive pancreatic cancers including undifferentiated pancreatic carcinomas are characterized by the loss of E-cadherin protein expression, which might explain the poor cohe-sion of many undifferentiated carcinomas.163

UndifferentiatedCarcinomawithOsteoclast-likeGiantCells

Osteoclast-like giant cells are not uncommon in sarco-matoid carcinomas, sarcomatoid mesotheliomas, and even some sarcomatoid melanomas.164 In undifferen-tiated carcinoma with osteoclast-like giant cells of the pancreas, these cells are strikingly abundant, forming a sea of giant cells that may, in some cases, obscure the epithelial component of the tumor.164 Recent studies confirmed that the osteoclast-like giant cells are in fact reactive in nature and that the malignant cells are actu-ally the smaller, atypical mononuclear cells in the back-ground.164

Immunohistochemically, the atypical mononuclear cells in the background, which are the true malignant cells representing the sarcomatoid carcinoma cells, almost always express vimentin, whereas only a minority expresses CKs (CAM 5.2, AE1/AE3 [Fig. 15.7A], or CK7); EMA; and CEA.28,160,161,165-170 In some cases, all of the epithelial markers are negative. The osteo-clast-like giant cells are positive for vimentin, leukocyte common antigen (LCA, CD45), histiocytic markers (CD68, KP1 [Fig. 15.7B]), and alpha-1-antichymo-trypsin, whereas they are nonreactive for CKs, EMA, or CEA.28,160,161,166-171 However, “tumor cannibalism” (i.e., presence of malignant cells in the benign giant cells) is fairly common in this entity and ought to be considered in evaluating these markers.

These neoplasms often have TP53 gene mutations, and immunolabeling for the p53 protein has been shown to label the pleomorphic mononuclear cells but not the osteoclast-like giant cells.140 Additionally, genetic anal-yses have demonstrated that the atypical mononuclear cells harbor KRAS mutations in about 90% of these neoplasms.28,161,167,169,172,173 By contrast, the osteoclast-like giant cells do not harbor KRAS mutations.169,170

Ductal Adenocarcinoma

Benign, noninvasive ducts versus ductal adenocarcinoma (DA):

▪ Strong cytoplasmic expression of MUC1 and CEA, which is often coupled with antigen leakage into stroma, is highly indicative of carcinoma (more common in high-grade areas).

▪ p53 and Ki-67 are significantly more abundant in carci-noma than in normal epithelium, although overlaps are common and should be used cautiously.

▪ Loss of DPC4 (SMAD4) is also a finding strongly in favor of adenocarcinoma, provided that in-built controls are working properly.

▪ Mesothelin, fascin, S-100, and PSCA are significantly more commonly expressed in carcinoma than in be-nign epithelium.62

Nonductal tumors versus DA:

▪ Colon versus DA: DA is consistently positive for CK7 diffusely and strongly. Approximately one third of DAs express CK20 and CDX2, but the expression is usually focal and weak. Colon carcinomas are negative for CK7 and they almost always express CK20 and CDX2. Additionally, although DA is MUC1+/MUC5AC+/MUC2−, colon carcinoma is more commonly MUC1−/MUC5AC−/MUC2+.

▪ Lung versus DA: TTF-1 and surfactant apoprotein A (PE-10) are usually positive in lung adenocarcinomas and are negative in DAs. In contrast, antibodies that can be focally positive in DAs such as CK20, CDX2, and CA125 are negative in lung carcinomas.

▪ Müllerian (gyn tract) versus DA: A panel composed of WT1, MUC5AC, and CK20 is advisable in distinguishing ovarian serous carcinomas from DAs in omental or peri-toneal biopsies, which often proves to be a challenging differential diagnosis. WT1−/MUC5AC+ phenotype would point toward DA, whereas WT1+/MUC5AC− is highly in favor of ovarian primary. If present, CK20 and, less reliably, CDX2 would also be more compatible with a diagnosis of DA.154 In one study, extensive CK17 reactivity has also been found to be supportive of a DA when the differential diagnosis includes ovarian serous and mucinous neoplasms.37

K E Y D I F F E R E N T I A L D I A G N O S I S


MedullaryCarcinoma

As in other organs (such as breast and tubular gas-trointestinal [GI] tract), medullary carcinoma in the pancreas is defined as syncytial growth of poorly differentiated epithelioid tumor cells, often accom-panied by dense lymphoplasmacytic inflammatory infil-trate.150-153 Desmoplastic reaction is minimal.150-153 The tumors may arise sporadically or in patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome.153 Current experience is too lim-ited to determine their biologic behavior and progno-sis; however, in one study, the patients were found to have an improved survival rate relative to those with DAs.150 Immunohistochemically, the epithelioid cells are labeled by antibodies to cytokeratins (CK),150-153 whereas trypsin, chymotrypsin, lipase, chromogranin, and synaptophysin are usually negative. CD3 antibody highlights the presence of numerous intratumoral T lymphocytes.152 Rare examples also contain Epstein-Barr virus RNA.152

Medullary carcinomas of the pancreas, like their colorectal counterparts, often show microsatellite insta-bility, which is usually caused by somatic hypermethyl-ation of the MLH1 promoter in sporadic cases174 and by an inherited mutation in MLH1 or MSH2 HNPCC syndrome.153 Immunolabeling for MLH1 and MSH2 reveals loss of expression of one of these DNA mis-match repair proteins in many cases.

AdenosquamousCarcinoma

In the pancreas, squamous cells can be encountered in injured ductal epithelium as a result of a meta plastic process. The same metaplastic phenomenon also seems to take place focally in some examples of DA. When this finding is prominent (>25% of the tumor is the cut-off the authors use), the tumor is classified as adenosquamous carcinoma, and if it is exclusively squamous, then squamous cell carcinoma.175 Most of the tumors express CKs (CAM 5.2, AE1/AE3, CK5/6, CK7, CK8, CK13, CK18, CK19, and CK20), EMA, CA19-9, CEA, and B72.3.175,176 Typically, CK5/6,

CK13, and p63 (see Fig. 15.4) are limited to the areas of squamous differentiation, whereas CK7, CK20, CA19-9, CEA, and B72.3 often label the glandular elements.175,177,178

The majority of the cases show nuclear p53 staining and loss of DPC4 protein similar to the molecular sig-nature found in DA.179 KRAS mutations are seen in the majority of cases.175,180

ColloidCarcinoma

Colloid carcinomas are characterized with well- delineated pools of stromal extracellular mucin con-taining scanty, floating carcinoma cells in clusters, strips, or as individual cells. By definition, mucin/epithelium ratio is typically high. Colloid carcinomas appear to have a distinctly better clinical course than other invasive carcinomas of ductal origin.181,182 Five-year survival of resected cases is 55% as opposed to 10% in DAs.181,182 The indolent behavior has been attributed to a combination of two factors: (1) There is inverse polarization of cells, which show secretory activity toward the “stroma-facing” surface of the cells, instead of the luminal surface, and (2) the mucin produced is a specific gel-forming mucin (MUC2) that acts as a containing factor, preventing the spread of the cells.181,182

Immunohistochemically, in addition to the conven-tional epithelial and ductal markers such as CKs, CEA, CA19-9, and B72.3, colloid carcinoma is unique among invasive carcinomas of the pancreas by expression of intestinal differentiation markers, MUC2, and CDX2 (Figs. 15.8 and 15.9, respectively).182 Also, in contrast with DAs, they are negative for MUC1. Furthermore, the pattern of accentuated CEA labeling in the stroma-facing surface of the cells (Fig. 15.10) is specific to col-loid carcinomas and is seldom seen in other invasive carcinomas of the pancreas.

As opposed to DAs, the expression of DPC4 is intact in almost all cases183 and only one third harbors KRAS oncogene mutations.182

A B

FIGURE 15.7 In undifferentiated carcinoma with osteoclast-like giant cells of the pancreas, the osteoclast-like giant cells are negative for AE1/AE3 (A) and positive for CD68 (B).

549PANCREAS

PANCREATICINTRAEPITHELIALNEOPLASIA

A spectrum of intraductal proliferative lesions is pre-sumed to be precursors of invasive carcinomas and is referred to as pancreatic intraepithelial neoplasia (PanIN).184 This spectrum is graded on a three-tiered scale as PanIN1A (the earliest step), progressing to 1B and 2, and finally to PanIN3, which is considered car-cinoma in situ (CIS). Lower-grade PanINs are relatively frequent incidental findings.3 As a rule of thumb, it is considered that nearly 50% of adults older than the age of 50 have foci of PanIN1 in their pancreas. However, higher-grade PanINs, particularly PanIN3 (carcinoma in situ), are seldom encountered in isolation without a concomitant invasive adenocarcinoma.2

The immunohistochemical labeling pattern of Pan-INs parallels that of DA. None of the lesions express MUC2,185 but most express MUC1, MUC4, MUC5AC, and MUC6.55,185 In the multistep progression of DA, MUC1 expression within normal intralobular and inter-lobular ducts appears to be decreased in the low-grade

PanINs and subsequently re-expressed in the advanced PanINs, increasing to 85% of PanIN3.185 Unlike the case with MUC1, MUC5AC is expressed relatively uni-formly throughout all grades of PanIN.185 Increasing Ki-67 labeling indices has been shown with increasing grades of “dysplasia” in PanINs.186

Most of the molecular abnormalities identified within DAs have also been detected within PanIN. Among these, p16 inactivation seems to be an “early” event. Its frequency increases with increasing grades of dyspla-sia (in 30% of PanIN1A lesions vs. in 85% of PanIN3 lesions), and it precedes both p53 mutation and DPC4 inactivation.93,185 On the basis of nuclear cyclin D1 expression seen in one third of PanIN2 lesions, cyclin D1 abnormalities would best be classified as an “inter-mediate” event, also preceding p53 mutation and DPC4 inactivation.185 p53 mutation, as assessed by nuclear overexpression of p53 protein (>25% nuclei), is a “late” event in the progression model of DA, occurring only in PanIN3 (57% of the lesions). Similar to the case of the p53 gene, inactivation of the DPC4 gene appears to be a “late” event, seen only in PanIN3 (28% of the lesions).187

Comparative molecular/genetic analysis of microdis-sected normal and neoplastic ducts combined by immu-nohistochemistry has disclosed the upregulation of a cluster of extrapancreatic foregut markers (pepsinogen C, MUC6, KLF4, and TFF1) and various gastric epithe-lial markers (Sox-2, gastrin, HoxA5, and others) in Pan-INs, whereas the intestinal markers (CDX1 and CDX2) are rarely expressed, if at all, in either PanIN lesions

FIGURE 15.8 Strong and often diffuse intracytoplasmic MUC2 labeling is specific for colloid carcinoma.

FIGURE 15.10 In contrast to MUC2, intracytoplasmic CEA label-ing is much weaker in colloid carcinoma; however, there is strong CEA labeling in the stroma-facing surface of the cells.

FIGURE 15.9 Colloid carcinomas also express CDX2 diffusely.


or invasive pancreatic cancer. These data suggest that PanIN development may involve Hedgehog-mediated conversion to a gastric epithelial differentiation pro-gram.188

INTRADUCTALPAPILLARYMUCINOUSNEOPLASM

Intraductal papillary mucinous neoplasms (IPMNs) are characterized by intraductal proliferation of neoplastic mucinous cells, which usually form papillae and lead to cystic dilation of the pancreatic ducts, forming clinically and macroscopically detectable masses.189 Microscopi-cally, papillae with three distinct morphologic patterns can be seen: (1) pattern reminiscent of gastric foveolar epithelium or resembling PanIN1A with scattered gob-let cells (gastric/foveolar), (2) pattern closely resembling colonic villous adenomas (villous/intestinal), (3) pat-tern characterized with more complex papillae lined by cuboidal cells (pancreatobiliary).189 There is also a spec-trum of cytoarchitectural atypia (IPMN with low-grade dysplasia, IPMN with moderate [borderline] dysplasia, and IPMN with high-grade dysplasia)4,162 and approxi-mately 30% of resected IPMNs have an associated invasive carcinoma. Gastric/foveolar and villous/intes-tinal-types IPMNs are usually associated with colloid carcinoma, and pancreatobiliary type is associated with tubular type invasion with all the morphologic features of DA.189 Ki-67 and proliferating cell nuclear antigen (PCNA) labeling demonstrates a progressive increase in cell proliferation from normal duct epithelium, to IPMN with low-grade dysplasia to IPMN with moderate dys-plasia and IPMN with high-grade dysplasia.190,191 Also immunostaining of p53 protein is seen only in IPMNs with moderate and high-grade dysplasia and in DAs.192

MUC expression profile of IPMNs has been instru-mental in delineating the differentiation and lineage of these neoplasms and in recognizing its subsets that are clinically significant55,61,193-195:

1. Gastric/foveolar type papillae appear to be full recapitulation of gastric mucosa, with more pap-illary areas expressing MUC5AC and only small glandular elements at the base labeling with MUC6. They are usually negative or only focally

positive for MUC1 (Fig. 15.11) and CDX2. Scat-tered goblet cells can be highlighted by MUC2 (Fig. 15.11).55,61,189,194,196

2. Villous/intestinal type papillae do, in fact, show molecular characteristics of intestinal differentia-tion as evidenced by diffuse MUC2 (Fig. 15.12) and CDX2 expression but not MUC1 (Fig. 15.12).55,61,189,194,196 Invasive carcinoma associ-ated with villous/intestinal type, which is typi-cally colloid carcinoma, also expresses MUC2 and CDX2 but not MUC1. In addition, villous/intestinal-type papillae are positive for MUC5AC and negative for MUC6.55,61,182,189

3. Pancreatobiliary type papillae typically do not express MUC2 and CDX2 but may instead express MUC1 (a “marker” of aggressive phenotype in the pancreas), as well as MUC5AC, and to a lesser degree, MUC6.17,61 The invasive component also expresses MUC1 but not MUC2.61 In general, IPMNs are reported to show weaker labeling for MUC4 (70%), MUC3 (60%), and MUC5B (35%)194 and are almost always negative for MUC7.4 IHC for IPMN is summarized in Table 15.2’s immunohistogram.

As expected, virtually all IPMNs express CKs. They are positive for CAM 5.2, AE1/AE3, CKs 7, 8, 18, 19, and variably for CK20.191,197,198 Some IPMNs, espe-cially villous/intestinal type, also express CA19-9 and CEA.198-200 In the villous/intestinal type, the degree of CEA expression increases with the degree of dysplasia.201 Twenty percent of IPMNs label for DUPAN-2.197,198,202 Scattered endocrine cells that are positive with chro-mogranin and synaptophysin are seen in most tumors but account for less than 5% of the tumor cells.108,203 Cyclooxygenase-2 is also expressed in 60% to 80% of IPMNs.204

KRAS oncogene mutations have been reported in 30% to 60% of IPMNs.90,192,197,205-216 The frequency of TP5390,190,207,209,211,212,217-223 and p16/CDKN2A tumor suppressor gene mutations varies greatly between reported series.90,209,217,223 However, in con-trast to DAs, the protein product of the SMAD4/DPC4 tumor suppressor gene is retained in virtually all cases

FIGURE 15.11 Intraductal papillary mucinous neoplasm with gas-tric/foveolar type papillae is usually negative for MUC1 (left). MUC2 expression is only focal, marking goblet cells (right).

FIGURE 15.12 Most intestinal-type intraductal papillary mucinous neoplasm papillae are negative for MUC1 (left). MUC2 expression is diffuse and strong (right).

551PANCREAS

including the invasive carcinomas, suggesting a sub-stantial difference in the pathogenesis of these two neo-plasms. 61,90,183,205,209,217 Recently, human cripto-1 protein, which is thought to have a role in tumor pro-gression, was reported to be expressed more abundantly in pancreatobiliary type than in other types.224

INTRADUCTALONCOCYTICPAPILLARYNEOPLASM

Many authors regard intraductal oncocytic papillary neoplasm (IOPN) as a special subtype of IPMN,225 although recent molecular findings suggest that it may be a distinct tumor type226 but very similar to IPMN in several aspects. Distinctive morphologic features of IOPNs include (1) exuberant, complexly branching papillae in a relatively clean background; (2) oncocytic cells caused by an abundance of mitochondria; and (3) intraepithelial lumina, which are round, punched-out spaces within the epithelium. These intraepithelial lumina often contain mucin. Scattered goblet cells may be identified.225

Immunohistochemically, IOPNs usually label for MUC161,194,226 and MUC6,17 whereas MUC2 and MUC5AC are largely restricted to goblet cells.61,194,226 CDX2 is rarely expressed.226 All cases are positive for B72.3, and some show focal, luminal staining for CEA. DPC4 is typically retained.226 CA19-9 and DUPAN-2 are rarely positive.225 IOPNs also label strongly with antibodies against mitochondrial antigens such as 111.3.225,226 Interestingly, there is consistent immunola-beling with hepatocyte paraffin-1 (Hep Par-1) antibod-ies; however, in situ hybridization for albumin, a more specific test for hepatocellular differentiation, is consis-tently negative.4

Most interestingly, IOPNs, typically lack KRAS2 mutations205,226 in stark contrast with any other tumor types in the pancreas characterized by ductal-mucinous differentiation including greater than 90% of invasive ductal carcinomas and almost half of IPMNs.

MUCINOUSCYSTICNEOPLASM

Mucinous cystic neoplasms (MCNs) are seen typi-cally in perimenopausal women (95% of the patients are women; mean age, 50 years) and present as a thick-walled cyst, often multilocular, in the tail of the pancreas. Microscopically the cysts are lined by mucin-producing epithelium. A distinctive ovarian-like stroma is invariably present in the septa of the cysts.227,228 Just as in IPMNs, MCNs also exhibit characteristics of an adenoma-carcinoma sequence (MCNs with low-grade dysplasia, MCNs with moderate [borderline] dysplasia, and MCNs with high-grade dysplasia or carcinoma in situ).4,162,227,228 Invasive carcinoma can be seen in asso-ciation with MCN in less than one fifth of MCNs. Inva-sive carcinoma is almost exclusively of tubular type with all the morphologic features of DA.229,230 On occasion, sarcomatoid neoplasms arise in MCNs. It is debated whether these are sarcomatoid carcinomas originating from the epithelial component or sarcomatous transfor-mation of the ovarian-type stroma.

The epithelial cells express immunoreactivity with CKs (CK7, 8, 18, and 19),42,52,197,231 as well as with EMA, CA19-9, CEA, DUPAN-2, and CA125.5,73,197,199,231-236 MUC1 is typically not expressed in noninvasive lesions of MCNs but is a marker of invasion observed in greater than 90% of cases with invasion, detected in both the in situ and invasive components.233,237 MUC2 and CDX2 positivity can be seen, especially in the interspersed goblet cells; however, in contrast with villous/ intestinal type IPMNs, the papillary dysplastic nodules are typi-cally negative or only focal for these markers.233,237 The lesions invariably express MUC5AC, which is also commonly expressed in DAs.42,233 Although the pap-illary components of MCN are mostly MUC6 nega-tive, the foveolar-like epithelium in nonpapillary areas is typically positive for MUC6.17 Chromogranin- or synaptophysin-positive scattered endocrine cells are fre-quently noted within the epithelium.203,232,235,238,239

The ovarian-like stroma is immunoreactive for vimen-tin, smooth muscle actin, muscle-specific actin, desmin, h-caldesmon, bcl-2, and CD99, usually in a patchy dis-tribution.229,232,240-245 Progesterone receptors are also expressed fairly consistently (Fig. 15.13) and, in subtle cases, may help establish the diagnosis by highlighting the presence of this pathognomonic finding. Estrogen receptors are often negative by the current antibod-ies available; however, it is suspected that this lack of detection is related to the receptor subunit specificities of these antibodies. If present, the luteinized cells label with antibodies to tyrosine hydroxylase, α-inhibin, and calretinin, which have been shown to recognize ovarian hilar cells and testicular Leydig cells.242,246

By molecular/genetic analysis, KRAS mutations appear early and increase in frequency proportional to the degree of dysplasia.218,247-249 p53 overexpression appears to occur relatively late in in situ and invasive mucinous cystadenocarcinomas.218,247-249 Approxi-mately half of the associated invasive carcinomas also show loss of DPC4 expression, which is not surpris-ing because most of these carcinomas are conventional DAs.183,232,233,241,250

TABLE 15.2 ImmunohistogramofIntraductalPapillaryMucinousNeoplasmwithSelectedAntibodies

100%90%80%70%60%50%40%30%20%10%0%

MUC1 MUC2 CDX2

Gastric type papillaeIntestinal type papillaePancreatobiliary type papillae


INTRADUCTALTUBULARCARCINOMA

Intraductal tubular carcinoma is a recently described entity, the clinicopathologic characteristics of which have yet to be fully characterized.18,251,252 It resembles IPMN by its intraductal nature and mimics acinar cell carcinoma (ACC) by its frequent acinar pattern. Approximately one third of the lesions have foci of inva-sion ranging from microscopic to larger foci.18

Immunohistochemically, intraductal tubular carcino-mas are positive for AE1/AE3 (100%), CK7 (85%), and CK19 (85%), but not for CK20 and CDX2.194,253-255 Most express CA19.9 (95%).18 Focal linear immu-noreactivity for CEA (40%) has been reported along the apical cytoplasm. The lesions also express MUC1 (90%) and MUC6 (60%), whereas MUC2 is nega-tive,194,253-256 placing them closer to the pancreatobili-ary type IPMNs. However, MUC4 and MUC5AC are negative. The lesions almost always (>90%) show intact DPC4 labeling (Fig. 15.14).18 Lack of acinar differentia-tion as demonstrated by negativity of trypsin and other acinar markers is essential in the differential diagnosis

of intraductal tubular carcinomas from acinar cell carci-nomas because the morphologic distinction of these two tumor types is often challenging.

SEROUSCYSTADENOMA

Serous cystadenoma (SCA) is the only nonmucinous example of ductal neoplasia in the pancreas and, unlike other ductal tumors, has virtually no tendency for malignant transformation.257 It is also known to have a well-established association with von Hippel-Lindau (vHL) syndrome.4,162

The tumor cells have a clear cytoplasm because of the abundant intracytoplasmic glycogen and label with CAM 5.2, AE1/AE3, CK7, CK8, CK18, and CK19 but usually not with CK20.257-263 A third of the cases are also positive for EMA.258,260,261,264 Even though CA19-9 and B72.3 expression261,265 is reported, immunolabeling for mucin markers is generally lacking with the exception of MUC6. CEA is uniformly nega-tive, as are insulin, glucagon, somatostatin, vasoactive intestinal polypeptide, and vimentin.260 Chromogranin- or synaptophysin-positive scattered endocrine cells are commonly detected.5,231,258,259,261,265-267 Molecules implicated in clear cell tumorigenesis (glucose uptake and transporter-1 “GLUT-1” [Fig. 15.15], hypoxia-inducible factor-1a “HIF-1α,” and carbonic anhydrase IX) are also consistently expressed.268 As in other vHL-related clear cell tumors, there is a prominent capil-lary network immediately adjacent to the epithelium confirming that the clear cell-angiogenesis association is also valid for this tumor type.268 This rich capillary

FIGURE 15.13 Progesterone receptors are expressed in the ovarian-like stroma cells and may be used to confirm the diagnosis in equivocal cases of mucinous cystic neoplasms.

Mucinous Cystic Neoplasm

▪ Mucinous cystic neoplasms (MCNs) are mucinous/ductal type neoplasia and thus the epithelial cells are positive for CKs, as well as for EMA, CA19-9, and CEA.

▪ All MCNs express MUC5AC, but MUC1 is seen primarily in cases with an invasive component.

▪ Progesterone receptors are expressed in the ovarian-like stroma cells and may be used to confirm the diagnosis in equivocal cases of MCNs.

▪ The ovarian-like stroma cells also label with antibodies to smooth muscle actin, muscle-specific actin, desmin, h-caldesmon, and also variably for alpha-inhibin and calretinin.


FIGURE 15.14 Intact DPC4 expression in the nuclei of the intra-ductal tubular carcinoma cells.

553PANCREAS

network, which can be highlighted by CD31 stain (Fig. 15.16) may be helpful for surgical pathologists, espe-cially as needle biopsies are becoming the norm for ini-tial workup of the patients.268 This finding may also be helpful in frozen sections where clear cell cytology is typically not evident owing to the preservation of gly-cogen.268

By molecular/genetic analysis, vHL gene (chromo-some 3p) allelic deletions are detected in SCAs from patients with vHL, providing further evidence of their neoplastic nature and integral association with vHL syndrome.269-271 Alterations of vHL gene may also be detected in sporadic cases.249,269 Frequent (in 50% of patients) loss of heterozygosity has also been reported on chromosome 10q.271 In contrast to DAs, activating mutations in the KRAS oncogene and inactivation of the TP53 tumor suppressor gene have not been reported in SCAs.218,249,265,270-272

ACINARCELLCARCINOMA

Acinar cell carcinomas (ACCs) are rare and fairly aggres-sive tumors, although prognostically not as dismal as DAs.3 They can be seen in any age group but are more common in elderly patients. They are typically solid, cellular, stroma-poor tumors characterized by sheets of relatively uniform cells. Variable amounts of endocrine elements in forms of scattered individual cells, large zones, and hybrid foci or even as separate well-established nod-ules are commonly present in most cases if searched care-fully. If the endocrine component is larger than 25% of the tumor, by convention the case is classified as “mixed.”

ACC cells are also almost always positive for CAM 5.2, AE1/AE3, CK8, and CK18,273,274 whereas CK7, CK19, and CK20 are generally negative. EMA is expressed in about half of the tumors.273 Glycoproteins, characteris-tic of ductal differentiation (MUC1, MUC5AC, CEA, CA19-9, DUPAN-2, B72.3, and CA125), are either negative or only focally positive. Immunohistochemical identification of pancreatic enzyme production is helpful in confirming the diagnosis.70,76,275-278 Both trypsin (Fig. 15.17) and chymotrypsin are detectable in more than 95% of cases, although some studies have shown less sensitivity for chymotrypsin.275 Lipase is less commonly identified, in approximately 70% to 85% of cases. Other enzymes that are reportedly positive in ACC are alpha-1-antitrypsin, alpha-1-antichymotrypsin, phospholipase A2, and pancreatic secretory trypsin inhibitor. In daily practice the most useful antibody applied is trypsin.

FIGURE 15.15 GLUT-1 expression in serous cystadenoma is detected predominantly in the cell membranes, but also in the cytoplasm.

Serous Cystadenoma (SCA)

▪ SCA is virtually the only ductal tumor that does not show the pancreatic ductal differentiation markers (mucins, mucin-related oncoproteins, and KRAS mutation).

▪ Instead, it commonly expresses the markers of centroacinar-cell/intercalated duct system such as MUC6, as well as markers of clear cell tumorigenesis such as GLUT-1, HIF-1α, and carbonic anhydrase IX.

▪ Epithelial differentiation markers (CKs and EMA) are always positive.

▪ The rich capillary network, which can be highlighted by CD31 stain, may be helpful, especially in needle biopsies and frozen sections.


Serous Cystadenoma (SCA)

▪ Clear cell variant of pancreatic endocrine neo-plasm versus serous cystadenoma: Diffuse immuno-reactivity for chromogranin and synaptophysin is highly in favor of an endocrine origin.

▪ PEComa (sugar tumor) versus SCA: HMB-45 and SMA are positive in PEComa, whereas the absence of such reactivity and immunolabeling for CKs and EMA is typical for SCA.


FIGURE 15.16 The remarkable intensity of capillary network im-mediately adjacent to the epithelium, highlighted by CD31, in se-rous cystadenoma.


The endocrine component shows immunoreactivity for chromogranin or synaptophysin76,275,279,280 and, rarely, peptide hormones such as glucagon or somatostatin are expressed. In some cases of ACC, even the most typical acinar areas may show positivity with endocrine mark-ers. Markers typically present in solid-pseudopapillary neoplasms (vimentin, CD56, and progesterone recep-tors) are negative.

Only rare cases exhibit abnormal nuclear accu-mulation of the p53 protein,76,281-283 and DPC4 is retained.90,284

By molecular/genetic analysis, ACCs rarely, if at all, show KRAS mutations76,90,281,283,285,286 in stark contrast with ductal cancers. Recent work has identified a high frequency of allelic loss on chromosomes 4q, 11p, and 16q.284,287,288 In addition, 25% of ACCs have muta-tions in the APC/β-catenin pathway, a pattern similar to that of pancreatoblastoma.76,283,284,287

PANCREATOBLASTOMA

Pancreatoblastoma is a rare pancreatic tumor showing differentiation toward all three lineages in the pancreas (acinar, ductal, and endocrine) in variable amounts.3 It is the most common pancreatic neoplasm of childhood, although one third of reported cases were in adults.

Microscopically, the tumors show large, solid, nest-ing, and acinar growth patterns and have characteristic squamoid corpuscles that occasionally have optically clear nuclei rich in biotin.289

Many cases show labeling for markers of acinar, ductal, and endocrine differentiation in the respective areas, although acinar differentiation is the most com-mon and the predominant pattern in the majority of the cases.289 The acinar component labels with anti-bodies to CAM 5.2, AE1/AE3, CKs 7, 8, 18, and 19. Positivity for trypsin and chymotrypsin is found in nearly every case; lipase is less common.70,290-295 The ductal elements, present in 50% to 65% of cases, express glycoprotein markers such as CEA, B72.3, and DUPAN-2.70,291,292,296,297 Finally, endocrine mark-ers chromogranin and synaptophysin are positive in two thirds of cases in a highly variable proportion of the cells.70,296,297 Staining for islet peptides (insulin, glucagon, or somatostatin) is generally not found.292 Immunohistochemical positivity for AFP has been detectable in cases with elevations in the serum levels of AFP.292,298

Morular formations that are referred as “squamoid corpuscles” are characteristic and entity-defining fea-tures of pancreatoblastoma, present in nearly every case. Immunohistochemical evaluation of the squamoid corpuscles has failed to define a reproducible line of differentiation for this component.292 In fact, both by morphology and immunophenotype, they show more striking similarities to morules seen in tumors like endometrial carcinoma, pulmonary endodermal tumor, and cribriform-morular variant of papillary thyroid carcinoma, all of which are associated with β-catenin alteration just like squamoid corpuscles of pancreato-blastomas. In fact, the abnormal nuclear immunolabel-ing pattern for β-catenin is most prominent in squamoid corpuscles than other cell types that occur in pancre-atoblastomas (Fig. 15.18). Squamoid corpuscles are also positive for EMA, which accentuates their simi-larities with meningothelial whorls. CEA can be focally

FIGURE 15.17 Immunohistochemical stains for acinar enzymes, in particular trypsin as shown here, are detectable in more than 95% of acinar cell carcinomas.

Acinar Cell Carcinoma (ACC)

▪ Immunohistochemical stains for acinar enzymes, particu-larly trypsin but also chymotrypsin, and lipases serve as highly specific markers of acinar differentiation.

▪ Pancreatic ductal differentiation markers (mucins and mucin-related oncoproteins) are either negative or only focally positive.

▪ Endocrine component is very commonly present and shows immunoreactivity for chromogranin or synapto-physin as well as other neuroendocrine markers.


Acinar Cell Carcinoma (ACC)

▪ Solid-pseudopapillary neoplasm (SPN) versus ACC: SPN typically expresses a nonspecific acinar marker, α1-antityripsin, similar to ACC; however, it also consistently expresses vimentin, CD56, β-catenin, CD10, and proges-terone receptors. Furthermore, in SPN, in contrast with ACC, more specific acinar markers (trypsin, chymotrypsin, and lipases) are not expressed and epithelial markers are usually either focal or weak.

▪ Pancreatic endocrine neoplasm versus ACC: Scat-tered endocrine cells or a focal endocrine component are common in ACC; however, diffuse and strong reactivity for the endocrine markers (chromogranin and synaptophysin) throughout the tumor is characteristic of PENs. Additionally, PENs do not show immunoreactivity for acinar markers.


555PANCREAS

positive292 and so can CK8, CK18, and CK19, but not CK7.293


The distribution pattern of the β-catenin immunolabel-ing is characteristic for pancreatoblastomas. The acinar/ductular elements show mostly membranous (normal) expression of β-catenin, whereas the squamous cor-puscles display diffuse nuclear/cytoplasmic (abnormal) expression (see Fig. 15.18) and overlapping cyclin D1 overexpression (>5% of tumor cells positive).299,300


The most common genetic alteration identified to date is LOH of the highly imprinted region of chromosome 11p near the WT2 gene locus.301 Additionally, altera-tions in the adenomatous polyposis coli (APC)/β-catenin pathway have been reported in 50% to 80% of pancre-atoblastomas.299 Most often, these involve the β-catenin gene (CTNNB1).299,300 Unlike DAs, TP53 and KRAS mutations have not been detected.284,299,301,302

SOLID-PSEUDOPAPILLARYNEOPLASM

Solid-pseudopapillary neoplasm (SPN) is a peculiar tumor of indeterminate lineage. Although they have been described in all age groups, the mean age is 30. They are seen almost exclusively in females; however, they can also occur in males on occasion. Histomorphologi-cally, SPNs typically show diffuse cellular proliferation of relatively bland cells admixed with variable degree of stroma. The preferential dyscohesiveness of the cells away from the microvasculature, presumably related to the alterations in cell adhesion molecules (β-catenin and E-cadherin), leads to the highly distinctive arrangement of cells that is referred as “pseudopapillary,” although it is not present in all cases.189 Eosinophilic globules composed of α1-antitrypsin might also be seen. SPNs are low-grade malignancies that are curable with com-plete removal in 85% of the cases. No reliable criteria recognize the remaining 15% that will spread to the peritoneum or liver, but typically even these patients experience a protracted clinical course.

Despite intensive study, the line of differentiation of these neoplasms remains uncertain.70,303-305 Both aci-nar and ductal markers, discussed previously, are con-sistently negative in SPN.70,306,307 The tumors are also consistently negative for chromogranin. In only 5% of SPNs, there is focal chromogranin expression. The literature reflects conflicting data on this issue, but all experts now agree that if a tumor shows substantial chromogranin expression, it is not an SPN.70,306,308,309 Peptide hormones are also usually negative or at most focally positive.70,303-305,310,311 SPNs also fail to show any convincing neurosecretory granules by electron microscopy, which further corroborates that these are nonendocrine neoplasms. However, these tumors com-monly react with some of the so-called neuroendocrine markers, synaptophysin, NSE, and neural cell adhesion molecule (NCAM or CD56).70,303-306,309,311-321

Even epithelial nature of SPN is dubious, although it has been referred to as “carcinoma” in the past. Cytokeratins (CAM 5.2, AE1/AE3) and other epithe-lial markers are typically either negative or only very focal in rare cases. Ultrastructural evidence of epithelial differentiation is also lacking. However, the neoplastic cells express vimentin and α1-antitrypsin diffusely and strongly.70,303-306,309,311-321 Another marker consis-tently expressed in SPN is CD10; however, this marker should be used cautiously in the differential diagnosis because DAs and PENs can also stain for CD10 (also known as common acute lymphocytic leukemia anti-gen, CALLA).24,322 Progesterone receptors are also expressed in SPNs, regardless of whether it is in women or men.314,317,323,324 Recently, C-kit (CD117)325 and FLI-1326 expressions have been reported in a portion of SPNs. SPNs usually do not stain with S-100,70 cal-retinin,327 or AFP.306 IHC is summarized by immuno-histogram in Table 15.3.


Greater than 90% of SPNs have mutations in exon 3 of the β-catenin gene (CTNNB1).2,67,68 In those cases not showing exon 3 mutations that would account

FIGURE 15.18 An abnormal cytoplasmic and nuclear immunola-beling pattern for the product of the β-catenin gene (CTNNB1) is seen in most pancreatoblastomas, predominantly in the squamoid corpuscles. Other areas usually display normal membranous immu-nolabeling.

Pancreatoblastoma

▪ Most pancreatoblastomas show at least some labeling for acinar enzymatic markers (trypsin, chymotrypsin, and lipases).

▪ Endocrine and ductal markers are also often positive, but to a lesser degree, and positivity may be more focal.

▪ An abnormal nuclear and cytoplasmic immunolabeling pattern for the product of the β-catenin gene (CTNNB1) and overexpression of its target gene cyclin D1 is seen in most cases, usually in the squamoid corpuscles.



for the remaining 10% of cases, it is likely that muta-tions are present in other exons.24 Therefore 100% of SPNs show an abnormal cytoplasmic/nuclear pattern of labeling with antibodies to the β-catenin protein (Fig. 15.19).314,318,326,328 This suggests a β-catenin gene (CTNNB1) mutation activating the Wnt-signaling path-way, which results in overexpression of cyclin D1 in the pathogenesis of these tumors.90,218,247,315,318,319,329 In fact, in more than two thirds of the cases, a concomi-tant cyclin D1 expression is also seen.318,319 In addition, through a mechanism that is not yet clear, the disrup-tion of β-catenin interferes with E-cadherin and, as a result, using an E-cadherin antibody to the extracellular domain of the molecule illustrates complete membrane staining loss, whereas the antibody directed to the cytoplasmic fragment produces distinct nuclear stain-ing of the tumor cells in virtually all cases.23,326,330 This loss of E-cadherin may be responsible for the distinc-tive dyscohesiveness of the cells that creates the char-acteristic (and entity/name-defining) pseudopapillary appearance. Thus the most common genetic alterations in SPNs, β-catenin gene mutations, both help explain the poor cohesion of the neoplastic cells and provide a useful diagnostic tool-immunolabeling for β-catenin protein.24,140

The expression of CD56, progesterone receptor, and FLI-1, all located on chromosome 11q, has also been interpreted as evidence that chromosome 11q might be involved in a translocation or mutation that leads to the expression of some or all of these three proteins in SPNs.326,331


In contrast to DAs, alterations in the KRAS, p16/CDKN2A, TP53, and SMAD4/DPC4 genes have not been reported in SPNs. Besides, SPNs almost always exhibit β-catenin gene (CTNNB1) muta-tions.218,247,313,315,318,328

TABLE 15.3 ImmunohistogramofSolid-PseudopapillaryNeoplasmwithSelectedAntibodies

100%90%80%70%60%50%40%30%20%10%

0%

Abnor

mal b c

aten

in

Vimen

tin

CD56 PR

a 1-A

ntitr

ypsin NSE

CD10

Cyclin

D1

Synap

toph

ysin

FLI-1

FIGURE 15.19 Solid-pseudopapillary neoplasm with diffuse cyto-plasmic and nuclear β-catenin labeling.

Solid-Pseudopapillary Neoplasm (SPN)

▪ Abnormal cytoplasmic/nuclear expression of β-catenin, as well as loss of membrane staining and/or abnormal nuclear staining for E-cadherin, combined with CD10 and progesterone receptor positivity, can be used to confirm the diagnosis of SPN even in small biopsy specimens.

▪ Diffuse and consistent immunoreactivity for vimentin and α1-antitrypsin is also helpful.

▪ Nonspecific endocrine markers synaptophysin, CD56, and NSE are consistently positive in SPNs; however, the most specific endocrine marker, chromogranin, is consistently negative in SPNs.

▪ Cytokeratins (CAM 5.2, AE1/AE3) are usually negative or very focal and/or weak.


Solid-Pseudopapillary Neoplasm (SPN)

SPN versus pancreatic endocrine neoplasia (PEN):

▪ Cytoplasmic/nuclear expression of β-catenin is consis-tent in SPN but uncommon in PENs. In contrast, most PENs show diffuse strong labeling for chromogranin and keratins, whereas virtually all SPNs are negative for these markers.

▪ Strong positivity of vimentin and progesterone recep-tors is also more common in SPNs than PENs.

▪ Synaptophysin, NSE, and CD56 are expressed consis-tently in both tumors and thus cannot be used in this differential diagnosis.

SPN versus ACC versus pancreatoblastoma:

▪ Expression of acinar enzymes, trypsin and chymotryp-sin, coupled with diffuse keratin positivity, are diagnos-tic of ACCs and also present in pancreatoblastomas.

▪ Nuclear β-catenin expression and positivity of α1-antitrypsin and antichymotrypsin (not to be confused with trypsin and chymotrypsin) are common to all three tumors and cannot be used in this differential.


557PANCREAS

Endocrine NeoplasmsFocal endocrine differentiation, especially in the form of scattered cells, is quite common in pancreatic neoplasia of ductal and acinar nature and is of no known biologic significance. However, if a tumor is predominantly com-posed of cells with endocrine lineage, it is classified as “endocrine.”3

PANCREATICENDOCRINENEOPLASMS

Pancreatic endocrine neoplasms (PENs) are the major-ity of the endocrine neoplasms in the pancreas.332 Most PENs are sporadic. However, these tumors also con-stitute one of the major components of the multiple endocrine neoplasia I (MEN1) syndrome or may arise in patients with vHL syndrome. Those that are associ-ated with increased serum levels of hormones and lead to corresponding symptoms are referred to as “func-tional” and are named according to which hormone they secrete (e.g., insulinoma—42% of all functional variants, gastrinoma—24%, glucagonoma—14%, VIPoma—10%, somatostatinoma—6%). Interestingly, the amount of hormone detected immunohistochemi-cally in the tumor cells does not necessarily correlate with the functionality status.289 Thus usage of hormone panel routinely in the diagnosis of PEN is a debated issue. Suffice it to say that serologic analysis or symp-toms and signs of the tumor override the immunohisto-chemical findings in this regard. The tumor cells mimic the islet cells by forming nests, trabecules, and gyriform patterns and show the typical endocrine cytologic fea-tures including round monotonous nuclei, salt and pep-per chromatin, and moderate amount of cytoplasm.4,162

Almost all PENs label for at least one of the endocrine differentiation markers such as chromogranin, synapto-physin, NSE, neural cell adhesion molecule (NCAM or CD56), and leu-7 (CD57).333-349 Among these, chromo-granin, the most specific endocrine marker, is detected in 85% to 95% of PENs. Synaptophysin is more consis-tently and diffusely expressed than chromogranin, but unfortunately it is less specific. For example, SPN, the main tumor in the differential diagnosis, is commonly positive for synaptophysin, as well as NSE and CD56.

Studies have shown that, in addition to the conven-tional peptide hormones of pancreatic islets (insulin, glu-cagon, somatostatin, and pancreatic polypeptide),350,351 these tumors can also secrete (and express) ectopic peptides, in particular gastrin350-352 and VIP (vaso-active intestinal peptide),350-352 but on occasion also ACTH (adrenal corticotropic hormone),353 antidiuretic hormone (ADH), MSH (melanocyte-stimulating hor-mone), calcitonin, neurotensin, secretoneurin,267,354-356 PTH (parathyroid hormone-like peptide),357 growth hormone and growth hormone releasing factor,358-360 secretogranin II,338 inhibin/activin,361 prohormone convertases 2 and 3,362 metallothionein,363 and soma-tostatin receptors.364 The pattern of labeling for these hormones varies widely.350,365,366 It is common to dem-onstrate the production of more than one hormone in a single PEN. However, only occasionally does a true PEN produce detectable serotonin.367 Therefore for

practical purposes, a serotonin-producing tumor should be regarded as a carcinoid, and if it is in the pancreas, the possibility of metastasis from the GI tract or else-where ought to be ruled out.

Most PENs also stain with CAM 5.2, CK8, and CK18 and approximately half with AE1/AE3. In general, the degree of keratin expression is weaker than it is in aci-nar and ductal tissue.368-370 CK7 and CK20 are usually either negative or stain rare cells.48,345,371,372 Scattered cells that are positive for acinar differentiation mark-ers such as trypsin or chymotrypsin are commonly seen PENs.373-375 If greater than 25% of the neoplastic cells in a predominantly endocrine neoplasm express mark-ers of acinar differentiation, the neoplasm is classified as a “mixed acinar-endocrine carcinoma” by conven-tion.376 Limited experience suggests that these are more aggressive neoplasms, behaving like acinar carcinomas. PENs may also show labeling for glycoprotein markers of ductal differentiation. This may be encountered even in PENs with classical morphology and is not sufficient evidence for a diagnosis of “mixed ductal-endocrine carcinoma” unless a morphologically separate compo-nent of ductal adenocarcinoma is recognized.4 Focal expression of DUPAN-2 or CA 19-9 is found in almost a quarter of conventional PENs.373,374,377 CEA is much less commonly expressed. Some oncocytic PENs also label with hepatocyte paraffin-1 (Hep Par-1), which may be important in the differential diagnosis because these oncocytic PENs do resemble hepatocellular carci-nomas.378 Normal islet cells express progesterone recep-tors and CD99, but only some PENs retain expression of these markers.373,379,380 The immunohistogram in Table 15.4 summarizes pancreatic endocrine neoplasms.

PENs are low- to intermediate-grade malignancies, but it is somewhat difficult to predict which examples are more prone for recurrence and metastasis. In the WHO classification, a constellation of clinical and micro-scopic findings (metastasis, extrapancreatic spread, size, mitotic activity, vascular/perineural/capsular invasion and Ki-67 [MIB-1] labeling index) is used to classify these tumors into prognostic categories as “tumor with

TABLE 15.4 ImmunohistogramofPancreaticEndocrineNeoplasmwithSelectedAntibodies

100%90%80%70%60%50%40%30%20%10%0%

NSE

Synap

toph

ysin

Chrom

ogra

nin

CAM5.

2CD56

AE1/AE3

CK19CD99 PR

a 1-A

ntitr

ypsin

DUPAN-2

CA19-9

mCEA


benign behavior,” “tumor with uncertain behavior” and “carcinoma.” A Ki-67 labeling index of more than 2% of the cells is considered an independent criterion to place the tumor into “uncertain behavior” rather than “benign behavior” category. Recently, it has been pro-posed to classify PENs on the basis of the Ki-67 labeling index of less than 2% as grade 1, 2% to 20% as grade 2, and greater than 20% as grade 3. If Ki-67 labeling index is high (>40%), the case ought to be reevalu-ated for the possibility of a different type of cancer or a poorly differentiated neuroendocrine carcinoma. Recently, several studies from various institutions have also shown that CK19 staining (Fig. 15.20) may serve as another reliable, independent predictor of aggres-sive behavior.368,369 This has not yet been incorporated into the classification schemes, but many experts per-form this stain routinely and report it in a comment. Functionality status has also been traditionally used as a prognostic parameter. It has been well documented that most insulinomas behave in a benign fashion and most glucagonomas exhibit a malignant course. However, it is now widely accepted that this association is through the stage of the tumor: Most insulinomas manifest early with symptoms and signs (Whipple triad: symptoms of hypoglycemia, low serum glucose, and relief of symp-toms with glucose administration) before they achieve a size of 2 cm, whereas many glucagonomas are large and metastatic at presentation.

Other markers are under intense scrutiny as predic-tors of outcome in PENs. Chetty and colleagues23,25 recently reported that aberrations of β-catenin (decrease in membranous staining compared with normal and/or abnormal cytoplasmic/nuclear staining) and E-cadherin expressions (decrease in membranous staining compared with normal and/or abnormal nuclear staining) occur in greater than 50% of PETs. They also showed that PENs with aberrant β-catenin and E-cadherin expressions tend to be larger than those with normal staining pat-terns and most of the cases with lymph node and liver involvement show concordant β-catenin and E-cadherin

abnormal immunoexpression.23,25,369 Whether or not these markers may be of use in identification of PENs with a potential for spread and hence poor outcome has yet to be fully characterized.


Cytogenetic and molecular genetic studies have iden-tified many chromosomal alterations in PENs (chro-mosomal losses are more common than gains).381-383 Furthermore, a dominantly inherited defect in the MEN1 gene has been described in patients with the MEN1 syndrome. Spontaneous MEN1 gene abnormali-ties also occur in about 20% of sporadic PENs,90,384-392 although a greater proportion show chromosomal losses in the same genetic region (11q13). It is believed that many of these PENs arise from the mutation in one locus of MEN1, followed by LOH. PENs arising in patients with the vHL syndrome also usually show bial-lelic inactivation of the vHL gene.393 The vHL gene is normal in sporadic PENs.90

In contrast to DAs, KRAS, TP53, and SMAD4/DPC4 are not mutated in most PENs with the exception of p16/CDKN2A abnormalities.90,281,386,394-396

FIGURE 15.20 CK19 staining in pancreatic endocrine neoplasms is considered, by some authors, to be an independent predictor of aggressive behavior.

Pancreatic Endocrine Neoplasms (PENs)

▪ Almost all PENs label for at least one of the endocrine dif-ferentiation markers such as chromogranin, synaptophy-sin, NSE, neural cell adhesion molecule (CD56), and leu-7 (CD57).

▪ They can express any of the pancreatic hormones, as well as ectopic peptides. In some cases, there is more than one hormone expressed. If an IHC “hormone panel” is to be used, the six hormones cover the vast majority of the cases: insulin, glucagon, somatostatin, PP, gastrin, and VIP; however, hormone IHC in the tumor does not necessarily correlate with the “functionality” status in all cases. Discrepancies do occur, and therefore serology and symptomatology are more reliable in classification of “functionality” status.

▪ For practical purposes, a serotonin-producing tumor should be regarded as a carcinoid, and if it is in the pan-creas, the possibility of metastasis from the gastrointesti-nal tract or elsewhere should be ruled out.

▪ If greater than 25% of the neoplastic cells in a predomi-nantly endocrine neoplasm express markers of acinar differentiation such as trypsin or chymotrypsin, the neo-plasm should be classified as a “mixed acinar-endocrine carcinoma.”

▪ Ki-67 labeling index is an important part of grading and classification of PENs.

▪ CK19 immunolabeling is also widely accepted to have prognostic significance.

▪ Normal islet cells are prone to bind antibodies nonspecifi-cally,21 and conceivably, the same problem may also exist for neoplastic endocrine cells. Therefore caution should be exercised before classifying islets (and PENs) as posi-tive for any marker that is not verified by Western blot or other confirmatory methods.


559EXTRAHEPATIC BILIARY TRACT (GALLBLADDER AND EXTRAHEPATIC BILE DUCTS)

POORLYDIFFERENTIATEDNEUROENDOCRINECARCINOMA

These highly aggressive and rapidly fatal tumors consti-tute less than 1% of all pancreatic endocrine neoplasms. They are so uncommon that some authors believe a poorly differentiated neuroendocrine carcinoma occur-ring in the pancreas is most likely a metastasis from an occult primary in the lung. However, there are proven cases of primary in this region.4,399-401 Many are in fact of ampullary origin, and few are pancreatic.4,399-401 Some are akin to small cell carcinomas as defined in the lung, but more commonly they resemble large cell neu-roendocrine carcinomas such as those common in the lung or elsewhere in the GI tract.162

Although defined by morphology and not reliant on the immunophenotype, immunohistochemical label-ing commonly reveals positivity for chromogranin and synaptophysin even in the most poorly differentiated neuroendocrine carcinomas of the pancreas. It may, however, be very focal (especially for chromogranin).4 CD56 and CD57 are frequently strongly positive in a membranous pattern.402,403 In some studies, in contrast to DAs and well-differentiated PENs, poorly differenti-ated endocrine carcinomas were found to express the cell adhesion molecule L1 (CD171).404,405 Parallel to the degree of mitotic activity and necrosis, the Ki-67 label-ing index also tends to be high in these tumors.

Primitive neuroectodermal tumor (PNET), a tumor that shares some cytologic features with small cell carci-noma but occurs in younger patients, stains immunohis-tochemically for CD99406 and FLI-1.

EXTRAHEPATIC BILIARY TRACT (GALLBLADDER AND EXTRAHEPATIC BILE DUCTS)

Epithelial NeoplasmsADENOMAANDPAPILLOMA

Most adenomas of the extrahepatic biliary tract (EHBT) occur in the gallbladder and are usually detected inci-dentally.407 In contrast, those in the extrahepatic bile ducts (EHBDs) present with signs and symptoms of obstruction. They can be multifocal, especially those with a papillary architecture. On the basis of the growth pattern, they have been classified traditionally as tubu-lar, papillary, or tubulopapillary,408-410 although the rel-evance of this classification independent of the degree of dysplasia is debatable.2

Another subclassification is based on the cytoarchi-tectural resemblance to different parts of the GI tract: Pyloric gland–type adenomas demonstrate glands that are virtually indistinguishable from pyloric glands of the stomach or Brunner glands of the duodenum. Intestinal-type adenomas are classified as such owing to their strik-ing histologic resemblance to colonic adenomas.408-411 Biliary-type adenomas are quite rare and less well char-acterized. Those papillary lesions that have more cuboi-dal-shaped cells than intestinal-type adenomas may also be included into this category.408-410

Another recently proposed classification system, which pertains mainly to adenoma in the EHBDs, divides them into two categories on the basis of the predominant cytologic features: “columnar-cell” and “cuboidal-cell” types.412 This is analogous to the villous/intestinal and pancreatobiliary subsets of pan-creatic IPMNs, respectively.196

The immunophenotype of adenomas corresponds to their particular lineage of cellular differentiation.2 Most adenomas are CK7 positive and some express mucin-related glycoproteins and oncoproteins such as MUC1 and CEA, which are typically confined to the apical membrane of the cells. MUC5AC is expressed in about 30% of cases.413 MUC2 expression paral-lels the degree of columnar-cell change and intestinal differentiation, whereas MUC6 is characteristically positive in the cuboidal-cell pattern and in cases with pyloric or biliary differentiation.412 Pyloric gland–type adenomas are reported to be consistently positive for MUC6, as well as other pyloric gland markers such as M-GGMC-1.413 Neuroendocrine markers such as chromogranin and synaptophysin highlight scattered endocrine cells, which also often show expression of serotonin and, on occasion, other hormones. Estro-gen receptors have been detected in more than 50% of adenomas.414

As in other organs, there is a spectrum of carcino-matous transformation that can occur in exophytic bili-ary neoplasms. Although adenomas, especially pyloric gland adenomas, are typically negative for p53, carcino-mas arising from these lesions may acquire p53 expres-sion. Ki-67 labeling index increases with the degree of dysplasia. Unfortunately, there are no established

Pancreatic Endocrine Neoplasms (PENS)

▪ Primary pancreas versus primary gastrointestinal tract versus primary lung neuroendocrine neo-plasms: Well-differentiated neuroendocrine (carcinoid) tumors of pulmonary and gastrointestinal origin show some morphologic similarities to PENs. Thus predicting the site of origin in a metastatic site may require aid from immunohistochemistry. The following facts have been reported397,398:

▪ Nuclear expression of pancreatic duodenal homeo-box-1 (PDX-1) and neuroendocrine secretory pro-tein-55 (a new addition to the chromogranin family, NESP-55), especially if coupled with negative CDX-2 and TTF-1, would favor pancreatic origin.

▪ Positivity of CDX-2 in the absence of PDX-1, NESP-55, and TTF-1 is highly in favor of a carcinoid of gastroin-testinal primary.

▪ TTF-1 positivity is compatible with pulmonary origin; however, it is not necessarily present in all lung neuro-endocrine neoplasms.

▪ Distinguishing full-blown PENs from reactive mega-islets (up to several millimeters) can be accomplished by hormone stains showing a more clonal distribution in PENs with one or two peptides, whereas non-neo-plastic islets often show more regional distribution of several hormones, similar to normal islets.



thresholds to assign the dysplasia into specific grades on the basis of immunohistochemistry alone. Furthermore, areas of regenerative atypia associated with ulceration also commonly express these markers at a higher level. The pattern and intensity of some membrane-bound glycoproteins such as MUC1 and CEA also change during carcinomatous transformation. Often, frank CIS or invasive carcinoma arising from these preinva-sive lesions displays more intense and intracytoplasmic labeling with these antibodies, as opposed to luminal-membranous labeling in lesser lesions.


Molecular alterations of gallbladder adenomas are fairly different than those observed in the conventional dys-plasia-carcinoma sequence. Mutations of the p53 gene are virtually nonexistent in gallbladder adenomas and only rarely detected in EHBD tumors.415 In contrast, p53 abnormalities are quite common in flat dysplasia and invasive carcinoma.416 Similarly, mutation of the KRAS oncogene is detected in only 25% of gallblad-der adenomas.415 In contrast, mutations of the β-catenin gene, which are uncommon in invasive biliary carcino-mas, have been detected in 60% of adenomas,417 mostly the pyloric gland type, and less commonly the papillary or intestinal types.417

MUCINOUSCYSTADENOMA

This adenoma represents the biliary counterpart of pan-creatic mucinous cystic neoplasm (see “Pancreas” ear-lier for details).418

DYSPLASIA

Dysplasia is reported in 40% to 60% of the patients with invasive adenocarcinoma in EHBT; however, the incidence of dysplasia outside the setting of invasive adenocarcinoma is difficult to determine. It is also clear that the frequency varies significantly between different populations and risk groups and parallels that of adeno-carcinoma.419,420 For example, incidence of gallbladder dysplasia is high in regions with a high incidence of car-cinoma, and incidence of bile duct dysplasia is higher in cases of primary sclerosing cholangitis or anomalous union of pancreatobiliary ducts or choledochal cysts.2

Microscopically, dysplasia is characterized by the dis-orderly intraepithelial proliferation of atypical colum-nar, cuboidal, or elongated biliary-type cells. However, biliary epithelium has tremendous capacity to develop marked cytologic atypia secondary to injury that may, at times, be impossible to distinguish from a true neo-plastic process.2

If used cautiously, immunohistochemistry can be employed as an adjunct in the differential diagnosis of reactive versus dysplastic lesions. Nuclear p53 expres-sion is present in more than 30% of all dysplasia in the EHBT (Fig. 15.21) and the incidence and the degree of expression is significantly higher in high-grade lesions,421 whereas p53 is relatively uncommon in non-neoplastic epithelium. On the other hand, it can be present in areas

of marked regenerative changes as well, which limits its value as a sole diagnostic marker. Similarly, although Ki-67 labeling index is often substantially greater in dys-plastic lesions and increases in quantity with increasing degrees of dysplasia, it can also be marked in areas of regenerative change.

INVASIVEADENOCARCINOMA

Carcinomas of the gallbladder and extrahepatic bile ducts are rare.2,422 They occur in elderly patients, seen predominantly in the seventh to eighth decades of life,410 although those associated with primary scleros-ing cholangitis tend to occur in younger patients. Most occur in the gallbladder, followed by the upper third of the EHBDs (above the cystic duct junction). There are well-established risk factors such as gallstones423 for gallbladder cancer and parasitic infections424 for EHBD carcinomas. The most common growth pattern is a scir-rhous, gray-white, firm mass due to the prominence of desmoplasia.410 Calculi are present in more than 80% of gallbladder cancers.1,2

EHBT carcinomas are similar, both morphologi-cally and immunophenotypically, to other foregut car-cinomas, namely pancreatic and gastro-esophageal cancers. Their similarity to pancreatic ductal carci-noma is such that they are often classified together as “pancreatobiliary-type” adenocarcinoma.1 Mucin, in particular sialomucin,425 nonsulfated, or neutral types, is demonstrable by histochemical or immunohistochemical stains in almost all cases and may be abundant.1

Immunohistochemically, CK7 is nearly always positive. CK20 may also be positive in some cases, which contrasts with intrahepatic cholangiocarcino-mas that tend to be negative for CK20. Many surface- glycoproteins such as MUC1 and CEA (Fig. 15.22), normally limited to the apical membrane of the benign cells, are commonly detected in the cytoplasm of the adenocarcinoma cells.1 For these markers, there appears to be a progressive increase in the level of expression from preinvasive to invasive and to poorly differentiated carcinomas, with dense intracytoplasmic expression detected mostly in advanced carcinomas. The tumors

FIGURE 15.21 Immunohistochemical overexpression of p53 pro-tein by dysplastic cells in gallbladder.

561EXTRAHEPATIC BILIARY TRACT (GALLBLADDER AND EXTRAHEPATIC BILE DUCTS)

also usually express MUC5AC, CA19-9, B72.3, epi-dermal growth factor receptor, and pepsinogen I and II.2,410,426 Estrogen receptors are only detectable in a small percentage of the cases.414 Scattered endocrine cells, positive for chromogranin and synaptophysin stains, may also be found.410


In approximately 65% of the cases, there is positive immunoreactivity for the protein product of the TP53 gene.416,427 Some studies have shown this to be fairly specific for carcinoma, as opposed to non-neoplastic changes associated with primary sclerosing cholangi-tis415; however, it should be used cautiously because of significant overlap. Loss of DPC4, present in approxi-mately half of pancreatic DAs, is almost as common in distal common bile duct carcinomas. However, DPC4 is retained in most proximal EHBD carcinomas.428


Deletion of p16 gene is observed in half of the gallblad-der cancers421,429,430 and is reported to be associated with a poor prognosis.431,432 Although the frequency of KRAS mutations has differed widely in different stud-ies, most investigators have found these mutations to be significantly higher in EHBD carcinomas than in gall-bladder carcinomas.427,433-437 Amplification of HER2/neu gene amplification is detected in half of the tumors as well.438 Overexpression of HER family receptors including EGFR and c-met has also been reported.439 Loss of pRB expression is rare in non–small cell gallblad-der carcinoma but is common in small cell carcinoma of the gallbladder.429 High-throughput microarrays have shown aberrant expression of several epithelial antigens such as mesothelin, prostate stem cell antigen, fascin, 14-3-3s, and topoisomerase II, as well as many peritu-moral stromal proteins.62 These have not yet been fully tested as diagnostic or prognostic markers.

OTHERINVASIVECARCINOMASOFEXTRAHEPATICBILIARYTRACT

Intestinal-type adenocarcinoma with all the features of conventional colonic adenocarcinomas is uncommon in the gallbladder; however, ordinary gallbladder adeno-carcinomas, on occasion, may exhibit foci of columnar cells and pseudostratification and thus resemble intesti-nal carcinomas. Further complicating the issue, intesti-nal markers such as MUC2 and CDX2 may show some positivity in these tumors1 and may be taken as further evidence of intestinal differentiation; however, it should be kept in mind that many classical foregut carcinomas also express these markers. It is advisable not to clas-sify such cases as intestinal-type adenocarcinoma unless they exhibit all the characteristic morphologic features of colonic adenocarcinomas.2

Pure mucinous (colloid) carcinomas as seen in the breast, skin, or pancreas182 are practically nonexistent in the gallbladder; however, mucinous carcinomas as described in the colon, in which the mucinous pattern constitute 50% to 90% of the tumor do occur.440

Hepatoid carcinomas showing many characteristics of hepatoid differentiation including positivity for hepatocyte-1 antigen (Hep Par-1), alpha feto-protein,441 and a canalicular pattern with polyclonal CEA or CD10 may be seen, although uncommonly. However, unlike true hepatocellular carcinomas, they also express CK19 and CK20, which are more characteristic of biliary dif-ferentiation.442,443

Squamous differentiation is not uncommon in EHBT carcinomas, especially in the gallbladder. Most are focal and not reported. If the squamous areas con-stitute greater than 25% of the tumor, then the term “adenosquamous” is employed. By convention, even a small glandular component qualifies the tumor as ade-nosquamous, not squamous. The term squamous cell carcinoma is reserved for those rare “pure” examples, in which glandular elements cannot be documented by extensive sampling. The areas of squamous differ-entiation are typically positive for CK5/6, CK13, and nuclear p63, whereas glandular areas show CEA and B72.3 positivity.2 In some studies, squamous/adeno-squamous carcinomas of the gallbladder are reported to be associated with a better prognosis,444,445 unlike those

FIGURE 15.22 Carcinoembryonic antigen, which is generally limited to the apical membrane of the benign cells (surface), often shows intracytoplasmic staining in invasive carcinoma cells.

Invasive Adenocarcinoma

Benign, noninvasive epithelium versus invasive carcinoma: Gallbladder adenocarcinomas may be deceptively bland appearing and, as such, may be difficult to distinguish from Aschoff-Rokitansky sinuses or Luschka’s ducts. Although p53 and Ki-67 are significantly more com-mon in neoplastic epithelium, overlaps are unfortunately too common for them to have a conclusive role in this distinc-tion. Similarly, dense cytoplasmic expression of MUC1 and CEA is more characteristic of invasive adenocarcinomas. Unfortunately, this feature is not as evident in the more problematic well-differentiated carcinomas, in which the expression is more typically luminal membranous.



of the pancreas. In contrast, some studies have noted a tendency for squamous carcinomas of the gallbladder to be of a higher stage at the time of diagnosis.446

Although the majority of EHBT carcinomas show overt glandular differentiation, in a substantial minor-ity gland formation may not be as evident. These are tumors that have a sheetlike growth pattern and lack gland formation.440 Some may have more monoto-nous rhabdoid-appearing cells. Immunohistochemistry is helpful in elucidating the epithelial nature of these malignancies by demonstrating keratins, EMA, and oth-ers and differentiating them from metastatic melanomas and lymphomas. Expression of mucin-related glycopro-teins such as MUC1, CA19-9, CEA, and others may also help further establish the identity of these tumors and sometimes also highlight the abortive glandular ele-ments confirming the diagnosis of adenocarcinoma.

Certain variants of poorly differenti ated/ undifferentiated carcinomas exist, and they should be recognized separately. For example, although rare, medullary-type carcinomas akin to those occurring in the other parts of the GI tract may be seen in EHBT. This pattern is typically associated with lymphoplasma-cytic infiltrates, nodular growth, and distinctive cytol-ogy including round to ovoid large cells with vesicular chromatin and single prominent nucleoli. In our experi-ence, some of these are associated with loss of one of the more common microsatellite instability markers such as MLH1 or MSH2.

Spindle cell (sarcomatoid) carcinomas also occur in the gallbladder.440,447 Similar to their counterparts in other organs, mesenchymal differentiation in these neo-plasms at the morphologic level is also accompanied by immunophenotypic transformation into mesenchymal type cells, manifested by increased expression of vimen-tin, as well as acquisition of aberrant expression of actin and others. Often, keratins are either weak or focal. A distinctive variant of sarcomatoid carcinoma is the “osteoclastic giant cell carcinoma,” in which there are abundant benign osteoclastic type giant cells positive for CD68 and other histiocytic lineage markers.

Endocrine NeoplasmsScattered endocrine cells may be identified in most gallbladder tumors including adenomas and carcino-mas, and a spectrum of endocrine differentiation may be encountered in other gallbladder neoplasia1,2; how-ever, if a tumor is predominantly composed of cells with endocrine lineage, it is classified as “endocrine.”

Carcinoid tumors, also called “well-differentiated neuroendocrine tumor” by the WHO 2004, are rela-tively uncommon in the EHBT.448-450 They have the typi-cal morphologic characteristics of carcinoids elsewhere (<10 mitosis per 10 high power field and minimal, or no, necrosis) and are commonly but not always positive for chromogranin (Fig. 15.23), synaptophysin, and CD56.1,2 Hormones commonly detected at the immunohistochem-ical level are serotonin and somatostatin, although these do not necessarily correlate with serologic findings.2

High-grade neuroendocrine carcinomas with necro-sis and/or greater than 10 mitosis per 10 high power

field should be distinguished from carcinoids. Most high-grade neuroendocrine carcinomas are of the small cell type, which are characterized by diffuse or nested growth patterns, cells with a high N/C ratio, high mitotic activity (typically > 30 per 10 high power fields), and necrosis. They are defined by their morphologic features, and immunohistochemical evidence of neuro-endocrine differentiation is not required; chromogranin may be focal or weak and appear only as fine granules within the cytoplasm. In addition, some tumors fit the description “large cell neuroendocrine carcinoma” as defined in the lung.429,451,452 For the diagnosis of these large cell neuroendocrine carcinomas, immunohisto-chemical support is desirable to differentiate them from their nonendocrine counterparts.

Nonepithelial NeoplasmsGranular cell tumors are not too uncommon in the EHBT, especially in the common bile duct, and may be multicentric or coexist with granular cell tumors in other sites such as skin. These have the characteristic morphologic features of granular cell tumors arising elsewhere and are immunoreactive to S-100, NSE, Leu-7 (CD57), and vimentin.1,410

Embryonal rhabdomyosarcoma is the most common malignant tumor of the biliary tract in children; however, it represents less than 1% of all rhabdomyosarcomas. It may arise from any segment of the EHBDs, as well as gallbladder, but the most common site is the common bile duct.1 Macroscopically, the tumor usually consists of a conglomerate of soft, mucosa-covered polyps. Micro-scopically, the polypoid fragments are usually covered by a layer of flattened biliary epithelium, which may be focally denuded. Beneath the surface is a dense zone of primitive spindle cells forming a cambium layer. Muscu-lar differentiation is demonstrable both by immunohisto-chemical labeling with muscle-specific actin, desmin, and muscle transcription factors (myogenin and myoD1).1 Genetic analyses of embryonal rhabdomyosarcoma have

FIGURE 15.23 Well-differentiated neuroendocrine tumor (carci-noid) in gallbladder. The tumor cells are diffusely and strongly posi-tive for chromogranin. Dysplastic glandular cells on the surface are negative.

563AMPULLA

shown expression of MYOD1 gene and loss of heterozy-gosity at loci on chromosome 11.410

AMPULLA

AdenocarcinomaMore than 80% of ampullary epithelial neoplasms are adenocarcinomas. They are seen predominantly in the seventh decade of life and may be associated with poly-posis syndromes and neurofibromatosis. Because ampul-lary carcinomas result in early symptoms and detection, they are often relatively small at the time of diagno-sis. Most have an exophytic component representing adenoma.1

The majority of lesions are “intestinal-type” adeno-carcinomas, exhibiting typical features of intestinal ade-nocarcinomas including large, elongated tubular units lined by columnar to cuboidal cells. The second most common type is “pancreatobiliary-type” adenocarcino-mas, characterized by smaller glandular units lined by cuboidal cells and surrounded by desmoplastic stroma. Because patients with intestinal-type ampullary adeno-carcinomas have been reported to have a significantly better prognosis than patients with pancreatobiliary-type ampullary adenocarcinomas,453 the differentiation between the two tumor types is important.1,2 Although immunohistochemistry has had only limited value in unequivocally differentiating these types, several authors have suggested that CDX2 and mucin (MUC) expression profile may be useful for this purpose.454-458 Recently, Sessa and colleagues459 showed that all (100%) intestinal-type ampullary adenocarcinomas are diffusely immunoreactive for CDX2 (Fig. 15.24A) compared with rare (30%) pancreatobiliary-type ampullary adeno-carcinomas exhibiting only focal CDX2 (Fig. 15.24B). In addition, a significantly higher frequency of MUC1 and MUC5AC expression is detected in pancreatobili-ary type than in intestinal type, whereas a significantly higher percentage of positivity for MUC2 was found among intestinal type compared with pancreatobiliary

type.454,459 Chu and colleagues455 reported that CDX2 and MUC2 can be used as positive markers for intes-tinal-type ampullary adenocarcinoma with a positive predictive value of 82%. According to the same study, CK17 and MUC1 can be used as positive markers for pancreatobiliary-type ampullary adenocarcinoma and for pancreatic DA and cholangiocarcinoma with posi-tive predictive values of 83%, 76%, and 58%, respec-tively.455 Moreover, CK7 is also often strongly positive and CK20 more focal in the pancreatobiliary type, whereas the intestinal type tends to have more CK20 and less CK7.459 All of these markers ought to be used in the context of morphology because none is entirely specific, and the overlaps are too common to allow their usage as a sole marker.

Additionally, in 75% of invasive carcinomas, there is a diffuse, strong cytoplasmic reaction for mono-clonal CEA, which is seen only in the luminal mem-brane of benign epithelium. Intestinal-type ampullary adenocarcinomas are more likely to stain than pan-creatobiliary type. Similarly, 60% of invasive carcino-mas express DUPAN-2; however, pancreatobiliary-type ampullary adenocarcinomas are more frequently posi-tive for DUPAN-2 than intestinal type. Most ampullary adenocarcinomas also stain for CA19-9. CA19-9 posi-tive cases are reported to be more aggressive.460

Endocrine cells are commonly found in ampullary adenocarcinomas.1,2 Although they may not be evident by routine microscopy, immunohistochemical stain-ing for chromogranin reveals scattered endocrine cells in one third of the cases.410 The distribution of these neuroendocrine cells is random; in some cases they are scattered evenly throughout the tumor, whereas in oth-ers they may be clustered. In general, they are more common in intestinal-type or mucinous adenocarcino-mas than in pancreatobiliary type.410

Other forms of adenocarcinoma that occur in the ampullary region include mucinous adenocarcinoma, signet-ring cell adenocarcinoma, and “invasive papillary carcinoma,”410 which may mimic in situ neoplasia because of its exophytic growth, papillae formation,

A B

FIGURE 15.24 Intestinal-type ampullary adenocarcinomas are diffusely immunoreactive for CDX2 (A). In contrast, pancreatobiliary-type ampullary adenocarcinomas either are negative (B) or exhibit only focal CDX2.


branching architecture, and relatively smooth con-tours. Adenosquamous and squamous carcinomas of this region are rare. Similarly, sarcomatoid carcinomas are exceedingly uncommon.1,410 The immunoprofile of these tumors is similar to their counterparts in the EHBT and pancreas as discussed in detail earlier.

GENOMICAPPLICATIONSOFIMMUNOHISTOCHEMISTRY

Mutations of p53 have been detected in the majority of ampullary carcinomas with corresponding accumulation of the abnormal product as detected immunohistochem-ically.461,462 EGFR is overexpressed in 50% to 65% of invasive ampullary carcinomas. Pancreatobiliary-type adenocarcinomas are more likely to overexpress EGFR than are intestinal-type tumors. Related growth factors c-erbB-2 and c-erbB-3 are also overexpressed in ampul-lary carcinoma.410

BEYONDIMMUNOHISTOCHEMISTRY:ANATOMICMOLECULARDIAGNOSTICAPPLICATIONS

Ampullary adenocarcinomas are less likely to show loss of DPC4 gene expression463 and KRAS gene muta-tions than pancreatic DAs,464 probably corresponding to the incidences of these mutations in intestinal ver-sus pancreatobiliary-type adenocarcinomas. Although rare, poorly differentiated ampullary carcinomas with morphologic features resembling medullary carcinomas of the large bowel have been reported to demonstrate microsatellite instability.459

Endocrine NeoplasmsA spectrum of endocrine differentiation may be seen in the ampullary region, which occurs in 3% of ampullary tumors.410

Most are carcinoids. Although the general charac-teristics of these are not too different from carcinoids elsewhere, there are some peculiarities worth mention-ing. The majority of the “functioning” somatostatinomas occur in the ampulla. Moreover, whether functional or not, somatostatin-positive tumors of this region, in addi-tion to the classical features of low-grade neuroendocrine neoplasms, also display tubule formation, focal intra-luminal mucin, and psammomatous calcifications and

may be associated with neurofibromatosis. This phe-nomenon is fairly specific to the ampulla.465 All express diffuse and strong chromogranin (Fig. 15.25), synapto-physin, and somatostatin; however, they are not associ-ated with the stigmata of somatostatin secretion, and therefore the term glandular psammomatous carcinoid of the ampulla is preferable to somatostatinomas. Other peptides may also be found. Even though lymph node metastasis is seen at presentation in more than 50% of the cases, surrogate signs of aggressiveness (high Ki-67 index and CK19 positivity) are not seen. Ki-67 index is typically low (<5%). Common S-100 expression is intriguing, especially considering the association with neurofibromatosis. Gland formation, intraluminal—but not intracytoplasmic—mucin, and infiltrative appear-ance may be mistaken as adenocarcinoma in small biop-sies. Moreover, many cases show focal CA19-9 and CEA positivity.465

The ampulla is also included in the “gastrinoma triangle”: Endocrine tumors associated with Zollinger-Ellison syndrome and multiple endocrine neoplasia-1 (MEN-1) are often localized in this region410 and may be microscopic, and thus difficult to identify preopera-tively and grossly.

High-grade neuroendocrine carcinomas may also be seen in the ampulla.1,2 Although these account for an exceedingly small percentage of malignancies in the gastrointestinal tract, they appear to occur at a rela-tively higher proportion in the ampulla. Whether this is related to the abundance of endocrine cells seen also in the adenomas of this region is not known. Both small and large cell variants are recognized.1

Most high-grade neuroendocrine carcinomas are of the “small cell” type, similar to pulmonary small cell carcinoma. They tend to have a diffuse growth pattern. The tumor cells have minimal cytoplasm, indistinct cell borders, and polygonal nuclei having finely stippled chromatin and indistinct nucleoli. Some cases have more “large cell” phenotype with a more nested pattern and a moderate amount of cytoplasm. The nuclei are round and vesicular with often prominent nucleoli. High-grade

Adenocarcinoma

▪ Most pancreatobiliary-type ampullary adenocarcinomas are CDX2−/MUC1+/MUC5AC+/MUC2−, and conversely, many intestinal-type ampullary adenocarcinomas are CDX2+/MUC1−/MUC5AC−/“MUC2+.”

▪ CK7 is often strongly positive and CK20 more focal in pancreatobiliary-type ampullary adenocarcinomas, whereas intestinal-type ampullary adenocarcinomas tend to have more CK20 and less CK7.


FIGURE 15.25 Diffuse and strong chromogranin positivity in am-pullary somatostatinoma (glandular psammomatous carcinoid).

565LIVER

neuroendocrine carcinomas are identified by brisk mito-sis and easily identifiable necrosis. Cytokeratin and endocrine markers including chromogranin, synapto-physin, and NSE are often positive but may be focal and weak. As with their counterparts in other organs, these tumors are mostly defined by morphologic char-acteristics rather than detectability of neuroendocrine markers. CEA may be positive. Typically, Ki-67 label-ing index is high, displaying positivity in the majority of the cells.410 Loss of retinoblastoma protein is reported in 60% of high-grade neuroendocrine carcinomas but not in nonendocrine carcinomas. In contrast, loss of p27 is common in nonendocrine carcinomas but not in high-grade neuroendocrine carcinomas.466

Duodenal Gangliocytic ParagangliomaDuodenal gangliocytic paraganglioma is a lesion that is fairly specific to this area. It is a peculiar tumor of unknown origin that exhibits a mixture of (1) epithelioid (paraganglioma-like or carcinoid-like) elements, which are positive for keratins and neuroendocrine markers, chromogranin, synaptophysin, and NSE; (2) ganglion-like cells; and (3) spindle cells of nerve sheath,10 which are negative for keratins, but express S100 instead. Spe-cific peptides may also be found, especially pancreatic polypeptide and somatostanin.410 The latter may be important in the differential from somatostatinomas because short of the ganglion-like and nerve sheath components, duodenal gangliocytic paragangliomas

may be difficult to distinguish from glandular carcinoids (somatostatinomas) of this region.

LIVERA wide variety of non-neoplastic (also referred to as “medical”) and neoplastic diseases may affect the liver. Proper diagnosis of these entities is important because effective therapeutic options are becoming increasingly available including medical therapy for non-neoplastic diseases and resection for neoplastic diseases. In addi-tion, liver transplantation has become an important modality for many chronic conditions. Immunohisto-chemistry (IHC) has an important role in the diagnosis of liver diseases. As discussed later, IHC can be useful in identifying hepatic infections, evaluating transplant biopsies, and classifying hepatic tumors. IHC has also been instrumental in elucidating the pathogenesis of many disorders of the liver.

Normal Hepatic ParenchymaHEPATOCYTES

Embryonal hepatocytes contain cytokeratins (CK) 8, 18, and 19; however, mature ones contain only CK8 and 18, with CK19 being negative by the tenth week of gestation. CAM5.2 stains hepatocytes in the peri-portal zones and adjacent to venules. CK7, 19, and 20 are negative, as is epithelial membrane antigen (EMA) and vimentin. Although often not detectable in nor-mal hepatocytes, α-fetoprotein can be positive in cir-rhotic nodules.46,468-474 Hepatocytes have significant amounts of biotin, which may lead to positive stain-ing with standard immunohistochemical techniques if the endogenous biotin activity is not blocked, which is a major potential problem of “false positiv-ity.”475-477 Hep Par-1 (human hepatocyte paraffin-1) stains hepatocytes in a diffuse granular cytoplasmic pattern without canalicular accentuation.478-480 Thy-roid transcription factor also stains the cytoplasm of hepatocytes in a coarsely granular pattern but does not stain the nuclei.480 Bile canaliculi can be high-lighted by a variety of antibodies against luminal-membranous glycoproteins such as polyclonal CEA and CD10.473,481

Bile DuctsIntrahepatic bile ducts and peribiliary glands stain for cytokeratins 7, 8, 18, 19, 34βH11, 34βH12, and AE1/AE3.468-470,479,482 CK20, CA19-9, and CEA are gener-ally negative.46,483

VasculatureThe expression of many endothelial markers includ-ing CD34, factor VIII, CD31, or Ulex europaeus lec-tin is fairly weak in normal vasculature, whereas it can become quite prominent in pathologic conditions such as chronic liver disease and hepatocellular carci-noma.473,484-486

Endocrine Neoplasms of the Ampulla

▪ High-grade neuroendocrine carcinoma versus carcinoid: In general, there is more diffuse positivity for general neuroendocrine markers in carcinoid tumors. By definition,467 proliferation markers such as Ki-67 are expressed significantly more commonly in high-grade carcinomas versus carcinoids.

▪ High-grade neuroendocrine carcinoma versus lymphoma: Immunohistochemical positivity for keratin and negative staining for lymphoid markers help exclude lymphoma.

▪ High-grade neuroendocrine carcinoma versus poorly differentiated nonendocrine cancer: By im-munohistochemistry, the high-grade neuroendocrine car-cinomas are positive for cytokeratins (AE1/AE3, CAM5.2, CK7, and in a lesser percentage CK20), similar to the pattern found in poorly differentiated nonendocrine carcinomas. However, if present, immunohistochemical expression of neuroendocrine markers is helpful in this regard. Also, loss of retinoblastoma protein expression, a characteristic finding in pulmonary small cell carcinomas, is present in almost half of ampullary high-grade neuro-endocrine carcinomas. In contrast, p27 expression is lost in poorly differentiated nonendocrine carcinomas and re-tained in most high-grade neuroendocrine carcinomas.466



InterstitiumThe interstitial matrix of the liver is composed of col-lagens, glycoproteins, proteoglycans, and glycosamino-glycans. Alterations in the extracellular matrix of the liver play an important role in fibrosis and the stromal milieu of both neoplastic and non-neoplastic processes. Type I collagen is the main collagen of portal tracts and in fibrotic liver and appears as thick, deep blue fibers on trichrome stains. Newly formed collagen is often com-posed of collagen type III and appears as fine, light blue fibers. Hepatic stellate cells have an important role in mediation of hepatic interstitial fibrosis and remodel-ing.487-500 In some studies, they have been shown to have features of neural/neuroectodermal differentiation and stain with antibodies to synaptophysin, glial fibrillary acidic protein (GFAP), and neural cell adhesion mol-ecule (N-CAM).473,501-503 Activated stellate cells often acquire myofibroblastic features and show expression of vimentin, desmin, and smooth muscle actin.473,504

Medical Liver DiseasesSTEATOHEPATITISANDMALLORY’SBODIES

First described in alcoholic patients by Frank B. Mallory in 1911, Mallory hyalines/bodies also appear in other chronic liver diseases.505 Sometimes they can be difficult to distinguish on biopsies, and ancillary immunohisto-chemistry with keratins CK18, 34βE12, and CAM5.2, as well as antibodies to ubiquitin (Fig. 15.26), may help by highlighting them. They are also occasionally posi-tive for CK7 and CK19.473,506,507

VIRALINFECTIONS

Viral infections may lead to hepatitis, which may become chronic, eventually leading to cirrhosis. In these infections, there is usually some degree of inflamma-tory mononuclear infiltrate in portal tracts, interface hepatitis, and lobular inflammation; however, since the inflammation is variable and does not always parallel

the infectious activity, immunohistochemistry may need to be employed in highlighting infected hepatocytes. In chronic hepatitis B (HBV), infected ground-glass hepa-tocytes can be appreciated and confirmed by cytoplas-mic staining for the hepatitis B surface antigen (HBsAg). Membranous staining for HBsAg is often indicative of active viral replication (Fig. 15.27A). Detection of HBsAg usually denotes chronic hepatitis because it is usually not detected in acute hepatitis. Hepatitis B core antigen (HBcAg) reactivity, on the other hand, which is usually appreciated in the nucleus (Fig. 15.27B) rather than in the cytoplasm, is used to measure the degree of viral replication. Patients who are immunocompro-mised or have received HBV through vertical transmis-sion have both membranous HBsAg and nuclear HBcAg staining without a great deal of inflammation.508-512 Hepatitis B immunohistochemistry may be particularly useful in identification of recurrent HBV after transplant for chronic HBV infection.513 Delta virus, or hepatitis D, can also be detected immunohistochemically with cyto-plasmic and nuclear staining.509,514,515 It has been dif-ficult to develop immunohistochemical antibodies that accurately detect hepatitis C virus (HCV) that can be applied on routine clinical specimens.509,516-521 In situ hybridization techniques to detect hepatitis B, C, and D have been developed and have shown some utility.522,523 Epstein-Barr virus (EBV) infection can be detected both immunohistochemically and through in situ hybrid-ization. This is of particular use in EBV-driven post- transplant lymphoproliferative disorders (PTLDs) and in lymphoepithelioma-like hepatocellular and cholan-giocarcinomas, which are associated with EBV.509,524-527 Cytomegalovirus (CMV) is another important infec-tion that can involve the liver and be detected with CMV immunohistochemical stains509,528,529 and in situ hybridization.522 Other infections of the liver that can be detected immunohistochemically include herpes sim-plex virus, herpes zoster virus, adenovirus, mycobacte-ria, and amoebiasis.509,530

CIRRHOSIS

Cirrhotic nodules may acquire some staining for α-fetoprotein (AFP); however, macroregenerative nod-ules are typically negative474,531 for this marker. In cir-rhotic regenerative nodules, peripheral hepatocytes may show ductular transformation and express both cyto-keratin 7 and 19.506,532-534 It is speculated that this is evidence of a stem-cell phenotype,535-538 and the focal expression of AFP is also interpreted to provide further evidence for this.474,539 Endothelial cells at the periphery of cirrhotic nodules adjacent to fibrous septa also have an altered phenotype, expressing CD31 and CD34, a feature that may be seen in other conditions including nodular regenerative hyperplasia.473,484-486

PRIMARYBILIARYCIRRHOSISANDSCLEROSINGCHOLANGITIS

Cytokeratin immunohistochemistry directed toward bil-iary epithelium can be useful in highlighting bile ductu-lar proliferations that occur in a variety of conditions

FIGURE 15.26 Ubiquitin immunohistochemistry demonstrating Mallory hyalines.

567LIVER

including primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), regardless of the mecha-nism, whether it is a result of “ductular metaplasia” of limiting plate hepatocytes or primary proliferation of native ducts.533,534 The utility of cytokeratin 7 in this context has been demonstrated.540,541 Immunohisto-chemistry for endothelial markers such as CD31, CD34, factor VIII-related antigen, Ulex europaeus, and other lectins may also reveal differences in the microvascula-ture of the portal tracts in primary sclerosing cholangitis and primary biliary cirrhosis with the overall common-ality to both disorders being a loss of capillary micro-vasculature. In PBC, vessels are often obliterated by granulomatous inflammation, whereas in PSC, the ves-sels are displaced by collagen deposits.542-544 Recently, investigators have demonstrated that immunostaining of plasma cells with antibodies for IgM and IgG may be useful in the distinction between PBC and autoimmune hepatitis (AIH) with most of the plasma cells staining for IgM in PBC and most of the plasma cells staining for IgG in AIH. This parallels an increase in serum IgG that can be seen in AIH and increase in serum IgM that can be seen in PBC. However, it must be noted that IgG positive plasma cells can be seen in the liver in a number of disorders.545

IGG4-RELATEDCHOLANGITIS

Recently lesions containing large numbers of immu-noglobulin G4 (IgG4)-bearing plasma cells have been identified in a number of organs. Much interest has focused on the ones in the pancreas546; however, lesions of the intrahepatic and extrahepatic bile ducts may also contain large numbers of IgG4-bearing plasma cells. They are referred to by a number of descriptive names, notably IgG4-associated cholangitis (IAC), and may be confused with other lesions, particularly primary scle-rosing cholangitis or autoimmune hepatitis. The lesions usually also contain prominent fibrous tissue in a stel-late pattern with admixed inflammatory cells includ-ing lymphocytes and eosinophils. Obliterative phlebitis

may be observed. The lesions may be quite florid with pseudotumor formation. Immunohistochemistry for IgG4 may be useful in properly categorizing these lesions.546-552 The numeric threshold of IgG4-positive plasma cells required to place a lesion into this cat-egory has yet to be established. In the pancreas, this number has been advocated as a minimum of 10 per high-power field, with some authors requiring 20 or even 30.553-558

FOCALNODULARHYPERPLASIA

Focal nodular hyperplasia (FNH) is considered to be a hyperplastic reactive proliferation of hepatocytes due to localized abnormalities in blood flow. Classically, FNH has a central scar with radiating septa and hyperplas-tic hepatocyte nodules but is devoid of bile ducts. The diagnosis of FNH can be aided by CD34 immunohisto-chemistry, which highlights sinusoidal endothelial cells in the vicinity of fibrous septa in a linear pattern.559,560 Hepatocytes at the periphery of cirrhotic-like nodules of FNH may stain with cytokeratins 7 and 19, and the cytokeratin 7 cells may be continuous with fibrous septa.468,534

Metabolic DisordersAlpha-1-antitrypsin (α1-AT) deficiency disease is associ-ated with PAS-positive, diastase-resistant globules in the hepatocytes (Fig.15.28A). Immunohistochemical stain-ing for α1-AT may be useful in verifying the nature of these granules (Fig. 15.28B). In addition, early in α1-AT deficiency, these globules may not be as visible and immunohistochemistry may be useful in highlighting α1-AT (Fig. 15.28B). The α1-AT stain may be positive as early as 19 weeks’ gestation.561-563

Afibrinogenemia and hypofibrinogenemia (type I fibrinogen deficiencies) are rare congenital disorders in which plasma fibrinogen levels are low or immeasur-able. Some cases of hypofibrinogenemia may actually be present as a “fibrinogen storage disease” in which

A B

FIGURE 15.27 A, Hepatitis B surface antigen (HBsAg) immunohistochemistry showing both cytoplasmic and membranous positivity. B, Hepatitis B core antigen (HBcAg) immunohistochemistry showing nuclear positivity.


fibrinogen is present in large quantities in hepatocytes, appreciable on routine stains as eosinophilic globules in hepatocytes, some of which have a dark core and some of which are vacuolated. Fibrinogen antibodies may be useful in determining that the intrahepatocytic material is fibrinogen.563,564

Liver TransplantationImmunohistochemistry has two important applications in the evaluation of transplant biopsies: (1) identifica-tion of infectious agents and (2) determination of the mechanism (and thus etiology) of the immune injury. Immunohistochemistry for various organisms, par-ticularly viral organisms, may be crucial in the inter-pretation of liver transplant biopsies. This may be particularly important for the identification of recurrent hepatitis B or C, as previously discussed.565 Diagnosing liver allograft rejection, particularly antibody-mediated (humoral) rejection, can unfortunately be problematic. Immunohistochemistry for C4d, a marker frequently used in the interpretation of renal allograft biopsies, has been investigated in liver allografts, with mixed results.566-581 Antibody-mediated rejection in the liver does appear to occur, as evidenced by antibody-medi-ated rejection of ABO incompatible livers. Deposition of C4d has been demonstrated in liver allografts and associated with decreased survival.566 However, stud-ies have pointed out different patterns of deposition of C4d in the liver including the following: stromal,566 portal,575,580 sinusoidal,572,574,580 and hepatocytic pat-tern.573,574

Even though the sinusoidal and portal capillary endothelium patterns (Figs. 15.29A and 29B) seem to be most promising,571,572,574 it is unclear which of these patterns actually has prognostic signifi-cance.566

This field, though yet imperfect, is in a rapid evolu-tion, and there is no question that IHC will have an

increasing role in the diagnosis of immune injury in the transplant setting in the future.

Neoplastic Liver DiseasesHEPATOCELLULARNEOPLASMS

HepaticAdenoma

Hepatic adenomas are benign tumors usually seen in women in their childbearing years. Long-term oral contraceptive steroid usage is a common risk factor. Grossly, the lesions are usually well demarcated and are yellow or tan, sometimes containing fibrosis, hem-orrhage, or necrosis.582 Microscopically, they are com-posed of plates of cells resembling normal hepatocytes separated by sinusoids. Immunohistochemically, estro-gen and progesterone receptors are present in 75% of the lesions.583 They might also express β-catenin.584-586 This has also been observed in hepatocellular carcinomas associated with hepatitis C virus infection582,587; how-ever, most hepatocellular carcinomas express glypican-3 and hepatic adenomas are negative for glypican-3.588,589 Additionally, hepatocellular carcinomas typically show “complete” CD34 staining pattern (most sinusoidal spaces are CD34 positive throughout the lesion) and hepatic adenomas usually show “incomplete” staining (portal and periportal sinusoids are CD34 positive).588

Hepatoblastoma

Hepatoblastoma, the most common primary hepatic tumor in children, is a malignant tumor with embryonal features and divergent differentiation including striated muscle, fibrous tissue, and material resembling osteoid. Approximately one third of the cases have pure fetal epithelial differentiation resembling developing hepa-tocytes. The keratins expressed are generally of low-molecular-weight type (CK8 and 18, but sometimes also 19 and 7). Most tumor cells are also positive for EMA, vimentin, pCEA, Hep Par-1 and α-fetoprotein.590,591

A B

FIGURE 15.28 A, PAS/D stain showing prominent intracytoplasmic diastase-resistant globules. B, α1-antitrypsin immunohistochemistry confirming that the globules contain α1-antitrypsin.

569LIVER

Kupffer cells and endothelial cells lining sinusoids can be marked for UEA-1 and anti-CD34 in a pattern simi-lar to HCC, more diffuse than normal liver.591-593

HepatocellularCarcinoma

Hepatocellular carcinoma (HCC) is the single most common histologic type of epithelial primary liver tumor. Architecturally, HCC may have a number of different patterns, the most common being a trabecu-lar or plate-like pattern. Other patterns include acinar, pseudoglandular, scirrhous, clear cell, spindle cell, and pleomorphic.582

cytokeratins HCC reacts with antibodies directed against a variety of cytokeratins (CKs), particularly low-molecular-weight cytokeratins (CK8, CK18). Thus most HCCs stain with CAM 5.2 and 35βH11. They are usually negative for CK7, CK19, and CK20 or show patchy staining. AE1/AE3 is also typically patchy, but poorly differentiated HCCs often show clusters of posi-tive cells.41,46,594-599

carcinoembryonic antigen A canalicular staining pattern for polyclonal carcinoembryonic antigen (pCEA) is seen in 60% to 90% of HCCs531,600 (Fig. 15.30) and is use-ful in discriminating hepatocellular tumors from other malignancies.531,586 However, it should be remembered that abortive lumina formation in poorly differentiated adenocarcinomas can mimic canalicular pattern.

Hepatocellular carcinomas are nonreactive with monoclonal CEA, an important factor in the differen-tial diagnosis with cholangiocarcinomas and metastatic carcinoma.601

hep par-1 Hep Par-1 is an antigen reflecting hepatocytic differentiation (Fig. 15.31) and yields a diffuse cytoplas-mic granular staining pattern in normal and neoplas-tic hepatocytes including approximately 80% to 90% of HCC cases. Therefore it is not useful for distinction of benign versus malignant hepatocellular lesions.599 Hep Par-1 stains conventional adult HCC, as well as fibrolamellar and clear cell variants but not sclerosing HCC. Some investigators have reported decreased stain-ing with more poorly differentiated HCC; however, this distinction is not completely clear. It should also be kept in mind that Hep Par-1 is not entirely specific for HCCs. It can be found in a variety of tumors, in particular those with oncocytoid morphology (abun-dant acidophilic, granular cytoplasm) including intra-ductal and cystic oncocytic neoplasia. Furthermore, rare carcinomas from the gastrointestinal tract and pancreas with hepatoid morphology (tumor cells with eosinophilic, granular cytoplasm) are positive for Hep Par-1.472,479,531,586,599,602,603

A

B

FIGURE 15.29 A, C4d immunohistochemical stain showing si-nusoidal pattern of positivity in a case of antibody-mediated (hu-moral) hepatic allograft rejection. C4d immunohistochemical stain-ing should be interpreted with caution in the liver because plasma proteins can show nonspecific staining with C4d. Donor-specific antibodies (DSAs) must also be considered. B, C4d immunohisto-chemical stain showing portal venule/capillary pattern of positivity in a case of antibody-mediated (humoral) hepatic allograft rejection. This has been referred to by some as a “Garland” pattern of staining.

FIGURE 15.30 Polyclonal carcinoembryonic antigen positivity in hepatocellular carcinoma. Note the canalicular staining pattern.


glypican-3 Glypican-3 is a placenta and hepatic hepa-ran sulfate proteoglycan, normally expressed in fetal liver but not in adult liver. Recently, much interest has been focused on its utility in the diagnosis of HCC. Because it is often negative in normal liver and adeno-mas,588,589 it can be useful in distinguishing benign versus malignant hepatocellular lesions.599 However, caution should be exercised in using glypican-3 in biopsy specimens because cirrhotic nodules can show positivity.588,589,604

Many studies have shown that glypican-3 is more sensitive than Hep Par-1 for HCC (Fig.15.32), particu-larly for poorly differentiated HCC,589 and can be use-ful in the identification of poorly differentiated HCC. It is also used in the differential diagnosis of HCC versus cholangiocarcinoma (positive in 60% to 90% of HCCs and usually negative in cholangiocarcino-mas).588,589,599,604-608

cd34 CD34, in conjunction with glypican-3, may be of some use in distinguishing HCC from its benign mimick-ers. Virtually all sinusoidal spaces in HCCs tend to have a “complete” staining pattern (most sinusoidal spaces are CD34 positive throughout the lesion), which is quite uncommon in benign lesions, with the exception of few hepatic adenomas and focal nodular hyperplasia.588 If used selectively, this CD34 staining pattern may also be helpful in the distinction of HCC from adenocarci-nomas. However, the sensitivity is low (20% to 40%) and because better antibodies are available, CD34 is not routinely used in this differential.599

albumin Albumin is a major serum transport protein synthesized by hepatocytes.602 In situ hybridization (ISH) for the messenger RNA (mRNA) that encodes albumin shows positivity in non-neoplastic hepatocytes, as well as in hepatic adenomas, hepatoblastomas, and HCCs. Unfortunately, immunohistochemistry is not amenable to the detection of albumin because albu-min is abundant in the serum, leading to nonspecific tissue staining. Up to 96% of HCC can show positivity

with ISH, although staining can be diffuse, patchy, or focal.592,602,609-613 One caveat is that other tumors including clear cell carcinoma of the ovary614 and hep-atoid carcinomas (e.g., hepatoid gastric and bladder carcinomas) are also positive.609,615-618 Combination of albumin in situ hybridization and Hep Par 1 can yield 100% sensitivity for diagnosis of HCC.613

alpha-fetoprotein α-Fetoprotein (AFP), an oncofetal gly-coprotein, is frequently elevated in the serum of patients with HCC. Although high levels of serum AFP levels are fairly specific for HCC, some increase can also be seen in hepatitis and cirrhosis.531,602,613,619 Its immunohisto-chemical expression in a tumor is specific for hepato-cellular differentiation, but staining tends to be patchy and sensitivity is fairly low, 30% to 50%.599 Moreover, serum increases and immunohistochemical staining of the tumor can be seen in cases of primary and meta-static hepatoid adenocarcinomas and yolk sac tumors as well.620-622 Therefore AFP is a less useful option for diagnosis.599,623

miscellaneous markers of use in hepatocellular carcinoma Thyroid transcription factor-1 (TTF-1), which is normally expressed in the nuclei of epithelial cells of thyroid and lung and in tumors that arise from them, shows cytoplasmic positivity in HCC (Fig. 15.33). Immunoreactivity varies with the antigen retrieval tech-nique and the antibody clone used.480,624,625 The signifi-cance of this cytoplasmic labeling and, in fact, whether it is “real” staining, is yet to be determined, but it can be of some value in identifying and differentiating HCC from some other carcinomas in select cases.

Epithelial membrane antigen (EMA) can be positive in 20% to 40% of HCC. There is some indication that higher-grade neoplasms have increased staining.29,626-629

CD10 (common acute lymphoblastic leukemia anti-gen, CALLA) can show a canalicular staining pattern similar to that of polyclonal-CEA in approximately half of HCCs.531,600

MOC-31 is a cell surface glycoprotein typically used as a marker of adenocarcinoma. Only a minority of HCC

FIGURE 15.32 Hepatocellular carcinoma with focal glypican-3 staining.FIGURE 15.31 Hepatocellular carcinoma with positive Hep Par-1

immunohistochemistry.

571LIVER

cases show labeling, often as weak staining, and studies show positivity in 0% to 20% of tumors.602,630-632

α1-Antitrypsin (α1-AT) can be positive in HCC with some studies indicating a high proportion (up to 86%) of HCC cases showing positivity.633 However, this marker often shows some nonspecific labeling, which significantly limits its usability.

Factor VIII (FVIII) related antigen (von Willebrand Factor) positivity can be seen in sinusoidal endothelium in nearly all HCC cases, whereas the staining can be focal and even interpreted as being absent in normal livers. Some authors advocate the use of FVIII in con-junction with CD34.486,634-638 Other studies have inves-tigated similar patterns of the vascular endothelium in HCC with markers such as CD31, Ulex europaeus lectin, and ABH blood group antigens.485,637,638 Unfor-tunately, these markers have not been found to be as reliable as CD34 in this regard.

Factor XIIIa (FXIIIa), a blood coagulation proen-zyme, can be positive in histiocytes/macrophages and benign hepatocytes, as well as in HCC. Some have sug-gested that FXIII can be useful in differentiating HCC

from cholangiocarcinoma, but other studies have indi-cated that FXIIIa is noncontributory in this differential diagnosis.479,531,532,633

HCC may also react with antibodies directed against erythropoietin, C-reactive protein (CRP), acidic isofer-ritin, thioredoxin (RX), alkaline phosphatase, inhibin, α1-antichymotripsin, and villin.586,602 These markers are not widely used in routine practice.

Table 15.5 is an immunohistogram of selected anti-bodies for hepatocellular carcinoma.

MolecularandGenomicApplicationsofImmunohistochemistry

Immunohistochemical staining with various mark-ers has been useful in elucidating potential explana-tions for the molecular pathogenesis of HCC. Nuclear accumulation of β-catenin, a component of the wing-less/Wnt pathway, has been observed by immunohis-tochemistry in HCC. This correlated with mutations in the β-catenin gene, which were detected in 26% to 41% of HCCs,582,587 particularly those associated with hepatitis C virus infections.587 In addition, phosphoino-sitol 3-kinase/mammalian target of rapamycin (mTOR) pathway has been investigated in HCC using a num-ber of methods including immunostaining. Investigators have also demonstrated the presence of microsatellite instability in HCC by both genetic molecular analysis and immunohistochemistry for the microsatellite mark-ers (MLH1, MSH2, MSH6, and PMS2).639-641

Molecular insights into the pathogenesis of HCC may eventually guide therapy. Using everolimus (a rapa-mycin analog) to block mTOR signaling decelerated HCC tumor growth in vitro and in a xenograft model and increased survival.642,643 Studies have shown that antibodies to molecules such as p53,644-646 Ki-67,644 and platelet-derived growth factor647 may be useful in pre-dicting prognosis in HCC.644-647 In one study, platelet-derived growth factor receptor alpha (PDGFR-α) was found to be overexpressed in the endothelium of hepa-tocellular carcinoma with a high metastatic potential. In this study, STI571 (Gleevec, or imatinib mesylate) inhib-ited tumor growth, apparently through antiangiogenesis

FIGURE 15.33 Hepatocellular carcinoma with granular cytoplas-mic TTF-1 staining.

TABLE 15.5 ImmunohistogramofHepatocellularCarcinomawithSelectedAntibodies

100%90%80%70%60%50%40%30%20%10%0%

CAM5.

2

Album

in IS

HFVIII

Hep P

ar-1

α 1-A

ntitr

ypsin

pCEA

Glypica

n-3

Cytopla

smic

TTF-1CD10

AFPCD34

EMA

CK7CK20


via inactivation of PDGFR-α, suggesting that PDGFR-α may serve as a biomarker for predicting metastasis and as a therapeutic target.647 Much of this information, however, has not yet been fully verified and translated into daily practice.

HepatocellularCarcinomaVariants

fibrolamellar hcc Fibrolamellar HCC is a distinct variant of HCC that occurs most often in noncirrhotic individu-als from adolescence to young adulthood. It is character-ized by sheets or trabeculae of neoplastic cells separated by collagen bundles in a lamellar configuration.582 Tumor cells have granular eosinophilic (oncocytoid) cytoplasm and are large and polygonal. Fibrolamellar HCC has immunophenotypic similarities to the usual type of HCC648; however, unlike usual HCC, it may show strong CK7 expression.649 Fibrolamellar HCC may sometimes stain with synaptophysin and on occa-sion focally even with chromogranin,599,650,651 which complicates the differential diagnosis from neuroendo-crine tumors. Increased expression of epidermal growth factor receptor (EGFR) may also be seen in fibrolamel-lar carcinoma, suggesting that treatment with EGFR antagonists may be considered in the future.642,652,653

spindle cell hcc High-grade/poorly differentiated HCC may have a spindle cell morphology.582 Poorly differ-entiated HCC may be frankly sarcomatous with some cases having heterologous differentiation such as chon-drosarcomatous.654 Sarcomatous component often loses its epithelial differentiation and becomes negative for the conventional epithelial markers. In addition, they show the appropriate lineage markers reflecting their differ-entiation at histomorphologic level. For example, areas with chondrosarcomatous differentiation stain with S-100 and vimentin. However, some cells of the sarco-matous component also retain keratin in some cases.654 Spindle cell carcinomas may also have osteoclast-like giant cells, in which case the osteoclast-like giant cells express histiocytic differentiation markers.655

clear cell hcc Clear cell HCC may be difficult to dis-tinguish from other clear cell tumors such as adrenal cortical carcinoma and renal cell carcinoma using rou-tine histology.586 Renal cell carcinoma usually shows reactivity with vimentin, whereas only high-grade hepatocellular carcinoma or hepatocellular carcinoma with spindled morphology shows reactivity with vimentin.586 Adrenal cortical carcinoma stains with Melan-A and inhibin, and hepatocellular carcinoma is usually negative with antibodies to these molecules. Furthermore, adrenal cortical carcinoma is negative for CAM 5.2.586,656 Neuroendocrine carcinoma can sometimes have clear cell morphology. Neuroendo-crine carcinomas are usually positive for synapto-physin and chromogranin, whereas HCC is usually negative for these markers.586

medullary (lymphoepithelioma-like) carcinoma Carci-noma with a medullary (lymphoepithelioma-like) mor-phology has been reported in the liver. These cases consist of poorly differentiated carcinoma (Hep Par-1

positive) admixed with abundant lymphoid stroma. The presence of Epstein-Barr virus has been demonstrated by in situ hybridization.525,527

biliary-type differentiation in hcc Some hepatocellular carcinomas may express markers that are otherwise considered specific for biliary-duct differentiation and do not stain hepatocellular carcinomas. The expression of these markers, by itself, is not considered enough by most authors to qualify the tumor as “combined HCC and cholangiocarcinoma.” Some authors refer to it as “biliary-type differentiation.” HCC with “biliary-type differentiation” stains with monoclonal CEA, CK 7, CK 19, and AE1/AE3.7,657,658

Hepatocellular Carcinoma (HCC)

▪ HCC is positive for certain cytokeratins, particularly low-molecular-weight cytokeratins (CK8, CK18).

▪ The markers Hep Par-1 and glypican-3 are useful for the diagnosis of HCC.

▪ Polyclonal carcinoembryonic antigen (pCEA) and CD10 usually show canalicular staining in HCC, but this may be difficult to distinguish from abortive glandular areas in poorly differentiated cholangiocarcinomas.

▪ CD34 may help in highlighting the distinctive, “com-plete” sinusoidal vasculature of HCC.

▪ In situ hybridization for albumin may also be useful in HCC.


Hepatocellular Carcinoma (HCC)

▪ HCC versus benign hepatic tissue: In the past, rou-tine histology and histochemical stains were the mainstay in the distinction between benign hepatic tissue and HCC. Reticulin outlines the normal sinusoidal architecture in benign hepatic tissue and highlights thickened hepato-cytic plates and abnormal nodules in HCC.602 Currently, dealing with this differential diagnosis is also aided by immunohistochemistry. Recently, glypican-3 has gained attention for its utility in this regard because antibodies directed against it stain mainly neoplastic liver and only rarely cirrhotic liver.482,588,589,602 In benign livers, CD34 shows a more selective (“incomplete”) pattern highlight-ing the portal and periportal sinusoids, whereas in HCC, it labels the sinusoids throughout the lesion, showing a more widely spread (“complete”) pattern.588,602 Molecu-lar studies that have been advocated to be of use include telomerase activity, comparative genomic hybridization, and measurement of proliferation status through count-ing of argyrophilic nucleolar organizer regions (AgNORs) with special silver staining,602 although these have not found their way to routine practice.

▪ HCC versus cholangiocarcinomas: Cholangiocarci-noma is positive for both CK7 and CK19, whereas HCC is usually (though not always) negative for these antibodies. Hep Par-1, a marker commonly used to identify HCC, is not typically expressed in cholangiocarcinomas.479,599


573LIVER

BILEDUCTLESIONS

BileDuctHamartomaandBileDuctAdenoma

Benign biliary ductular proliferations are seen in a vari-ety of conditions associated with hepatic injury and subsequent metaplastic changes. Bile duct hamartomas (von Meyenburg complexes) are small collections of bile ductules displaying dilated ductules with contour irreg-ularities, single layer of bland, cuboidal epithelium, and intraluminal bile.

Bile duct adenomas, on the other hand, are well circumscribed, firm, tan-white nodules resembling metastatic carcinoma. Nearly 85% are solitary. Micro-scopically, they contain numerous small tubular structures with small lumina, an important finding in differentiating from hamartomas. Lining epithelium lacks nuclear pleomorphism and hyperchromasia. Immunophenotype is that of peribiliary glands: There is reactivity for CK19, CEA and EMA. KRAS mutations have been reported in small percentage of the lesions.659

These benign biliary proliferations can be difficult to distinguish from well-differentiated carcinomas of pancreatobiliary type (cholangiocarcinomas or ductal adenocarcinomas). CD56 often shows labeling in these benign proliferations (except von Meyenburg com-plexes) but is lacking in adenocarcinomas (Fig. 15.36). Similarly, expression of p53, Ki-67, and B72.3 is signifi-cantly more common in malignant than in benign, but

unfortunately overlaps are common and therefore, these ought to be used cautiously.

Cholangiocarcinoma

Cholangiocarcinoma is a malignant tumor composed of cells recapitulating the biliary ductal cells, described as being intrahepatic (or peripheral) when arising in the liver or hilar when arising from the right or left hepatic ducts near their junction. It is a member of the generic category of “pancreatobiliary-type” adenocarcinomas, histologically similar, if not identical, to gallbladder car-cinomas and pancreatic ductal adenocarcinomas. Many characteristics of these carcinomas are already discussed in detail in earlier sections. Cholangiocarcinoma typi-cally shows malignant glands in a tubular configuration embedded in a fibrous stroma. There may be areas of adenosquamous, mucinous, and signet ring cell carci-noma. Variants include clear cell, mucinous, pleomor-phic, and spindle cell types.660 The lesions usually react with CK7, CK19, CAM5.2, AE1/AE3 (Fig. 15.34), CEA (both monoclonal and polyclonal in a noncanalicular pattern), and MOC-31. Cholangiocarcinoma is more likely to react with CK7 than other pancreatobiliary car-cinomas and less likely to react with CK20, CK17, and p53.586 CK19 is expressed in 85% to 100% of cholangio-carcinomas, whereas most HCCs either are negative or show patchy staining.599 MOC-31 is consistently (80% to 100%) expressed in cholangiocarcinoma.599 Ber-EP4 stains in a similar pattern to MOC-31.632 Although not always included in diagnostic panels of cholangiocarci-noma, CA19-9 can be positive in up to 85% to 100% of cholangiocarcinomas (Fig. 15.35).627,661-663 Mucins (e.g., MUC4, MUC5AC, MUC5B, MUC6) may be use-ful in classifying cholangiocarcinomas and predicting prognosis. A gastric mucin phenotype has been found in some studies to be associated with a worse progno-sis.457,586,664,665 α1-Antitrypsin is typically negative with reports indicating that 0% to 10% of tumors stain.633

Cholangiocarcinoma may stain with parathyroid hormone-related peptide.545,602 Table 15.6 summarizes select antibodies for cholangiocarcinoma.

▪ HCC versus other adenocarcinomas: In the differ-ential diagnosis of HCC from other adenocarcinomas, generic adenocarcinoma markers such as MOC-31 B72.3, mCEA (cytoplasmic or diffuse noncanalicular positivity), CA19-9, MUC1, CK7, and CK20, which tend to be much more commonly positive in adenocarcinomas than in HCC, can be combined with the HCC markers. Depend-ing on the clinico-morphologic differential diagnosis, more specific adenocarcinoma antibodies such as com-bination of mammoglobulin, breast-2, ER, PR, and HER2/neu for breast, or PSA for prostate (although metastasis to liver from prostate is exceedingly uncommon, it can occur) or others can be employed.586,599,602

▪ HCC versus metastatic neuroendocrine tumors: This is one differential in which immunohistochemistry can be of utmost importance. Numerous cytologic and architectural similarities exist between HCC and metastatic well-differentiated neuroendocrine neoplasia (carcinoids and pancreatic endocrine neoplasia): They are both cellular, stroma-poor tumors that have a delicate sinusoidal vasculature and relatively monotonous cells with fair amount of cytoplasm and round nuclei. A panel of “neuroendocrine markers” (chromogranin, synapto-physin, and CD56) combined with “hepatocytic” markers (Hep Par-1 and polyclonal-CEA, and FISH for albumin) can be helpful.

▪ HCC and melanomas: Melanomas can metastasize to liver. Because they can morphologically mimic HCC by showing monotonous cells with abundant cytoplasm, round nuclei, and prominent nucleoli, immunohistochemistry (e.g., keratins and melanoma markers including S-100, HMB45, Mart-1) is often crucial in this differential diagnosis.

FIGURE 15.34 AE1/AE3 staining in cholangiocarcinoma. Note the interface of the tumor with normal liver.


CholangiocarcinomaVariants

spindle cell (sarcomatoid) cholangiocarcinoma Spindle cell (sarcomatoid) cholangiocarcinoma usually occurs as a component of a more conventional or poorly differen-tiated cholangiocarcinoma. The spindled cell areas may be negative or stain only focally for cytokeratins.671-674 Cases with a variety of sarcomatous differentiation have been reported including chondrosarcomatous ele-ments675 and rhabdoid.75,676 Cells of the sarcomatous

elements stain in a typical pattern for those sarco-mas.671,673,674 Rhabdoid elements are typically positive for vimentin,674,676 which is typically true of other spin-dled/sarcomatous elements.673 CEA positivity may be seen.672,673 AFP is usually negative.671,672

MixedHepatocellularandCholangiocarcinoma

Existence of tumors that have both HCC and cholangio-carcinoma components has been well established in the literature. The proper classification and terminology of these tumors, however, which can exhibit a spectrum of cross differentiation, has been somewhat problematic. Although some cases have clearly distinct and easily iden-tifiable HCC and cholangiocarcinoma components (with all the characteristic morphologic and immunopheno-typical features of the respective tumors), in many, there are subtle transdifferentiation and a spectrum of hybrid phenotypes. The interpretation of this spectrum has also been variable among authors. Some employ the term “cholangiocellular carcinoma” for those that morpho-logically consist of biliary adenocarcinomas that have the immunohistochemical staining pattern of HCC.677 In general, studies have shown that their molecular sig-nature is closer to cholangiocarcinoma, suggesting they

FIGURE 15.35 CA 19-9 positivity in cholangiocarcinoma.

TABLE 15.6 ImmunohistogramofCholangiocarcinomawithSelectedAntibodies

100%90%80%70%60%50%40%30%20%10%

0%

CAM5.

2EM

ApC

EACK7

CK19

MOC-3

1

Ber-E

P4

CD15

CA19-9

CA125

B72.3

CK20

Cholangiocarcinoma

▪ Cholangiocarcinoma is commonly positive for certain subtypes of cytokeratins (notably CK7 and CK19).

▪ Cholangiocarcinoma is positive for both monoclonal and polyclonal CEA in a noncanalicular pattern.

▪ Cholangiocarcinoma is also positive for various conventional “markers of adenocarcinoma” (e.g., MOC-31, Ber-EP4).


Cholangiocarcinoma

Cholangiocarcinoma versus benign biliary ductular prolifera-tions:

▪ If von Meyenburg complexes are excluded, CD56 can be used to differentiate benign biliary ductular proliferations from neoplastic proliferations. Reactive proliferating bile ductules are CD56 positive, whereas most cholangiocarcinomas are CD56 negative (Fig. 15.36).666 However, exceptions do exist including cholangiocarcinomas with clear cell differentiation that are CD56 positive.667

▪ p53, Ki-67, and B72.3 can be employed but should be used cautiously because overlaps are common.

▪ Cholangiocarcinoma versus HCC: The distinction of cholangiocarcinoma from HCC can be aided by cy-tokeratin staining because cholangiocarcinoma shows staining for cytokeratins such as CK7 and CK19, whereas HCC is usually negative. Cholangiocarcinoma is usu-ally negative for TTF-1, whereas HCC is often positive (cytoplasmic, not nuclear as it normally is in the lung or thyroid). Claudins, which are positive in cholangiocar-cinoma and typically negative in HCC, have also been proposed as useful in the distinction of cholangiocarci-noma from HCC.586,625,668,669

▪ Cholangiocarcinoma versus metastatic lesions: Cholangiocarcinoma can be difficult to differentiate from adenocarcinomas metastatic from other sites, particularly gastrointestinal primaries (e.g., stomach, gallbladder, extrahepatic biliary tree, pancreas) using immunohistochemistry because cholangiocarcinoma shows an overlapping staining pattern with carcinomas of many sites.599,602 Cholangiocarcinoma may react with CA125, making distinction from müllerian carcino-mas difficult. However, cholangiocarcinoma does not usually react with estrogen receptor.586,670


575LIVER

may arise from cholangiocarcinoma. It should be kept in mind that some antibodies can produce seemingly discrepant results. For example, Hep Par-1 may stain some cholangiocarcinomas.479,586,665,678-681 Overall, the cholangiocarcinoma component is usually more aggres-sive and appears to dictate the prognosis.

BiliaryCystadenomaandCystadenocarcinoma

Biliary cystadenomas and cystadenocarcinomas have an immunohistochemical profile similar to pancreatobiliary cystadenomas and cystadenocarcinomas.418,610,682-685 The epithelium typically shows staining for CK7, CK20, AE1/AE3, CEA, CA 19-9, and CA125.418 Ovarian-type stroma may be present and may show staining for pro-gesterone receptors.686,687

POORLYDIFFERENTIATEDANDUNDIFFERENTIATEDCARCINOMAS

Carcinosarcoma of the liver can occur, although it is rare.675,688,689 In a series from China in which all of the patients were hepatitis B surface antigen positive, con-ventional hepatocellular carcinoma merged with areas of rhabdomyosarcoma, “malignant fibrous histiocy-toma,” and fibrosarcoma; and immunohistochemistry supported the diagnosis of carcinosarcoma.689

NEUROENDOCRINENEOPLASMS

The overwhelming majority of neuroendocrine neo-plasms occurring in the liver are metastatic. Well to mod-erately differentiated neuroendocrine neoplasms (e.g., carcinoids, pancreatic endocrine neoplasms, medullary thyroid carcinomas) may mimic primary hepatocellular processes, and thus immunohistochemistry with antibod-ies for chromogranin, synaptophysin, and CD56 can be extremely helpful, as discussed earlier. They may also be positive for MOC-31 but not for Hep Par-1. In contrast, HCC may occasionally show focal neuroendocrine differ-entiation and may stain for CD56,690,691 synaptophysin,

and chromogranin.599,692 In particular, the fibrolamellar variant of HCC is often synaptophysin positive; how-ever, HCCs are also positive for Hep Par-1.599

Metastatic high-grade neuroendocrine carcinomas ought to be differentiated from other high-grade malignan-cies, metastatic or primary. Wide-spectrum keratins are typically positive and helpful in confirming the epithelial nature of the tumor. Neuroendocrine markers (synapto-physin, chromogranin and CD56) can be fairly focal and thus ought to be evaluated carefully in high power. It should be remembered that these tumors are defined by morpho-logic features, and lack of neuroendocrine markers does not rule out this diagnosis by any means. If the differential diagnosis is with the lesser grade (well-moderately differ-entiated) endocrine lesions, Ki-67 labeling index, which is usually greater than 20% of the cells, can be helpful.

OTHERNEOPLASMS

Hemangioma is the most common primary hepatic tumor, usually an incidental finding at autopsy. Micro-scopically, most lesions are of cavernous type (see other chapters for details).

Epithelioid hemangioendothelioma of liver is often multifocal with involvement of both right and left liver lobes. Although it is generally regarded as a low-grade malignant neoplasm, in some cases it follows an aggres-sive clinical course. Nevertheless, the overall 5-year sur-vival is estimated to be 50%, starkly different than that of cholangiocarcinoma for which it is often mistaken. Furthermore, transplant can be an effective treatment of this tumor type.693 Histologically, subtle examples are typically dismissed as a non-neoplastic fibrosing disor-der. More prominent and vessel-forming examples are misdiagnosed as metastatic adenocarcinoma or cho-langiocarcinoma. Immunohistochemically, cytokeratins are often positive, further accentuating the challenge in distinguishing these lesions from glandular-epithelial lesions. Endothelial markers, CD34, CD31 (Fig. 15.37),

FIGURE 15.37 Endothelial markers (CD31 is shown here) are commonly expressed in epithelioid hemangioendothelioma and are extremely helpful in the differential diagnosis between adenocarci-nomas and HCC.

FIGURE 15.36 CD56 can be useful in distinguishing benign bili-ary ductular proliferations from neoplastic proliferations. Cholangio-carcinomas (left) are more commonly negative for CD56, whereas benign bile ductules (right) are positive.


and/or factor VIII, are commonly expressed and can be extremely helpful.619,694 Less than 50% react with smooth muscle actin586 as well.

Angiomyolipoma is a benign tumor thought to arise from perivascular epithelioid cells (also known as PEC cells) and consist of variable combinations of blood ves-sels, adipose tissue, and smooth muscle. Blood vessels may have thick walls and may be hyalinized. Extramed-ullary hematopoiesis may be present. The lesions are strongly positive for HMB-45 and Melan-A,694,695 as well as ER, and weakly positive for actin and desmin.696

Embryonal (undifferentiated) sarcoma of the liver, also known as mesenchymal sarcoma or malignant mes-enchymoma, occurs predominantly in children. Grossly, it is usually a large solitary and well-circumscribed mass measuring up to 30 cm in diameter, with degenerative changes including necrosis, hemorrhage, and cystic degeneration. Microscopically, the lesions are composed of highly atypical spindle to stellate cells and giant cells embedded in myxoid stroma. PAS-positive and diastase-resistant, large eosinophilic hyaline globules within the cytoplasm of tumor cells and in the stroma are char-acteristic. Scattered entrapped ducts are seen in most cases. Data regarding the immunophenotype of this rare tumor type are conflicting. Generally, they are consid-ered to be positive for vimentin, smooth muscle actin, desmin, and keratin697-699 but not myoglobin.700

SUMMARYThe array of medical and neoplastic diseases of the pan-creas, extrahepatic and intrahepatic biliary tree, and liver are vast. Morphologic impressions remain the bed-rock for proper interpretation of IHC and other special-ized molecular anatomic tissue testing.

ACKNOWLEDGMENTS

The authors express their gratitude to Dr. Ipek Coban, Dr. Nevra Dursun, and Ms. Rhonda Everett for their assis-tance in preparing this chapter.

REFERENCES 1. Adsay NV. Gallbladder, Extrahepatic Biliary Tree and Ampulla.

In: Mills SE, ed. Sternberg’s Diagnostic Surgical Pathology. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2009.

2. Adsay NV, Klimstra DS. Benign and Malignant Tumors of the Gallbladder and Extrahepatic Biliary Tract. In: Odze RD, Goldblum JR, eds. Surgical Pathology of the GI tract, Liver, Biliary Tract, and Pancreas. Philadelphia: Elsevier; 2009: 845-875.

3. Klimstra DS, Adsay NV. Tumors of the Pancreas and Ampulla of Vater. In: Odze RD, Goldblum JR, eds. Surgical Pathology of the GI tract, Liver, Biliary Tract, and Pancreas. Philadelphia: Elsevier; 2009:909-960.

4. Hruban RH, Pitman MB, Klimstra DS. Tumors of the Pancreas. 4th ed. Washington, DC: ARP Press; 2007.

5. Batge B, Bosslet K, Sedlacek HH, et al. Monoclonal antibodies against CEA-related components discriminate between pancre-atic duct type carcinomas and nonneoplastic duct lesions as well as nonduct type neoplasias. Virchows Arch A Pathol Anat His-topathol. 1986;408:361-374.

6. Klimstra DS, Hameed MR, Marrero AM, et al. Ductal Prolifera-tive Lesions Associated with Infiltrating Ductal Adenocarcinoma of the Pancreas. Int J Pancreatol. 1994;16:224-225.

7. Ma CK, Zarbo RJ, Frierson Jr HF, Lee MW. Comparative im-munohistochemical study of primary and metastatic carcinomas of the liver. Am J Clin Pathol. 1993;99:551-557.

8. Yamaguchi K, Enjoji M. Carcinoma of the pancreas: a clinico-pathologic study of 96 cases with immunohistochemical obser-vations. Jpn J Clin Oncol. 1989;19:14-22.

9. Luttges J, Vogel I, Menke M, et al. Clear cell carcinoma of the pancreas: an adenocarcinoma with ductal phenotype. Histopa-thology. 1998;32:444-448.

10. Yamaguchi K, Enjoji M, Tsuneyoshi M. Pancreatoduodenal car-cinoma: a clinicopathologic study of 304 patients and immuno-histochemical observation for CEA and CA19-9. J Surg Oncol. 1991;47:148-154.

11. Moniaux N, Escande F, Porchet N, et al. Structural organiza-tion and classification of the human mucin genes. Front Biosci. 2001;6:D1192-D1206.

12. Nagata K, Horinouchi M, Saitou M, et al. Mucin expression profile in pancreatic cancer and the precursor lesions. J Hepato-biliary Pancreat Surg. 2007;14:243-254.

13. Levi E, Klimstra DS, Andea A, et al. MUC1 and MUC2 in pan-creatic neoplasia. J Clin Pathol. 2004;57:456-462.

14. Yonezawa S, Goto M, Yamada N, et al. Expression profiles of MUC1, MUC2, and MUC4 mucins in human neoplasms and their relationship with biological behavior. Proteomics. 2008;8:3329-3341.

15. Yonezawa S, Nakamura A, Horinouchi M, Sato E. The expres-sion of several types of mucin is related to the biological be-havior of pancreatic neoplasms. J Hepatobiliary Pancreat Surg. 2002;9:328-341.

16. Saitou M, Goto M, Horinouchi M, et al. MUC4 expression is a novel prognostic factor in patients with invasive ductal carci-noma of the pancreas. J Clin Pathol. 2005;58:845-852.

17. Khayyata S, Basturk O, Klimstra DS, et al. MUC6 Expression Distinguishes Oncocytic and Pancreatobiliary Type from Intes-tinal Type Papillae in Pancreatic Neoplasia: Delineation of a Pyloropancreatic Pathway. Mod Pathol. 2006;19:275A.

18. Klimstra DS, Adsay NV, Dhall D, et al. Intraductal tubular car-cinoma of the pancreas: Clinicopathologic and immunohisto-chemical analysis of 18 cases. Mod Pathol. 2007;20:285A.

19. Koshikawa N, Hasegawa S, Nagashima Y, et al. Expression of trypsin by epithelial cells of various tissues, leukocytes, and neu-rons in human and mouse. Am J Pathol. 1998;153:937-944.

20. Portela-Gomes GM, Hacker GW, Weitgasser R. Neuroendo-crine cell markers for pancreatic islets and tumors. Appl Immu-nohistochem Mol Morphol. 2004;12:183-192.

21. Nalbantoglu I, Basturk O, Thirabanjasak D, et al. Islets, the king of antigens? Non-specific reactivity of (especially the) islets is a common pitfall in immunohistochemical evaluation of the pancreas. Mod Pathol. 2007;20:288A.

22. Pignatelli M, Vessey CJ. Adhesion molecules: novel molecular tools in tumor pathology. Hum Pathol. 1994;25:849-856.

23. Chetty R, Serra S. Nuclear E-cadherin immunoexpression: from biology to potential applications in diagnostic pathology. Adv Anat Pathol. 2008;15:234-240.

24. Serra S, Chetty R. Revision 2: an immunohistochemical ap-proach and evaluation of solid pseudopapillary tumour of the pancreas. J Clin Pathol. 2008;61:1153-1159.

25. Chetty R, Serra S, Asa SL. Loss of membrane localization and aberrant nuclear E-cadherin expression correlates with invasion in pancreatic endocrine tumors. Am J Surg Pathol. 2008;32:413-419.

26. Evans DB, Abruzzese JL, Rich TA. Cancer of the pancreas. In: Devita VT, Hellman S, Rosenberg SA, eds. Cancer: Principles and practice of oncology. 6th ed. Philadelphia: Lippincott- Raven; 2001:1126-1161.

27. Moll R. Cytokeratins as markers of differentiation in the diag-nosis of epithelial tumors. Subcell Biochem. 1998;31:205-262.

28. Hoorens A, Prenzel K, Lemoine NR, Kloppel G. Undifferenti-ated carcinoma of the pancreas: analysis of intermediate fila-ment profile and Ki-ras mutations provides evidence of a ductal origin. J Pathol. 1998;185:53-60.

29. Thomas P, Battifora H. Keratins versus epithelial membrane antigen in tumor diagnosis: an immunohistochemical com-parison of five monoclonal antibodies. Hum Pathol. 1987;18:728-734.

577REFERENCES

30. Shimonishi T, Miyazaki K, Nakanuma Y. Cytokeratin profile relates to histological subtypes and intrahepatic location of intrahepatic cholangiocarcinoma and primary sites of metastatic adenocarcinoma of liver. Histopathology. 2000;37:55-63.

31. Alexander J, Krishnamurthy S, Kovacs D, Dayal Y. Cytokeratin profile of extrahepatic pancreatobiliary epithelia and their carci-nomas. Appl Immunohistochem Mol Morphol. 1997;5:216-222.

32. Real FX, Vila MR, Skoudy A, et al. Intermediate filaments as differentiation markers of exocrine pancreas. II. Expres-sion of cytokeratins of complex and stratified epithelia in nor-mal pancreas and in pancreas cancer. Int J Cancer. 1993;54:720-727.

33. Vlasoff DM, Baschinsky DY, Frankel WL. Cytokeratin 5/6 im-munostaining in hepatobiliary and pancreatic neoplasms. Appl Immunohistochem Mol Morphol. 2002;10:147-151.

34. Duval JV, Savas L, Banner BF. Expression of cytokeratins 7 and 20 in carcinomas of the extrahepatic biliary tract, pancreas, and gallbladder. Arch Pathol Lab Med. 2000;124:1196-1200.

35. Goldstein NS, Bassi D. Cytokeratins 7, 17, and 20 reactivity in pancreatic and ampulla of vater adenocarcinomas. Percentage of positivity and distribution is affected by the cut-point threshold. Am J Clin Pathol. 2001;115:695-702.

36. Lee MJ, Lee HS, Kim WH, et al. Expression of mucins and cytokeratins in primary carcinomas of the digestive system. Mod Pathol. 2003;16:403-410.

37. Goldstein NS, Bassi D, Uzieblo A. WT1 is an integral compo-nent of an antibody panel to distinguish pancreaticobiliary and some ovarian epithelial neoplasms. Am J Clin Pathol. 2001;116:246-252.

38. Ramaekers F, van Niekerk C, Poels L, et al. Use of monoclonal antibodies to keratin 7 in the differential diagnosis of adenocar-cinomas. Am J Pathol. 1990;136:641-655.

39. Tot T. Adenocarcinomas metastatic to the liver: the value of cy-tokeratins 20 and 7 in the search for unknown primary tumors. Cancer. 1999;85:171-177.

40. Baars JH, De Ruijter JL, Smedts F, et al. The applicability of a keratin 7 monoclonal antibody in routinely Papanicolaou-stained cytologic specimens for the differential diagnosis of car-cinomas. Am J Clin Pathol. 1994;101:257-261.

41. Chu P, Wu E, Weiss LM. Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 2000;13:962-972.

42. Ji H, Isacson C, Seidman JD, et al. Cytokeratins 7 and 20, Dpc4, and MUC5AC in the distinction of metastatic mucinous car-cinomas in the ovary from primary ovarian mucinous tumors: Dpc4 assists in identifying metastatic pancreatic carcinomas. Int J Gynecol Pathol. 2002;21:391-400.

43. Tot T. Cytokeratins 20 and 7 as biomarkers: usefulness in dis-criminating primary from metastatic adenocarcinoma. Eur J Cancer. 2002;38:758-763.

44. Rullier A, Le Bail B, Fawaz R, et al. Cytokeratin 7 and 20 ex-pression in cholangiocarcinomas varies along the biliary tract but still differs from that in colorectal carcinoma metastasis. Am J Surg Pathol. 2000;24:870-876.

45. Tot T. The value of cytokeratins 20 and 7 in discriminating met-astatic adenocarcinomas from pleural mesotheliomas. Cancer. 2001;92:2727-2732.

46. Moll R, Lowe A, Laufer J, Franke WW. Cytokeratin 20 in hu-man carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol. 1992;140:427-447.

47. Kaufmann O, Deidesheimer T, Muehlenberg M, et al. Immu-nohistochemical differentiation of metastatic breast carcinomas from metastatic adenocarcinomas of other common primary sites. Histopathology. 1996;29:233-240.

48. Miettinen M. Keratin 20: immunohistochemical marker for gastrointestinal, urothelial, and Merkel cell carcinomas. Mod Pathol. 1995;8:384-388.

49. Wauters CC, Smedts F, Gerrits LG, et al. Keratins 7 and 20 as diagnostic markers of carcinomas metastatic to the ovary. Hum Pathol. 1995;26:852-855.

50. Schussler MH, Skoudy A, Ramaekers F, Real FX. Intermedi-ate filaments as differentiation markers of normal pancreas and pancreas cancer. Am J Pathol. 1992;140:559-568.

51. Chen J, Baithun SI. Morphological study of 391 cases of exocrine pancreatic tumours with special reference to the classification of exocrine pancreatic carcinoma. J Pathol. 1985;146:17-29.

52. Cathro HP, Stoler MH. Expression of cytokeratins 7 and 20 in ovarian neoplasia. Am J Clin Pathol. 2002;117:944-951.

53. Chu PG, Weiss LM. Expression of cytokeratin 5/6 in epithelial neoplasms: an immunohistochemical study of 509 cases. Mod Pathol. 2002;15:6-10.

54. Di Como CJ, Urist MJ, Babayan I, et al. p63 expression pro-files in human normal and tumor tissues. Clin Cancer Res. 2002;8:494-501.

55. Adsay NV, Merati K, Andea A, et al. The dichotomy in the pre-invasive neoplasia to invasive carcinoma sequence in the pan-creas: differential expression of MUC1 and MUC2 supports the existence of two separate pathways of carcinogenesis. Mod Pathol. 2002;15:1087-1095.

56. Monges GM, Mathoulin-Portier MP, Acres RB, et al. Differen-tial MUC 1 expression in normal and neoplastic human pancre-atic tissue. An immunohistochemical study of 60 samples. Am J Clin Pathol. 1999;112:635-640.

57. Swartz MJ, Batra SK, Varshney GC, et al. MUC4 expression increases progressively in pancreatic intraepithelial neoplasia. Am J Clin Pathol. 2002;117:791-796.

58. Masaki Y, Oka M, Ogura Y, et al. Sialylated MUC1 mucin expression in normal pancreas, benign pancreatic lesions, and pancreatic ductal adenocarcinoma. Hepatogastroenterology. 1999;46:2240-2245.

59. Andrianifahanana M, Moniaux N, Schmied BM, et al. Mu-cin (MUC) gene expression in human pancreatic adenocarci-noma and chronic pancreatitis: a potential role of MUC4 as a tumor marker of diagnostic significance. Clin Cancer Res. 2001;7:4033-4040.

60. Kim GE, Bae HI, Park HU, et al. Aberrant expression of MU-C5AC and MUC6 gastric mucins and sialyl Tn antigen in in-traepithelial neoplasms of the pancreas. Gastroenterology. 2002;123:1052-1060.

61. Luttges J, Zamboni G, Longnecker D, Kloppel G. The immu-nohistochemical mucin expression pattern distinguishes differ-ent types of intraductal papillary mucinous neoplasms of the pancreas and determines their relationship to mucinous noncys-tic carcinoma and ductal adenocarcinoma. Am J Surg Pathol. 2001;25:942-948.

62. Swierczynski SL, Maitra A, Abraham SC, et al. Analysis of novel tumor markers in pancreatic and biliary carcinomas using tissue microarrays. Hum Pathol. 2004;35:357-366.

63. Terada T, Ohta T, Sasaki M, et al. Expression of MUC apo-mucins in normal pancreas and pancreatic tumours. J Pathol. 1996;180:160-165.

64. Yonezawa S, Sueyoshi K, Nomoto M, et al. MUC2 gene expres-sion is found in noninvasive tumors but not in invasive tumors of the pancreas and liver: its close relationship with prognosis of the patients. Hum Pathol. 1997;28:344-352.

65. Zhang H, Maitra A, Tabaczka P, et al. Differential MUC1, MUC2 and MUC5AC expression in colorectal, ampullary and pancreatobiliary carcinomas: Potential biologic and diagnostic implications (abstract). Mod Pathol. 2003;16:180A.

66. Werling RW, Yaziji H, Bacchi CE, Gown AM. CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am J Surg Pathol. 2003;27:303-310.

67. Moskaluk CA, Zhang H, Powell SM, et al. Cdx2 protein ex-pression in normal and malignant human tissues: an immuno-histochemical survey using tissue microarrays. Mod Pathol. 2003;16:913-919.

68. Barbareschi M, Murer B, Colby TV, et al. CDX-2 homeobox gene expression is a reliable marker of colorectal adenocarcino-ma metastases to the lungs. Am J Surg Pathol. 2003;27:141-149.

69. Li MK, Folpe AL. CDX-2, a new marker for adenocarcinoma of gastrointestinal origin. Adv Anat Pathol. 2004;11:101-105.

70. Morohoshi T, Kanda M, Horie A, et al. Immunocytochemi-cal markers of uncommon pancreatic tumors. Acinar cell car-cinoma, pancreatoblastoma, and solid cystic (papillary-cystic) tumor. Cancer. 1987;59:739-747.


71. Ichihara T, Nagura H, Nakao A, et al. Immunohistochemical localization of CA 19-9 and CEA in pancreatic carcinoma and associated diseases. Cancer. 1988;61:324-333.

72. Osako M, Yonezawa S, Siddiki B, et al. Immunohistochemical study of mucin carbohydrates and core proteins in human pan-creatic tumors. Cancer. 1993;71:2191-2199.

73. Sessa F, Bonato M, Frigerio B, et al. Ductal cancers of the pan-creas frequently express markers of gastrointestinal epithelial cells. Gastroenterology. 1990;98:1655-1665.

74. Takeda S, Nakao A, Ichihara T, et al. Serum concentration and immunohistochemical localization of SPan-1 antigen in pancre-atic cancer. A comparison with CA19-9 antigen. Hepatogastro-enterology. 1991;38:143-148.

75. Simeone DM, Ji B, Banerjee M, et al. CEACAM1, a novel serum biomarker for pancreatic cancer. Pancreas. 2007;34:436-443.

76. Hoorens A, Lemoine NR, McLellan E, et al. Pancreatic acinar cell carcinoma. An analysis of cell lineage markers, p53 expres-sion, and Ki-ras mutation. Am J Pathol. 1993;143:685-698.

77. Kamisawa T, Fukayama M, Tabata I, et al. Neuroendocrine dif-ferentiation in pancreatic duct carcinoma special emphasis on duct-endocrine cell carcinoma of the pancreas. Pathol Res Pract. 1996;192:901-908.

78. Pour PM, Permert J, Mogaki M, et al. Endocrine aspects of exo-crine cancer of the pancreas. Their patterns and suggested bio-logic significance. Am J Clin Pathol. 1993;100:223-230.

79. Bommer G, Friedl U, Heitz PU, Kloppel G. Pancreatic PP cell distribution and hyperplasia. Immunocytochemical morphol-ogy in the normal human pancreas, in chronic pancreatitis and pancreatic carcinoma. Virchows Arch A Pathol Anat Histol. 1980;387:319-331.

80. Ademmer K, Ebert M, Muller-Ostermeyer F, et al. Effector T lymphocyte subsets in human pancreatic cancer: detection of CD8+CD18+ cells and CD8+CD103+ cells by multi-epitope im-aging. Clin Exp Immunol. 1998;112:21-26.

81. Yen TW, Aardal NP, Bronner MP, et al. Myofibroblasts are responsible for the desmoplastic reaction surrounding human pancreatic carcinomas. Surgery. 2002;131:129-134.

82. Koshiba T, Hosotani R, Wada M, et al. Involvement of matrix metalloproteinase-2 activity in invasion and metastasis of pan-creatic carcinoma. Cancer. 1998;82:642-650.

83. Maitra A, Iacobuzio-Donahue C, Rahman A, et al. Immuno-histochemical validation of a novel epithelial and a novel stro-mal marker of pancreatic ductal adenocarcinoma identified by global expression microarrays: sea urchin fascin homolog and heat shock protein 47. Am J Clin Pathol. 2002;118:52-59.

84. Yamamoto H, Itoh F, Iku S, et al. Expression of matrix me-talloproteinases and tissue inhibitors of metalloproteinases in human pancreatic adenocarcinomas: clinicopathologic and prognostic significance of matrilysin expression. J Clin Oncol. 2001;19:1118-1127.

85. Zhou W, Sokoll LJ, Bruzek DJ, et al. Identifying markers for pancreatic cancer by gene expression analysis. Cancer Epide-miol Biomarkers Prev. 1998;7:109-112.

86. Furukawa T, Horii A. Molecular pathology of pancreatic can-cer: in quest of tumor suppressor genes. Pancreas. 2004;28:253-256.

87. Hruban RH, Iacobuzio-Donahue C, Wilentz RE, et al. Molecu-lar pathology of pancreatic cancer. Cancer J. 2001;7:251-258.

88. Rozenblum E, Schutte M, Goggins M, et al. Tumor-suppressive pathways in pancreatic carcinoma. Cancer Res. 1997;57:1731-1734.

89. Moore PS, Sipos B, Orlandini S, et al. Genetic profile of 22 pan-creatic carcinoma cell lines. Analysis of K-ras, p53, p16 and DPC4/Smad4. Virchows Arch. 2001;439:798-802.

90. Moore PS, Orlandini S, Zamboni G, et al. Pancreatic tumours: molecular pathways implicated in ductal cancer are involved in ampullary but not in exocrine nonductal or endocrine tumori-genesis. Br J Cancer. 2001;84:253-262.

91. DiGiuseppe JA, Hruban RH, Goodman SN, et al. Overexpres-sion of p53 protein in adenocarcinoma of the pancreas. Am J Clin Pathol. 1994;101:684-688.

92. van Heek T, Rader AE, Offerhaus GJ, et al. K-ras, p53, and DPC4 (MAD4) alterations in fine-needle aspirates of the pancre-as: a molecular panel correlates with and supplements cytologic diagnosis. Am J Clin Pathol. 2002;117:755-765.

93. Wilentz RE, Geradts J, Maynard R, et al. Inactivation of the p16 (INK4A) tumor-suppressor gene in pancreatic duct lesions: loss of intranuclear expression. Cancer Res. 1998;58:4740-4744.

94. Boschman CR, Stryker S, Reddy JK, Rao MS. Expression of p53 protein in precursor lesions and adenocarcinoma of human pan-creas. Am J Pathol. 1994;145:1291-1295.

95. Li Y, Bhuiyan M, Vaitkevicius VK, Sarkar FH. Molecular analy-sis of the p53 gene in pancreatic adenocarcinoma. Diagn Mol Pathol. 1998;7:4-9.

96. Redston MS, Caldas C, Seymour AB, et al. p53 mutations in pancreatic carcinoma and evidence of common involvement of homocopolymer tracts in DNA microdeletions. Cancer Res. 1994;54:3025-3033.

97. Schutte M. DPC4/SMAD4 gene alterations in human cancer, and their functional implications. Ann Oncol. 1999;10(Suppl 4):56-59.

98. Hahn SA, Schutte M, Hoque AT, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science. 1996;271:350-353.

99. Wilentz RE, Su GH, Dai JL, et al. Immunohistochemical labeling for dpc4 mirrors genetic status in pancreatic adenocarcinomas: a new marker of DPC4 inactivation. Am J Pathol. 2000;156:37-43.

100. Iacobuzio-Donahue CA, Song J, Parmiagiani G, et al. Missense mutations of MADH4: characterization of the mutational hot spot and functional consequences in human tumors. Clin Can-cer Res. 2004;10:1597-1604.

101. Tascilar M, Offerhaus GJ, Altink R, et al. Immunohistochemi-cal labeling for the Dpc4 gene product is a specific marker for adenocarcinoma in biopsy specimens of the pancreas and bile duct. Am J Clin Pathol. 2001;116:831-837.

102. Ali S, Cohen C, Little JV, et al. The utility of SMAD4 as a diagnostic immunohistochemical marker for pancreatic adenocarcinoma, and its expression in other solid tumors. Diagn Cytopathol. 2007;35:644-648.

103. Bartsch D, Shevlin DW, Callery MP, et al. Reduced survival in patients with ductal pancreatic adenocarcinoma associated with CDKN2 mutation. J Natl Cancer Inst. 1996;88:680-682.

104. Schutte M, Hruban RH, Geradts J, et al. Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carci-nomas. Cancer Res. 1997;57:3126-3130.

105. Caldas C, Hahn SA, da Costa LT, et al. Frequent somatic mu-tations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nat Genet. 1994;8:27-32.

106. Geradts J, Hruban RH, Schutte M, et al. Immunohistochemi-cal p16INK4a analysis of archival tumors with deletion, hypermethylation, or mutation of the CDKN2/MTS1 gene. A comparison of four commercial antibodies. Appl Immunohis-tochem Mol Morphol. 2000;8:71-79.

107. Almoguera C, Shibata D, Forrester K, et al. Most human carci-nomas of the exocrine pancreas contain mutant c-K-ras genes. Cell. 1988;53:549-554.

108. Satoh K, Sasano H, Shimosegawa T, et al. An immunohisto-chemical study of the c-erbB-2 oncogene product in intraductal mucin-hypersecreting neoplasms and in ductal cell carcinomas of the pancreas. Cancer. 1993;72:51-56.

109. Day JD, Digiuseppe JA, Yeo C, et al. Immunohistochemical evaluation of HER-2/neu expression in pancreatic adenocarci-noma and pancreatic intraepithelial neoplasms. Hum Pathol. 1996;27:119-124.

110. Yamanaka Y, Friess H, Kobrin MS, et al. Overexpression of HER2/neu oncogene in human pancreatic carcinoma. Hum Pathol. 1993;24:1127-1134.

111. Dancer J, Takei H, Ro JY, Lowery-Nordberg M. Coexpression of EGFR and HER-2 in pancreatic ductal adenocarcinoma: a comparative study using immunohistochemistry correlated with gene amplification by fluorescencent in situ hybridization. Oncol Rep. 2007;18:151-155.

112. Cheng JQ, Ruggeri B, Klein WM, et al. Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proc Natl Acad Sci U S A. 1996;93:3636-3641.

113. Ruggeri BA, Huang L, Wood M, et al. Amplification and over-expression of the AKT2 oncogene in a subset of human pancre-atic ductal adenocarcinomas. Mol Carcinog. 1998;21:81-86.

579REFERENCES

114. Iacobuzio-Donahue CA, Maitra A, Shen-Ong GL, et al. Discov-ery of novel tumor markers of pancreatic cancer using global gene expression technology. Am J Pathol. 2002;160:1239-1249.

115. Iacobuzio-Donahue CA, Ryu B, Hruban RH, Kern SE. Explor-ing the host desmoplastic response to pancreatic carcinoma: gene expression of stromal and neoplastic cells at the site of pri-mary invasion. Am J Pathol. 2002;160:91-99.

116. Ryu B, Jones J, Hollingsworth MA, et al. Invasion-specific genes in malignancy: serial analysis of gene expression comparisons of primary and passaged cancers. Cancer Res. 2001;61:1833-1838.

117. Ryu B, Jones J, Blades NJ, et al. Relationships and differen-tially expressed genes among pancreatic cancers examined by large-scale serial analysis of gene expression. Cancer Res. 2002;62:819-826.

118. Argani P, Iacobuzio-Donahue C, Ryu B, et al. Mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (SAGE). Clin Cancer Res. 2001;7:3862-3868.

119. Armengol G, Knuutila S, Lluis F, et al. DNA copy number changes and evaluation of MYC, IGF1R, and FES amplifica-tion in xenografts of pancreatic adenocarcinoma. Cancer Genet Cytogenet. 2000;116:133-141.

120. Nichols LS, Ashfaq R, Iacobuzio-Donahue CA. Claudin 4 protein expression in primary and metastatic pancreatic can-cer: support for use as a therapeutic target. Am J Clin Pathol. 2004;121:226-230.

121. Crnogorac-Jurcevic T, Missiaglia E, Blaveri E, et al. Molecular alterations in pancreatic carcinoma: expression profiling shows that dysregulated expression of S100 genes is highly prevalent. J Pathol. 2003;201:63-74.

122. Rosty C, Ueki T, Argani P, et al. Overexpression of S100A4 in pancreatic ductal adenocarcinomas is associated with poor differentiation and DNA hypomethylation. Am J Pathol. 2002;160:45-50.

123. Vimalachandran D, Greenhalf W, Thompson C, et al. High nuclear S100A6 (Calcyclin) is significantly associated with poor survival in pancreatic cancer patients. Cancer Res. 2005;65:3218-3225.

124. Argani P, Rosty C, Reiter RE, et al. Discovery of new markers of cancer through serial analysis of gene expression: prostate stem cell antigen is overexpressed in pancreatic adenocarcinoma. Cancer Res. 2001;61:4320-4324.

125. Ligato S, Zhao H, Mandich D, Cartun RW. KOC (K homol-ogy domain containing protein overexpressed in cancer) and S100A4-protein immunoreactivity improves the diagnostic sen-sitivity of biliary brushing cytology for diagnosing pancreatico-biliary malignancies. Diagn Cytopathol. 2008;36:561-567.

126. Iacobuzio-Donahue CA, Hruban RH. Gene expression in neo-plasms of the pancreas: applications to diagnostic pathology. Adv Anat Pathol. 2003;10:125-134.

127. Porschen R, Remy U, Bevers G, et al. Prognostic significance of DNA ploidy in adenocarcinoma of the pancreas. A flow cytometric study of paraffin-embedded specimens. Cancer. 1993;71:3846-3850.

128. Yoshimura T, Manabe T, Imamura T, et al. Flow cytometric analysis of nuclear DNA content of duct cell carcinoma of the pancreas. Cancer. 1992;70:1069-1074.

129. Allison DC, Piantadosi S, Hruban RH, et al. DNA content and other factors associated with ten-year survival after resection of pancreatic carcinoma. J Surg Oncol. 1998;67:151-159.

130. Borg A, Sandberg T, Nilsson K, et al. High frequency of mul-tiple melanomas and breast and pancreas carcinomas in CDK-N2A mutation-positive melanoma families. J Natl Cancer Inst. 2000;92:1260-1266.

131. Griffin CA, Hruban RH, Long PP, et al. Chromosome abnor-malities in pancreatic adenocarcinoma. Genes Chromosomes Cancer. 1994;9:93-100.

132. Griffin CA, Hruban RH, Morsberger LA, et al. Consistent chro-mosome abnormalities in adenocarcinoma of the pancreas. Can-cer Res. 1995;55:2394-2399.

133. Johansson B, Bardi G, Pandis N, et al. Karyotypic pattern of pancreatic adenocarcinomas correlates with survival and tu-mour grade. Int J Cancer. 1994;58:8-13.

134. Coppola D, Lu L, Fruehauf JP, et al. Analysis of p53, p21WAF1, and TGF-beta1 in human ductal adenocarcinoma of the pancre-as: TGF-beta1 protein expression predicts longer survival. Am J Clin Pathol. 1998;110:16-23.

135. van der Heijden MS, Yeo CJ, Hruban RH, Kern SE. Fanconi anemia gene mutations in young-onset pancreatic cancer. Can-cer Res. 2003;63:2585-2588.

136. Su GH, Hruban RH, Bansal RK, et al. Germline and somatic mutations of the STK11/LKB1 Peutz-Jeghers gene in pancreatic and biliary cancers. Am J Pathol. 1999;154:1835-1840.

137. Su GH, Bansal R, Murphy KM, et al. ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma. Proc Natl Acad Sci U S A. 2001;98:3254-3257.

138. Su GH, Hilgers W, Shekher MC, et al. Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically tar-geted tumor suppressor gene. Cancer Res. 1998;58:2339-2342.

139. Goggins M, Shekher M, Turnacioglu K, et al. Genetic alterations of the transforming growth factor beta receptor genes in pancre-atic and biliary adenocarcinomas. Cancer Res. 1998;58:5329-5332.

140. Shi C, Daniels JA, Hruban RH. Molecular characterization of pancreatic neoplasms. Adv Anat Pathol. 2008;15:185-195.

141. Hruban RH, Adsay NV. Molecular Classification of Neoplasms of the Pancreas (in press) Human Pathology. 2009.

142. Hruban RH, van Mansfeld AD, Offerhaus GJ, et al. K-ras on-cogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonu-cleotide hybridization. Am J Pathol. 1993;143:545-554.

143. Luttges J, Diederichs A, Menke MA, et al. Ductal lesions in pa-tients with chronic pancreatitis show K-ras mutations in a fre-quency similar to that in the normal pancreas and lack nuclear immunoreactivity for p53. Cancer. 2000;88:2495-2504.

144. Terhune PG, Phifer DM, Tosteson TD, Longnecker DS. K-ras mutation in focal proliferative lesions of human pancreas. Can-cer Epidemiol Biomarkers Prev. 1998;7:515-521.

145. Moskaluk CA, Hruban RH, Kern SE. p16 and K-ras gene muta-tions in the intraductal precursors of human pancreatic adeno-carcinoma. Cancer Res. 1997;57:2140-2143.

146. Aguirre AJ, Brennan C, Bailey G, et al. High-resolution charac-terization of the pancreatic adenocarcinoma genome. Proc Natl Acad Sci U S A. 2004;101:9067-9072.

147. Calhoun ES, Jones JB, Ashfaq R, et al. BRAF and FBXW7 (CDC4, FBW7, AGO, SEL10) mutations in distinct subsets of pancreatic cancer: potential therapeutic targets. Am J Pathol. 2003;163:1255-1260.

148. Miwa W, Yasuda J, Murakami Y, et al. Isolation of DNA se-quences amplified at chromosome 19q13.1-q13.2 including the AKT2 locus in human pancreatic cancer. Biochem Biophys Res Commun. 1996;225:968-974.

149. Wallrapp C, Muller-Pillasch F, Solinas-Toldo S, et al. Charac-terization of a high copy number amplification at 6q24 in pan-creatic cancer identifies c-myb as a candidate oncogene. Cancer Res. 1997;57:3135-3139.

150. Goggins M, Offerhaus GJ, Hilgers W, et al. Pancreatic adeno-carcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+. Am J Pathol. 1998;152:1501-1507.

151. Montgomery E, Wilentz RE, Argani P, et al. Analysis of ana-phase figures in routine histologic sections distinguishes chro-mosomally unstable from chromosomally stable malignancies. Cancer Biol Ther. 2003;2:248-252.

152. Wilentz RE, Goggins M, Redston M, et al. Genetic, immuno-histochemical, and clinical features of medullary carcinoma of the pancreas: a newly described and characterized entity. Am J Pathol. 2000;156:1641-1651.

153. Yamamoto H, Itoh F, Nakamura H, et al. Genetic and clini-cal features of human pancreatic ductal adenocarcinomas with widespread microsatellite instability. Cancer Res. 2001;61:3139-3144.

154. Ali-Fehmi R, Basturk O, Cheng JD, et al. Immunohistochemis-try as a helpful adjunct in the differential diagnosis of intraab-dominal carcinomatosis originating from the pancreas versus ovary. Mod Pathol. 2005;18:273A.


155. Alguacil-Garcia A, Weiland LH. The histologic spectrum, prog-nosis, and histogenesis of the sarcomatoid carcinoma of the pan-creas. Cancer. 1977;39:1181-1189.

156. Paal E, Thompson LD, Frommelt RA, et al. A clinicopathologic and immunohistochemical study of 35 anaplastic carcinomas of the pancreas with a review of the literature. Ann Diagn Pathol. 2001;5:129-140.

157. Motoo Y, Kawashima A, Watanabe H, et al. Undifferenti-ated (anaplastic) carcinoma of the pancreas showing sarcoma-tous change and neoplastic cyst formation. Int J Pancreatol. 1997;21:243-248.

158. Goldstein NS, Bosler DS. Immunohistochemistry of the Gastro-intestinal Tract, Pancreas, Bile Ducts, Gall Bladder and Liver. In: Dabbs D, ed. Diagnostic Immunohistchemistry. 2nd ed. Philadephia: Elsevier; 2006:442-508.

159. Nishihara K, Katsumoto F, Kurokawa Y, et al. Anaplastic car-cinoma showing rhabdoid features combined with mucinous cystadenocarcinoma of the pancreas. Arch Pathol Lab Med. 1997;121:1104-1107.

160. Gatteschi B, Saccomanno S, Bartoli FG, et al. Mixed pleomorphic-osteoclast-like tumor of the pancreas. Light microscopical, immunohistochemical, and molecular biological studies. Int J Pancreatol. 1995;18:169-175.

161. Deckard-Janatpour K, Kragel S, Teplitz RL, et al. Tumors of the pancreas with osteoclast-like and pleomorphic giant cells: an immunohistochemical and ploidy study. Arch Pathol Lab Med. 1998;122:266-272.

162. Klimstra DS, Adsay NV. Benign and malignant tumors of the pancreas. In: Odze RD, Goldblum JR, Crawford JM, eds. Surgi-cal pathology of the GI tract, liver, biliary tract and pancreas. Philadelphia: Saunders; 2004:699-731.

163. Winter JM, Ting AH, Vilardell F, et al. Absence of E-cadherin expression distinguishes noncohesive from cohesive pancreatic cancer. Clin Cancer Res. 2008;14:412-418.

164. Dhall D, Klimstra DS. The cellular composition of osteoclastlike giant cell-containing tumors of the pancreatobiliary tree. Am J Surg Pathol. 2008;32:335-337; author response 337.

165. Berendt RC, Shnitka TK, Wiens E, et al. The osteoclast-type giant cell tumor of the pancreas. Arch Pathol Lab Med. 1987;111:43-48.

166. Dizon MA, Multhaupt HA, Paskin DL, Warhol MJ. Osteoclas-tic giant cell tumor of the pancreas: an immunohistochemical study. Arch Pathol Lab Med. 1996;120:306-309.

167. Lukas Z, Dvorak K, Kroupova I, et al. Immunohistochemical and genetic analysis of osteoclastic giant cell tumor of the pan-creas. Pancreas. 2006;32:325-329.

168. Molberg KH, Heffess C, Delgado R, et al. Undifferentiated car-cinoma with osteoclast-like giant cells of the pancreas and peri-ampullary region. Cancer. 1998;82:1279-1287.

169. Sakai Y, Kupelioglu AA, Yanagisawa A, et al. Origin of giant cells in osteoclast-like giant cell tumors of the pancreas. Hum Pathol. 2000;31:1223-1229.

170. Westra WH, Sturm P, Drillenburg P, et al. K-ras oncogene mu-tations in osteoclast-like giant cell tumors of the pancreas and liver: genetic evidence to support origin from the duct epithe-lium. Am J Surg Pathol. 1998;22:1247-1254.

171. Shiozawa M, Imada T, Ishiwa N, et al. Osteoclast-like giant cell tumor of the pancreas. Int J Clin Oncol. 2002;7:376-380.

172. Gocke CD, Dabbs DJ, Benko FA, Silverman JF. KRAS onco-gene mutations suggest a common histogenetic origin for pleo-morphic giant cell tumor of the pancreas, osteoclastoma of the pancreas, and pancreatic duct adenocarcinoma. Hum Pathol. 1997;28:80-83.

173. Imai Y, Morishita S, Ikeda Y, et al. Immunohistochemical and molecular analysis of giant cell carcinoma of the pancreas: a report of three cases. Pancreas. 1999;18:308-315.

174. Ueki T, Toyota M, Sohn T, et al. Hypermethylation of mul-tiple genes in pancreatic adenocarcinoma. Cancer Res. 2000;60:1835-1839.

175. Kardon DE, Thompson LD, Przygodzki RM, Heffess CS. Ad-enosquamous carcinoma of the pancreas: a clinicopathologic series of 25 cases. Mod Pathol. 2001;14:443-451.

176. Ylagan LR, Scholes J, Demopoulos R. Cd44: a marker of squa-mous differentiation in adenosquamous neoplasms. Arch Pathol Lab Med. 2000;124:212-215.

177. Adsay NV, Hasteh F, Sarkar F, et al. Squamous cell and ad-enosquamous carcinomas of the pancreas: a clinicopathologic analysis of 11 cases. Mod Pathol. 2000;13:179a.

178. Basturk O, Khanani F, Sarkar F, et al. DeltaNp63 expression in pancreas and pancreatic neoplasia. Mod Pathol. 2005;18:1193-1198.

179. Witkiewicz AK, Brody JB, Constantino CL, et al. Adenosqua-mous carcinoma of the pancreas harbors KRAS2, DPC4 and TP53 molecular alterations similar to pancreatic ductal adeno-carcinoma. Mod Pathol. 2009;22:325A.

180. Madura JA, Jarman BT, Doherty MG, et al. Adenosquamous carcinoma of the pancreas. Arch Surg. 1999;134:599-603.

181. Adsay NV, Merati K, Nassar H, et al. Pathogenesis of colloid (pure mucinous) carcinoma of exocrine organs: Coupling of gel-forming mucin (MUC2) production with altered cell polarity and abnormal cell-stroma interaction may be the key factor in the morphogenesis and indolent behavior of colloid carci-noma in the breast and pancreas. Am J Surg Pathol. 2003;27:571-578.

182. Adsay NV, Pierson C, Sarkar F, et al. Colloid (mucinous non-cystic) carcinoma of the pancreas. Am J Surg Pathol. 2001;25:26-42.

183. Iacobuzio-Donahue CA, Klimstra DS, Adsay NV, et al. Dpc-4 protein is expressed in virtually all human intraductal papillary mucinous neoplasms of the pancreas: comparison with conven-tional ductal adenocarcinomas. Am J Pathol. 2000;157:755-761.

184. Hruban RH, Takaori K, Klimstra DS, et al. An illustrated con-sensus on the classification of pancreatic intraepithelial neopla-sia and intraductal papillary mucinous neoplasms. Am J Surg Pathol. 2004;28:977-987.

185. Maitra A, Adsay NV, Argani P, et al. Multicomponent analy-sis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray. Mod Pathol. 2003;16:902-912.

186. Klein WM, Hruban RH, Klein-Szanto AJ, Wilentz RE. Direct correlation between proliferative activity and dysplasia in pan-creatic intraepithelial neoplasia (PanIN): additional evidence for a recently proposed model of progression. Mod Pathol. 2002;15:441-447.

187. Adsay NV, Basturk O, Cheng JD, Andea AA. Ductal neoplasia of the pancreas: nosologic, clinicopathologic, and biologic as-pects. Semin Radiat Oncol. 2005;15:254-264.

188. Prasad NB, Biankin AV, Fukushima N, et al. Gene expression profiles in pancreatic intraepithelial neoplasia reflect the effects of Hedgehog signaling on pancreatic ductal epithelial cells. Can-cer Res. 2005;65:1619-1626.

189. Basturk O, Coban I, Adsay NV. Cystic Neoplasms of the Pan-creas. Arch Pathol Lab Med. 2009; in press.

190. Mukawa K, Kawa S, Aoki Y, et al. Reduced expression of p53 and cyclin A in intraductal mucin-hypersecreting neoplasm of the pancreas compared with usual pancreatic ductal adenocarci-noma. Am J Gastroenterol. 1999;94:2263-2267.

191. Terada T, Ohta T, Kitamura Y, et al. Cell proliferative activity in intraductal papillary-mucinous neoplasms and invasive duc-tal adenocarcinomas of the pancreas: an immunohistochemical study. Arch Pathol Lab Med. 1998;122:42-46.

192. Tomaszewska R, Okon K, Nowak K, Stachura J. HER-2/Neu expression as a progression marker in pancreatic intraepithelial neoplasia. Pol J Pathol. 1998;49:83-92.

193. Nakamura A, Horinouchi M, Goto M, et al. New classification of pancreatic intraductal papillary-mucinous tumour by mu-cin expression: its relationship with potential for malignancy. J Pathol. 2002;197:201-210.

194. Terris B, Dubois S, Buisine MP, et al. Mucin gene expression in intraductal papillary-mucinous pancreatic tumours and related lesions. J Pathol. 2002;197:632-637.

195. Fukushima N, Sato N, Sahin F, et al. Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms. Br J Cancer. 2003;89:338-343.

196. Adsay NV, Merati K, Basturk O, et al. Pathologically and bi-ologically distinct types of epithelium in intraductal papillary mucinous neoplasms: delineation of an “intestinal” pathway of carcinogenesis in the pancreas. Am J Surg Pathol. 2004;28:839-848.

581REFERENCES

197. Paal E, Thompson LD, Przygodzki RM, et al. A clinicopatho-logic and immunohistochemical study of 22 intraductal papil-lary mucinous neoplasms of the pancreas, with a review of the literature. Mod Pathol. 1999;12:518-528.

198. Terada T, Ohta T, Nakanuma Y. Expression of oncogene products, anti-oncogene products and oncofetal antigens in intraductal papillary-mucinous neoplasm of the pancreas. His-topathology. 1996;29:355-361.

199. Nagai E, Ueki T, Chijiiwa K, et al. Intraductal papillary muci-nous neoplasms of the pancreas associated with so-called “mu-cinous ductal ectasia.” Histochemical and immunohistochemi-cal analysis of 29 cases. Am J Surg Pathol. 1995;19:576-589.

200. Nagasaka T, Nakashima N. Problems in histological diagnosis of intraductal papillary-mucinous tumor (IPMT). Hepatogas-troenterology. 2001;48:972-976.

201. Adsay NV, Pierson C, Sarkar F, et al. Comparative Immuno-histochemical Analysis of Oncoprotein Expression in Various Components of IPMNs. Pancreatology. 2000;28:103A.

202. Tobi M, Hatfield J, Adsay V, et al. Prognostic Significance of the Labeling of Adnab-9 in Pancreatic Intraductal Papillary Mucinous Neoplasms. Int J Gastrointest Cancer. 2001;29:141-150.

203. Terada T, Ohta T, Kitamura Y, et al. Endocrine cells in intra-ductal papillary-mucinous neoplasms of the pancreas. A his-tochemical and immunohistochemical study. Virchows Arch. 1997;431:31-36.

204. Niijima M, Yamaguchi T, Ishihara T, et al. Immunohisto-chemical analysis and in situ hybridization of cyclooxygenase-2 expression in intraductal papillary-mucinous tumors of the pan-creas. Cancer. 2002;94:1565-1573.

205. Patel SA, Adams R, Goldstein M, Moskaluk CA. Genetic analysis of invasive carcinoma arising in intraductal onco-cytic papillary neoplasm of the pancreas. Am J Surg Pathol. 2002;26:1071-1077.

206. Fujii H, Inagaki M, Kasai S, et al. Genetic progression and het-erogeneity in intraductal papillary-mucinous neoplasms of the pancreas. Am J Pathol. 1997;151:1447-1454.

207. Hoshi T, Imai M, Ogawa K. Frequent K-ras mutations and ab-sence of p53 mutations in mucin-producing tumors of the pan-creas. J Surg Oncol. 1994;55:84-91.

208. Izawa T, Obara T, Tanno S, et al. Clonality and field canceriza-tion in intraductal papillary-mucinous tumors of the pancreas. Cancer. 2001;92:1807-1817.

209. Nakata B, Yashiro M, Nishioka N, et al. Very low incidence of microsatellite instability in intraductal papillary-mucinous neo-plasm of the pancreas. Int J Cancer. 2002;102:655-659.

210. Satoh K, Sawai T, Shimosegawa T, et al. The point mutation of c-Ki-ras at codon 12 in carcinoma of the pancreatic head region and in intraductal mucin-hypersecreting neoplasm of the pan-creas. Int J Pancreatol. 1993;14:135-143.

211. Satoh K, Shimosegawa T, Moriizumi S, et al. K-ras mutation and p53 protein accumulation in intraductal mucin-hypersecret-ing neoplasms of the pancreas. Pancreas. 1996;12:362-368.

212. Sessa F, Solcia E, Capella C, et al. Intraductal papillary- mucinous tumours represent a distinct group of pancreatic neoplasms: an investigation of tumour cell differentiation and K-ras, p53 and c-erbB-2 abnormalities in 26 patients. Virchows Arch. 1994;425:357-367.

213. Tada M, Omata M, Ohto M. Ras gene mutations in intraductal papillary neoplasms of the pancreas. Analysis in five cases. Can-cer. 1991;67:634-637.

214. Yamaguchi K, Chijiiwa K, Noshiro H, et al. Ki-ras codon 12 point mutation and p53 mutation in pancreatic diseases. Hepa-togastroenterology. 1999;46:2575-2581.

215. Yanagisawa A, Kato Y, Ohtake K, et al. c-Ki-ras point muta-tions in ductectatic-type mucinous cystic neoplasms of the pan-creas. Jpn J Cancer Res. 1991;82:1057-1060.

216. Yoshizawa K, Nagai H, Sakurai S, et al. Clonality and K-ras mutation analyses of epithelia in intraductal papillary mucinous tumor and mucinous cystic tumor of the pancreas. Virchows Arch. 2002;441:437-443.

217. Biankin AV, Biankin SA, Kench JG, et al. Aberrant p16(INK4A) and DPC4/Smad4 expression in intraductal papillary mucinous tumours of the pancreas is associated with invasive ductal ad-enocarcinoma. Gut. 2002;50:861-868.

218. Flejou JF, Boulange B, Bernades P, et al. p53 protein expres-sion and DNA ploidy in cystic tumors of the pancreas. Pancreas. 1996;13:247-252.

219. Islam HK, Fujioka Y, Tomidokoro T, et al. Immunohistochemi-cal analysis of expression of molecular biologic factors in intra-ductal papillary-mucinous tumors of pancreas—diagnostic and biologic significance. Hepatogastroenterology. 1999;46:2599-2605.

220. Islam HK, Fujioka Y, Tomidokoro T, et al. Immunohistochemi-cal study of genetic alterations in intraductal and invasive ductal tumors of the pancreas. Hepatogastroenterology. 2001;48:879-883.

221. Kawahira H, Kobayashi S, Kaneko K, et al. p53 protein expres-sion in intraductal papillary mucinous tumors (IPMT) of the pancreas as an indicator of tumor malignancy. Hepatogastroen-terology. 2000;47:973-977.

222. Kitagawa Y, Unger TA, Taylor S, et al. Mucus is a predictor of better prognosis and survival in patients with intraductal papillary mucinous tumor of the pancreas. J Gastrointest Surg. 2003;7:12-18; discussion 18-19.

223. Sasaki S, Yamamoto H, Kaneto H, et al. Differential roles of alterations of p53, p16, and SMAD4 expression in the progres-sion of intraductal papillary-mucinous tumors of the pancreas. Oncol Rep. 2003;10:21-25.

224. Hong SP, Lee EK, Park JY, et al. Cripto-1 overexpression is in-volved in the tumorigenesis of gastric-type and pancreatobiliary-type intraductal papillary mucinous neoplasms of the pancreas. Oncol Rep. 2009;21:19-24.

225. Adsay NV, Adair CF, Heffess CS, Klimstra DS. Intraductal on-cocytic papillary neoplasms of the pancreas. Am J Surg Pathol. 1996;20:980-994.

226. Chung SM, Hruban RH, Iacobuzio-Donahue CA, et al. Analy-sis of molecular alterations and differentiation pathways in in-traductal oncocytic papillary neoplasm of the pancreas. Mod Pathol. 2005;18:277A.

227. Kloppel G, Luttges J. WHO-classification 2000: exocrine pan-creatic tumors. Verh Dtsch Ges Pathol. 2001;85:219-228.

228. Kloppel G, Kosmahl M. Cystic lesions and neoplasms of the pancreas. The features are becoming clearer. Pancreatology. 2001;1:648-655.

229. Fukushima N, Mukai K. ‘Ovarian-type’ stroma of pancreatic mucinous cystic tumor expresses smooth muscle phenotype. Pathol Int. 1997;47:806-808.

230. Sarr MG, Carpenter HA, Prabhakar LP, et al. Clinical and pathologic correlation of 84 mucinous cystic neoplasms of the pancreas: can one reliably differentiate benign from malignant (or premalignant) neoplasms? Ann Surg. 2000;231:205-212.

231. Yamaguchi K, Enjoji M. Cystic neoplasms of the pancreas. Gas-troenterology. 1987;92:1934-1943.

232. Thompson LD, Becker RC, Przygodzki RM, et al. Mucinous cystic neoplasm (mucinous cystadenocarcinoma of low-grade malignant potential) of the pancreas: a clinicopathologic study of 130 cases. Am J Surg Pathol. 1999;23:1-16.

233. Luttges J, Feyerabend B, Buchelt T, et al. The mucin profile of noninvasive and invasive mucinous cystic neoplasms of the pan-creas. Am J Surg Pathol. 2002;26:466-471.

234. Ohta T, Nagakawa T, Fukushima W, et al. Immunohistochemi-cal study of carcinoembryonic antigen in mucinous cystic neo-plasm of the pancreas. Eur Surg Res. 1992;24:37-44.

235. van den Berg W, Tascilar M, Offerhaus GJ, et al. Pancreatic mucinous cystic neoplasms with sarcomatous stroma: molecu-lar evidence for monoclonal origin with subsequent divergence of the epithelial and sarcomatous components. Mod Pathol. 2000;13:86-91.

236. Yu HC, Shetty J. Mucinous cystic neoplasm of the pancreas with high carcinoembryonic antigen. Arch Pathol Lab Med. 1985;109:375-377.

237. Khalifeh I, Basturk O, Zamboni G, et al. Villous-intestinal dif-ferentiation and progression to colloid carcinoma, characteristic of a major subset of IPMNs, are not features of mucinous cystic neoplasms. Mod Pathol. 2005;18:281A.

238. Albores-Saavedra J, Nadji M, Henson DE, et al. Entero-endo-crine cell differentiation in carcinomas of the gallbladder and mucinous cystadenocarcinomas of the pancreas. Pathol Res Pract. 1988;183:169-175.


239. Albores-Saavedra J, Angeles-Angeles A, Nadji M, et al. Mucinous cystadenocarcinoma of the pancreas. Morphologic and immunocytochemical observations. Am J Surg Pathol. 1987;11:11-20.

240. Khalifeh I, Qureshi F, Jacques S, et al. The nature of “ovarian-like” mesenchyme of pancreatic and hepatic mucinous cystic neoplasms: A recapitulation of the periductal fetal mesenchme?. Mod Pathol. 2004;17:304A.

241. Wilentz RE, Albores-Saavedra J, Hruban RH. Mucinous cystic neoplasms of the pancreas. Semin Diagn Pathol. 2000;17:31-42.

242. Zamboni G, Scarpa A, Bogina G, et al. Mucinous cystic tumors of the pancreas: clinicopathological features, prognosis, and re-lationship to other mucinous cystic tumors. Am J Surg Pathol. 1999;23:410-422.

243. Izumo A, Yamaguchi K, Eguchi T, et al. Mucinous cystic tumor of the pancreas: immunohistochemical assessment of “ovarian-type stroma. Oncol Rep. 2003;10:515-525.

244. Weihing RR, Shintaku IP, Geller SA, Petrovic LM. Hepatobi-liary and pancreatic mucinous cystadenocarcinomas with mes-enchymal stroma: analysis of estrogen receptors/progesterone receptors and expression of tumor-associated antigens. Mod Pathol. 1997;10:372-379.

245. Ridder GJ, Maschek H, Klempnauer J. Favourable prognosis of cystadeno- over adenocarcinoma of the pancreas after curative resection. Eur J Surg Oncol. 1996;22:232-236.

246. Zheng W, Sung CJ, Hanna I, et al. Alpha and beta subunits of inhibin/activin as sex cord-stromal differentiation markers. Int J Gynecol Pathol. 1997;16:263-271.

247. Bartsch D, Bastian D, Barth P, et al. K-ras oncogene mutations indicate malignancy in cystic tumors of the pancreas. Ann Surg. 1998;228:79-86.

248. Jimenez RE, Warshaw AL, Z’Graggen K, et al. Sequential ac-cumulation of K-ras mutations and p53 overexpression in the progression of pancreatic mucinous cystic neoplasms to malig-nancy. Ann Surg. 1999;230:501-509; discussion 509-511.

249. Kim SG, Wu TT, Lee JH, et al. Comparison of epigenetic and genetic alterations in mucinous cystic neoplasm and serous mi-crocystic adenoma of pancreas. Mod Pathol. 2003;16:1086-1094.

250. Iacobuzio-Donahue CA, Wilentz RE, Argani P, et al. Dpc4 pro-tein in mucinous cystic neoplasms of the pancreas: frequent loss of expression in invasive carcinomas suggests a role in genetic progression. Am J Surg Pathol. 2000;24:1544-1548.

251. Tajiri T, Tate G, Inagaki T, et al. Intraductal tubular neoplasms of the pancreas: histogenesis and differentiation. Pancreas. 2005;30:115-121.

252. Tajiri T, Tate G, Kunimura T, et al. Histologic and immuno-histochemical comparison of intraductal tubular carcinoma, intraductal papillary-mucinous carcinoma, and ductal adeno-carcinoma of the pancreas. Pancreas. 2004;29:116-122.

253. Albores-Saavedra J, Sheahan K, O’Riain C, Shukla D. Intraduct-al tubular adenoma, pyloric type, of the pancreas: additional observations on a new type of pancreatic neoplasm. Am J Surg Pathol. 2004;28:233-238.

254. Bakotic BW, Robinson MJ, Sturm PD, et al. Pyloric gland adenoma of the main pancreatic duct. Am J Surg Pathol. 1999;23:227-231.

255. Kato N, Akiyama S, Motoyama T. Pyloric gland-type tubular adenoma superimposed on intraductal papillary mucinous tu-mor of the pancreas. Pyloric gland adenoma of the pancreas. Virchows Arch. 2002;440:205-208.

256. Nakayama Y, Inoue H, Hamada Y, et al. Intraductal tubular adenoma of the pancreas, pyloric gland type: a clinicopathologic and immunohistochemical study of 6 cases. Am J Surg Pathol. 2005;29:607-616.

257. Compton CC. Serous cystic tumors of the pancreas. Semin Di-agn Pathol. 2000;17:43-55.

258. Alpert LC, Truong LD, Bossart MI, Spjut HJ. Microcystic ade-noma (serous cystadenoma) of the pancreas. A study of 14 cases with immunohistochemical and electron-microscopic correla-tion. Am J Surg Pathol. 1988;12:251-263.

259. Egawa N, Maillet B, Schroder S, et al. Serous oligocystic and ill-demarcated adenoma of the pancreas: a variant of serous cystic adenoma. Virchows Arch. 1994;424:13-17.

260. Kosmahl M, Wagner J, Peters K, et al. Serous cystic neoplasms of the pancreas: an immunohistochemical analysis revealing al-pha-inhibin, neuron-specific enolase, and MUC6 as new mark-ers. Am J Surg Pathol. 2004;28:339-346.

261. Shorten SD, Hart WR, Petras RE. Microcystic adenomas (serous cystadenomas) of pancreas. A clinicopathologic investigation of eight cases with immunohistochemical and ultrastructural stud-ies. Am J Surg Pathol. 1986;10:365-372.

262. Sperti C, Pasquali C, Perasole A, et al. Macrocystic serous cyst-adenoma of the pancreas: clinicopathologic features in seven cases. Int J Pancreatol. 2000;28:1-7.

263. Yasuhara Y, Sakaida N, Uemura Y, et al. Serous microcystic adenoma (glycogen-rich cystadenoma) of the pancreas: study of 11 cases showing clinicopathological and immunohistochemical correlations. Pathol Int. 2002;52:307-312.

264. Perez-Ordonez B, Naseem A, Lieberman PH, Klimstra DS. Solid serous adenoma of the pancreas. The solid variant of serous cystadenoma? Am J Surg Pathol. 1996;20:1401-1405.

265. Ishikawa T, Nakao A, Nomoto S, et al. Immunohistochemical and molecular biological studies of serous cystadenoma of the pancreas. Pancreas. 1998;16:40-44.

266. Helpap B, Vogel J. Immunohistochemical studies on cystic pan-creatic neoplasms. Pathol Res Pract. 1988;184:39-45.

267. Solcia E, Capella C, Kloppel G. Tumors of the Pancreas. 3rd ed. Washington, DC: American Registry of Pathology; 1997.

268. Thirabanjasak D, Basturk O, Altinel D, et al. Is serous cystad-enoma of pancreas a model of clear cell associated angiogenesis and tumorigenesis? Pancreatology. 2008;9:182-188.

269. Mohr VH, Vortmeyer AO, Zhuang Z, et al. Histopathology and molecular genetics of multiple cysts and microcystic (serous) ad-enomas of the pancreas in von Hippel-Lindau patients. Am J Pathol. 2000;157:1615-1621.

270. Vortmeyer AO, Lubensky IA, Fogt F, et al. Allelic deletion and mutation of the von Hippel-Lindau (VHL) tumor sup-pressor gene in pancreatic microcystic adenomas. Am J Pathol. 1997;151:951-956.

271. Moore PS, Zamboni G, Brighenti A, et al. Molecular charac-terization of pancreatic serous microcystic adenomas: evidence for a tumor suppressor gene on chromosome 10q. Am J Pathol. 2001;158:317-321.

272. Santos LD, Chow C, Henderson CJ, et al. Serous oligocystic adenoma of the pancreas: a clinicopathological and immuno-histochemical study of three cases with ultrastructural findings. Pathology. 2002;34:148-156.

273. Ordonez NG. Pancreatic acinar cell carcinoma. Adv Anat Pathol. 2001;8:144-159.

274. Hartman GG, Ni H, Pickleman J. Acinar cell carcinoma of the pancreas. Arch Pathol Lab Med. 2001;125:1127-1128.

275. Klimstra DS, Heffess CS, Oertel JE, Rosai J. Acinar cell carci-noma of the pancreas. A clinicopathologic study of 28 cases. Am J Surg Pathol. 1992;16:815-837.

276. Skacel M, Ormsby AH, Petras RE, et al. Immunohistochemistry in the differential diagnosis of acinar and endocrine pancreatic neo-plasms. Appl Immunohistochem Mol Morphol. 2000;8:203-209.

277. Kuerer H, Shim H, Pertsemlidis D, Unger P. Functioning pancre-atic acinar cell carcinoma: immunohistochemical and ultrastruc-tural analyses. Am J Clin Oncol. 1997;20:101-107.

278. Ishihara A, Sanda T, Takanari H, et al. Elastase-1-secreting aci-nar cell carcinoma of the pancreas. A cytologic, electron micro-scopic and histochemical study. Acta Cytol. 1989;33:157-163.

279. Ohike N, Kosmahl M, Kloppel G. Mixed acinar-endocrine carcinoma of the pancreas. A clinicopathological study and comparison with acinar-cell carcinoma. Virchows Arch. 2004;445:231-235.

280. Basturk O, Zamboni G, Klimstra DS, et al. Intraductal and pap-illary variants of acinar cell carcinomas: a new addition to the challenging differential diagnosis of intraductal neoplasms. Am J Surg Pathol. 2007;31:363-370.

281. Pellegata NS, Sessa F, Renault B, et al. K-ras and p53 gene muta-tions in pancreatic cancer: ductal and nonductal tumors progress through different genetic lesions. Cancer Res. 1994;54:1556-1560.

282. Terhune PG, Heffess CS, Longnecker DS. Only wild-type c- Ki-ras codons 12, 13, and 61 in human pancreatic acinar cell carcinomas. Mol Carcinog. 1994;10:110-114.

583REFERENCES

283. Terhune PG, Memoli VA, Longnecker DS. Evaluation of p53 mutation in pancreatic acinar cell carcinomas of humans and transgenic mice. Pancreas. 1998;16:6-12.

284. Abraham SC, Wu TT, Hruban RH, et al. Genetic and immu-nohistochemical analysis of pancreatic acinar cell carcinoma: frequent allelic loss on chromosome 11p and alterations in the APC/beta-catenin pathway. Am J Pathol. 2002;160:953-962.

285. Longnecker DS. Molecular pathology of invasive carcinoma. Ann N Y Acad Sci. 1999;880:74-82.

286. Moore PS, Beghelli S, Zamboni G, Scarpa A. Genetic abnor-malities in pancreatic cancer. Mol Cancer. 2003;2:7.

287. Rigaud G, Moore PS, Zamboni G, et al. Allelotype of pancreatic acinar cell carcinoma. Int J Cancer. 2000;88:772-777.

288. Taruscio D, Paradisi S, Zamboni G, et al. Pancreatic acinar car-cinoma shows a distinct pattern of chromosomal imbalances by comparative genomic hybridization. Genes Chromosomes Can-cer. 2000;28:294-299.

289. Klimstra DS. Nonductal neoplasms of the pancreas. Mod Pathol. 2007;20(Suppl 1):S94-112.

290. Henke AC, Kelley CM, Jensen CS, Timmerman TG. Fine-needle aspiration cytology of pancreatoblastoma. Diagn Cytopathol. 2001;25:118-121.

291. Kawamoto K, Matsuo T, Jubashi T, et al. Primary pancreatic carcinoma in childhood. Pancreatoblastoma. Acta Pathol Jpn. 1985;35:137-143.

292. Klimstra DS, Wenig BM, Adair CF, Heffess CS. Pancreatoblas-toma: A clinicopathologic study and review of the literature. Am J Surg Pathol. 1995;19:1371-1389.

293. Nishimata S, Kato K, Tanaka M, et al. Expression pattern of keratin subclasses in pancreatoblastoma with special emphasis on squamoid corpuscles. Pathol Int. 2005;55:297-302.

294. Silverman JF, Holbrook CT, Pories WJ, et al. Fine needle aspira-tion cytology of pancreatoblastoma with immunocytochemical and ultrastructural studies. Acta Cytol. 1990;34:632-640.

295. Ohaki Y, Misugi K, Fukuda J, et al. Immunohistochemical study of pancreatoblastoma. Acta Pathol Jpn. 1987;37:1581-1590.

296. Cooper JE, Lake BD. Use of enzyme histochemistry in the diag-nosis of pancreatoblastoma. Histopathology. 1989;15:407-414.

297. He L, Li P, Liu S, et al. [A clinicopathological study of pan-creatoblastoma]. Zhonghua Bing Li Xue Za Zhi. 1999;28:337-339.

298. Iseki M, Suzuki T, Koizumi Y, et al. Alpha-fetoprotein-produc-ing pancreatoblastoma. A case report. Cancer. 1986;57:1833-1835.

299. Abraham SC, Wu TT, Klimstra DS, et al. Distinctive molecular genetic alterations in sporadic and familial adenomatous polyp-osis-associated pancreatoblastomas: frequent alterations in the APC/beta-catenin pathway and chromosome 11p. Am J Pathol. 2001;159:1619-1627.

300. Tanaka Y, Kato K, Notohara K, et al. Significance of aberrant (cytoplasmic/nuclear) expression of beta-catenin in pancreato-blastoma. J Pathol. 2003;199:185-190.

301. Kerr NJ, Chun YH, Yun K, et al. Pancreatoblastoma is asso-ciated with chromosome 11p loss of heterozygosity and IGF2 overexpression. Med Pediatr Oncol. 2002;39:52-54.

302. Wiley J, Posekany K, Riley R, et al. Cytogenetic and flow cy-tometric analysis of a pancreatoblastoma. Cancer Genet Cyto-genet. 1995;79:115-118.

303. Balercia G, Zamboni G, Bogina G, Mariuzzi GM. Solid-cystic tumor of the pancreas. An extensive ultrastructural study of fourteen cases. J Submicrosc Cytol Pathol. 1995;27:331-340.

304. Martin RC, Klimstra DS, Brennan MF, Conlon KC. Solid-pseu-dopapillary tumor of the pancreas: a surgical enigma? Ann Surg Oncol. 2002;9:35-40.

305. Miettinen M, Partanen S, Fraki O, Kivilaakso E. Papillary cys-tic tumor of the pancreas. An analysis of cellular differentiation by electron microscopy and immunohistochemistry. Am J Surg Pathol. 1987;11:855-865.

306. Yamaguchi K, Miyagahara T, Tsuneyoshi M, et al. Papillary cys-tic tumor of the pancreas: an immunohistochemical and ultra-structural study of 14 patients. Jpn J Clin Oncol. 1989;19:102-111.

307. Wunsch LP, Flemming P, Werner U, et al. Diagnosis and treat-ment of papillary cystic tumor of the pancreas in children. Eur J Pediatr Surg. 1997;7:45-47.

308. von Herbay A, Sieg B, Otto HF. Solid-cystic tumour of the pan-creas. An endocrine neoplasm? Virchows Arch A Pathol Anat Histopathol. 1990;416:535-538.

309. Pettinato G, Manivel JC, Ravetto C, et al. Papillary cystic tu-mor of the pancreas. A clinicopathologic study of 20 cases with cytologic, immunohistochemical, ultrastructural, and flow cy-tometric observations, and a review of the literature. Am J Clin Pathol. 1992;98:478-488.

310. Lieber MR, Lack EE, Roberts Jr JR, et al. Solid and papillary epithelial neoplasm of the pancreas. An ultrastructural and immunocytochemical study of six cases. Am J Surg Pathol. 1987;11:85-93.

311. Stommer P, Kraus J, Stolte M, Giedl J. Solid and cystic pancre-atic tumors. Clinical, histochemical, and electron microscopic features in ten cases. Cancer. 1991;67:1635-1641.

312. Yoon DY, Hines OJ, Bilchik AJ, et al. Solid and papillary epi-thelial neoplasms of the pancreas: aggressive resection for cure. Am Surg. 2001;67:1195-1199.

313. Lam KY, Lo CY, Fan ST. Pancreatic solid-cystic-papillary tu-mor: clinicopathologic features in eight patients from Hong Kong and review of the literature. World J Surg. 1999;23:1045-1050.

314. Kosmahl M, Seada LS, Janig U, et al. Solid-pseudopapillary tumor of the pancreas: its origin revisited. Virchows Arch. 2000;436:473-480.

315. Lee WY, Tzeng CC, Chen RM, et al. Papillary cystic tumors of the pancreas: assessment of malignant potential by analysis of progesterone receptor, flow cytometry, and ras oncogene muta-tion. Anticancer Res. 1997;17:2587-2591.

316. Notohara K, Hamazaki S, Tsukayama C, et al. Solid-pseudo-papillary tumor of the pancreas: immunohistochemical localiza-tion of neuroendocrine markers and CD10. Am J Surg Pathol. 2000;24:1361-1371.

317. Zamboni G, Bonetti F, Scarpa A, et al. Expression of pro-gesterone receptors in solid-cystic tumour of the pancreas: a clinicopathological and immunohistochemical study of ten cases. Virchows Arch A Pathol Anat Histopathol. 1993;423:425-431.

318. Abraham SC, Klimstra DS, Wilentz RE, et al. Solid-pseudo-papillary tumors of the pancreas are genetically distinct from pancreatic ductal adenocarcinomas and almost always harbor beta-catenin mutations. Am J Pathol. 2002;160:1361-1369.

319. Muller-Hocker J, Zietz CH, Sendelhofert A. Deregulated ex-pression of cell cycle-associated proteins in solid pseudopapil-lary tumor of the pancreas. Mod Pathol. 2001;14:47-53.

320. Chott A, Kloppel G, Buxbaum P, Heitz PU. Neuron specific eno-lase demonstration in the diagnosis of a solid-cystic (papillary cystic) tumour of the pancreas. Virchows Arch A Pathol Anat Histopathol. 1987;410:397-402.

321. Kloppel G, Maurer R, Hofmann E, et al. Solid-cystic (papillary-cystic) tumours within and outside the pancreas in men: report of two patients. Virchows Arch A Pathol Anat Histopathol. 1991;418:179-183.

322. Kim MJ, Jang SJ, Yu E. Loss of E-cadherin and cytoplasmic-nuclear expression of beta-catenin are the most useful immuno-profiles in the diagnosis of solid-pseudopapillary neoplasm of the pancreas. Hum Pathol. 2008;39:251-258.

323. Tien YW, Ser KH, Hu RH, et al. Solid pseudopapillary neo-plasms of the pancreas: is there a pathologic basis for the ob-served gender differences in incidence? Surgery. 2005;137:591-596.

324. Nishihara K, Tsuneyoshi M, Ohshima A, Yamaguchi K. Papil-lary cystic tumor of the pancreas. Is it a hormone-dependent neoplasm? Pathol Res Pract. 1993;189:521-526.

325. Cao D, Antonescu C, Wong G, et al. Positive immunohisto-chemical staining of KIT in solid-pseudopapillary neoplasms of the pancreas is not associated with KIT/PDGFRA mutations. Mod Pathol. 2006;19:1157-1163.

326. Tiemann K, Heitling U, Kosmahl M, Kloppel G. Solid pseudo-papillary neoplasms of the pancreas show an interruption of the Wnt-signaling pathway and express gene products of 11q. Mod Pathol. 2007;20:955-960.

327. Doglioni C, Tos AP, Laurino L, et al. Calretinin: a novel im-munocytochemical marker for mesothelioma. Am J Surg Pathol. 1996;20:1037-1046.


328. Tanaka Y, Kato K, Notohara K, et al. Frequent beta-catenin mutation and cytoplasmic/nuclear accumulation in pancreatic solid-pseudopapillary neoplasm. Cancer Res. 2001;61:8401-8404.

329. Yamaue H, Tanimura H, Shono Y, et al. Solid and cystic tumor of the pancreas: clinicopathologic and genetic studies of four cases. Int J Pancreatol. 2000;27:69-76.

330. Chetty R, Serra S. Membrane loss and aberrant nuclear local-ization of E-cadherin are consistent features of solid pseudo-papillary tumour of the pancreas. An immunohistochemical study using two antibodies recognizing different domains of the E-cadherin molecule. Histopathology. 2008;52:325-330.

331. Tiemann K, Kosmahl M, Ohlendorf J, et al. Solid pseudopapil-lary neoplasms of the pancreas are associated with FLI-1 ex-pression, but not with EWS/FLI-1 translocation. Mod Pathol. 2006;19:1409-1413.

332. Heitz PU, Komminoth P, Perren A. Pancreatic endocrine tu-mors: Introduction. In: Delellis RA, Lloyd RV, Heitz PU, Eng C, eds. WHO Classification of Tumors. Pathology and Genetics of Tumours od Endocrine Organs. Lyon, France: IARC Press; 2004:177-182.

333. Lloyd RV, Mervak T, Schmidt K, et al. Immunohistochemical detection of chromogranin and neuron-specific enolase in pan-creatic endocrine neoplasms. Am J Surg Pathol. 1984;8:607-614.

334. Thomas RM, Baybick JH, Elsayed AM, Sobin LH. Gastric carci-noids. An immunohistochemical and clinicopathologic study of 104 patients. Cancer. 1994;73:2053-2058.

335. Al-Khafaji B, Noffsinger AE, Miller MA, et al. Immunohistolog-ic analysis of gastrointestinal and pulmonary carcinoid tumors. Hum Pathol. 1998;29:992-999.

336. Bordi C. Gastric carcinoids: an immunohistochemical and clini-copathologic study of 104 patients. Cancer. 1995;75:129-130.

337. Bordi C, Yu JY, Baggi MT, et al. Gastric carcinoids and their precursor lesions. A histologic and immunohistochemical study of 23 cases. Cancer. 1991;67:663-672.

338. Le Gall F, Vallet VS, Thomas D, et al. Immunohistochemical study of secretogranin II in 62 neuroendocrine tumours of the digestive tract and of the pancreas in comparison with other granins. Pathol Res Pract. 1997;193:179-185.

339. Burke AP, Federspiel BH, Sobin LH, et al. Carcinoids of the duodenum. A histologic and immunohistochemical study of 65 tumors. Am J Surg Pathol. 1989;13:828-837.

340. Makhlouf HR, Burke AP, Sobin LH. Carcinoid tumors of the ampulla of Vater: a comparison with duodenal carcinoid tu-mors. Cancer. 1999;85:1241-1249.

341. Lam KY, Lo CY. Pancreatic endocrine tumour: a 22-year clin-ico-pathological experience with morphological, immunohisto-chemical observation and a review of the literature. Eur J Surg Oncol. 1997;23:36-42.

342. Azzoni C, Doglioni C, Viale G, et al. Involvement of BCL-2 on-coprotein in the development of enterochromaffin-like cell gas-tric carcinoids. Am J Surg Pathol. 1996;20:433-441.

343. Hayashi H, Nakagawa M, Kitagawa S, et al. Immunohisto-chemical analysis of gastrointestinal carcinoids. Gastroenterol Jpn. 1993;28:483-490.

344. Rindi G, Ubiali A, Villanacci V. The phenotype of gut endocrine tumours. Dig Liver Dis. 2004;36(Suppl 1):S26-30.

345. Erickson LA, Lloyd RV. Practical markers used in the diagnosis of endocrine tumors. Adv Anat Pathol. 2004;11:175-189.

346. Chetty R, Asa SL. Pancreatic endocrine tumors: an update. Adv Anat Pathol. 2004;11:202-210.

347. Bostwick DG, Null WE, Holmes D, et al. Expression of opioid peptides in tumors. N Engl J Med. 1987;317:1439-1443.

348. Chejfec G, Falkmer S, Grimelius L, et al. Synaptophysin. A new marker for pancreatic neuroendocrine tumors. Am J Surg Pathol. 1987;11:241-247.

349. Simpson S, Vinik AI, Marangos PJ, Lloyd RV. Immunohistochem-ical localization of neuron-specific enolase in gastroenteropancre-atic neuroendocrine tumors. Correlation with tissue and serum levels of neuron-specific enolase. Cancer. 1984;54:1364-1369.

350. Heitz PU, Kasper M, Polak JM, Kloppel G. Pancreatic endocrine tumors. Hum Pathol. 1982;13:263-271.

351. Mukai K. Functional pathology of pancreatic islets: immunocy-tochemical exploration. Pathol Annu. 1983;18(Pt 2):87-107.

352. Mukai K, Grotting JC, Greider MH, Rosai J. Retrospective study of 77 pancreatic endocrine tumors using the immunoper-oxidase method. Am J Surg Pathol. 1982;6:387-399.

353. Clark ES, Carney JA. Pancreatic islet cell tumor associated with Cushing’s syndrome. Am J Surg Pathol. 1984;8:917-924.

354. Schmid KW, Brink M, Freytag G, et al. Expression of chromo-granin A and B and secretoneurin immunoreactivity in neoplas-tic and nonneoplastic pancreatic alpha cells. Virchows Arch. 1994;425:127-132.

355. Delis S, Bakoyiannis A, Giannakou N, et al. Asymptomatic calcitonin-secreting tumor of the pancreas. A case report. JOP. 2006;7:70-73.

356. Osamura RY, Oberg K, Perren A. Pancreatic endocrine tu-mours: ACTH and other ectopic hormone producing tumours. In: Delellis RA, Lloyd RV, Heitz PU, Eng C, eds. WHO Clas-sification of Tumours. Pathology and Genetics of Tumours of Endocrine Organs. Lyon, France: IARC Press; 2004:199-200.

357. Miraliakbari BA, Asa SL, Boudreau SF. Parathyroid hor-mone-like peptide in pancreatic endocrine carcinoma and ad-enocarcinoma associated with hypercalcemia. Hum Pathol. 1992;23:884-887.

358. Berger G, Trouillas J, Bloch B, et al. Multihormonal carcinoid tumor of the pancreas. Secreting growth hormone-releasing fac-tor as a cause of acromegaly. Cancer. 1984;54:2097-2108.

359. Bostwick DG, Quan R, Hoffman AR, et al. Growth-hormone-releasing factor immunoreactivity in human endocrine tumors. Am J Pathol. 1984;117:167-170.

360. Dayal Y, Lin HD, Tallberg K, et al. Immunocytochemical demonstration of growth hormone-releasing factor in gastro-intestinal and pancreatic endocrine tumors. Am J Clin Pathol. 1986;85:13-20.

361. La Rosa S, Uccella S, Billo P, et al. Immunohistochemical local-ization of alpha- and betaA-subunits of inhibin/activin in human normal endocrine cells and related tumors of the digestive sys-tem. Virchows Arch. 1999;434:29-36.

362. Kimura N, Pilichowska M, Okamoto H, et al. Immunohisto-chemical expression of chromogranins A and B, prohormone convertases 2 and 3, and amidating enzyme in carcinoid tumors and pancreatic endocrine tumors. Mod Pathol. 2000;13:140-146.

363. Tomita T. Metallothionein in pancreatic endocrine neoplasms. Mod Pathol. 2000;13:389-395.

364. Papotti M, Bongiovanni M, Volante M, et al. Expression of so-matostatin receptor types 1-5 in 81 cases of gastrointestinal and pancreatic endocrine tumors. A correlative immunohistochemi-cal and reverse-transcriptase polymerase chain reaction analysis. Virchows Arch. 2002;440:461-475.

365. Larsson LI, Grimelius L, Hakanson R, et al. Mixed endocrine pancreatic tumors producing several peptide hormones. Am J Pathol. 1975;79:271-284.

366. Liu TH, Tseng HC, Zhu Y, et al. Insulinoma. An immuno-cytochemical and morphologic analysis of 95 cases. Cancer. 1985;56:1420-1429.

367. Osamura RY, Oberg K, Speel EJ, et al. Pancreatic endocrine tumours: serotonin-secreting tumour. In: Delellis RA, Lloyd RV, Heitz PU, Eng C, eds. WHO Classification of Tumours. Pathology and Genetics of Tumours of Endocrine Organs. Lyon, France: IARC Press; 2004:198.

368. Deshpande V, Fernandez-del Castillo C, Muzikansky A, et al. Cytokeratin 19 is a powerful predictor of survival in pancreatic endocrine tumors. Am J Surg Pathol. 2004;28:1145-1153.

369. Schmitt AM, Anlauf M, Rousson V, et al. WHO 2004 criteria and CK19 are reliable prognostic markers in pancreatic endo-crine tumors. Am J Surg Pathol. 2007;31:1677-1682.

370. Ali A, Serra S, Asa SL, Chetty R. The predictive value of CK19 and CD99 in pancreatic endocrine tumors. Am J Surg Pathol. 2006;30:1588-1594.

371. Cai YC, Banner B, Glickman J, Odze RD. Cytokeratin 7 and 20 and thyroid transcription factor 1 can help distinguish pulmo-nary from gastrointestinal carcinoid and pancreatic endocrine tumors. Hum Pathol. 2001;32:1087-1093.

372. d’Amore ES, Manivel JC, Pettinato G, et al. Intestinal ganglio-neuromatosis: mucosal and transmural types. A clinicopatho-logic and immunohistochemical study of six cases. Hum Pathol. 1991;22:276-286.

585REFERENCES

373. Hochwald SN, Zee S, Conlon KC, et al. Prognostic factors in pancreatic endocrine neoplasms: an analysis of 136 cases with a proposal for low-grade and intermediate-grade groups. J Clin Oncol. 2002;20:2633-2642.

374. Kamisawa T, Tu Y, Egawa N, et al. Ductal and acinar dif-ferentiation in pancreatic endocrine tumors. Dig Dis Sci. 2002;47:2254-2261.

375. Yantiss RK, Chang HK, Farraye FA, et al. Prevalence and prog-nostic significance of acinar cell differentiation in pancreatic en-docrine tumors. Am J Surg Pathol. 2002;26:893-901.

376. Klimstra DS, Rosai J, Heffess CS. Mixed acinar-endocrine carci-nomas of the pancreas. Am J Surg Pathol. 1994;18:765-778.

377. Arihiro K, Inai K. Malignant islet cell tumor of the pancreas with multiple hormone production and expression of CEA and CA19-9. Report of an autopsy case. Acta Pathol Jpn. 1991;41:150-157.

378. Hussain S, Arwini A, Chetty R, Klimstra DS. Oncocytic pancre-atic endocrine neoplasms: a clinicopathologic and immnunohis-tochemical analysis of 21 cases. Mod Pathol. 2005;18:279A.

379. Goto A, Niki T, Terado Y, et al. Prevalence of CD99 protein ex-pression in pancreatic endocrine tumours (PETs). Histopathol-ogy. 2004;45:384-392.

380. Viale G, Doglioni C, Gambacorta M, et al. Progesterone recep-tor immunoreactivity in pancreatic endocrine tumors. An im-munocytochemical study of 156 neuroendocrine tumors of the pancreas, gastrointestinal and respiratory tracts, and skin. Can-cer. 1992;70:2268-2277.

381. Speel EJ, Richter J, Moch H, et al. Genetic differences in en-docrine pancreatic tumor subtypes detected by comparative ge-nomic hybridization. Am J Pathol. 1999;155:1787-1794.

382. Stumpf E, Aalto Y, Hoog A, et al. Chromosomal alterations in human pancreatic endocrine tumors. Genes Chromosomes Can-cer. 2000;29:83-87.

383. Zhao J, Moch H, Scheidweiler AF, et al. Genomic imbalances in the progression of endocrine pancreatic tumors. Genes Chromo-somes Cancer. 2001;32:364-372.

384. Asteria C, Anagni M, Fugazzola L, et al. MEN1 gene muta-tions are a rare event in patients with sporadic neuroendocrine tumors. Eur J Intern Med. 2002;13:319-323.

385. Cupisti K, Hoppner W, Dotzenrath C, et al. Lack of MEN1 gene mutations in 27 sporadic insulinomas. Eur J Clin Invest. 2000;30:325-329.

386. Gortz B, Roth J, Krahenmann A, et al. Mutations and allelic deletions of the MEN1 gene are associated with a subset of spo-radic endocrine pancreatic and neuroendocrine tumors and not restricted to foregut neoplasms. Am J Pathol. 1999;154:429-436.

387. Hessman O, Lindberg D, Einarsson A, et al. Genetic alterations on 3p, 11q13, and 18q in nonfamilial and MEN 1-associated pancreatic endocrine tumors. Genes Chromosomes Cancer. 1999;26:258-264.

388. Komminoth P. Review: multiple endocrine neoplasia type 1, sporadic neuroendocrine tumors, and MENIN. Diagn Mol Pathol. 1999;8:107-112.

389. Moore PS, Missiaglia E, Antonello D, et al. Role of disease-caus-ing genes in sporadic pancreatic endocrine tumors: MEN1 and VHL. Genes Chromosomes Cancer. 2001;32:177-181.

390. Shan L, Nakamura Y, Nakamura M, et al. Somatic mutations of multiple endocrine neoplasia type 1 gene in the sporadic endo-crine tumors. Lab Invest. 1998;78:471-475.

391. Wang EH, Ebrahimi SA, Wu AY, et al. Mutation of the ME-NIN gene in sporadic pancreatic endocrine tumors. Cancer Res. 1998;58:4417-4420.

392. Zhuang Z, Vortmeyer AO, Pack S, et al. Somatic mutations of the MEN1 tumor suppressor gene in sporadic gastrinomas and insulinomas. Cancer Res. 1997;57:4682-4686.

393. Lubensky IA, Pack S, Ault D, et al. Multiple neuroendocrine tumors of the pancreas in von Hippel-Lindau disease patients: histopathological and molecular genetic analysis. Am J Pathol. 1998;153:223-231.

394. Goebel SU, Iwamoto M, Raffeld M, et al. Her-2/neu expression and gene amplification in gastrinomas: correlations with tumor biology, growth, and aggressiveness. Cancer Res. 2002;62:3702-3710.

395. Chung DC, Smith AP, Louis DN, et al. A novel pancreatic en-docrine tumor suppressor gene locus on chromosome 3p with clinical prognostic implications. J Clin Invest. 1997;100:404-410.

396. Scarpa A, Orlandini S, Moore PS, et al. Dpc4 is expressed in virtually all primary and metastatic pancreatic endocrine carci-nomas. Virchows Arch. 2002;440:155-159.

397. Srivastava A, Hornick JL. Immunohistochemical staining for CDX-2, PDX-1, NESP-55, and TTF-1 can help distinguish gastrointestinal carcinoid tumors from pancreatic endocrine and pulmonary carcinoid tumors. Am J Surg Pathol. 2009; 33:626-632.

398. Srivastava A, Padilla O, Fischer-Colbrie R, et al. Neuroendo-crine secretory protein-55 (NESP-55) expression discriminates pancreatic endocrine tumors and pheochromocytomas from gastrointestinal and pulmonary carcinoids. Am J Surg Pathol. 2004;28:1371-1378.

399. Sellner F, Sobhian B, De Santis M, et al. Well or poorly differ-entiated nonfunctioning neuroendocrine carcinoma of the pan-creas: a single institution experience with 17 cases. Eur J Surg Oncol. 2008;34:191-195.

400. Nilsson O, Van Cutsem E, Delle Fave G, et al. Poorly differenti-ated carcinomas of the foregut (gastric, duodenal and pancre-atic). Neuroendocrinology. 2006;84:212-215.

401. Stukavec J, Jirasek T, Mandys V, et al. Poorly differentiated endocrine carcinoma and intraductal papillary-mucinous neo-plasm of the pancreas: description of an unusual case. Pathol Res Pract. 2007;203:879-884.

402. Kaufmann O, Georgi T, Dietel M. Utility of 123C3 monoclonal antibody against CD56 (NCAM) for the diagnosis of small cell carcinomas on paraffin sections. Hum Pathol. 1997;28:1373-1378.

403. Capella C, Heitz PU, Hofler H, et al. Revised classification of neuroendocrine tumours of the lung, pancreas and gut. Vir-chows Arch. 1995;425:547-560.

404. Kaifi JT, Heidtmann S, Schurr PG, et al. Absence of L1 in pancreatic masses distinguishes adenocarcinomas from poorly differentiated neuroendocrine carcinomas. Anticancer Res. 2006;26:1167-1170.

405. Kaifi JT, Zinnkann U, Yekebas EF, et al. L1 is a potential marker for poorly-differentiated pancreatic neuroendocrine carcinoma. World J Gastroenterol. 2006;12:94-98.

406. Movahedi-Lankarani S, Hruban RH, Westra WH, Klimstra DS. Primitive neuroectodermal tumors of the pancreas: a report of seven cases of a rare neoplasm. Am J Surg Pathol. 2002;26:1040-1047.

407. O’Hara BJ, McCue PA, Miettinen M. Bile duct adenomas with endocrine component. Immunohistochemical study and comparison with conventional bile duct adenomas. Am J Surg Pathol. 1992;16:21-25.

408. Albores-Saavedra J, Vardaman CJ, Vuitch F. Non-neoplastic polypoid lesions and adenomas of the gallbladder. Pathol Annu. 1993;28(Pt 1):145-177.

409. Christensen AH, Ishak KG. Benign tumors and pseudotumors of the gallbladder. Report of 180 cases. Arch Pathol. 1970;90:423-432.

410. Albores-Saavedra J, Henson DE, Klimstra DS. Tumors of the gallbladder, extrahepatic bile ducts and ampulla of Vater. 3rd ed. Washington D.C: American Registry of Pathology; 2000.

411. Nakatani Y, Masudo K, Nozawa A, et al. Biotin-rich, optically clear nuclei express estrogen receptor-beta: tumors with morules may develop under the influence of estrogen and aberrant beta-catenin expression. Hum Pathol. 2004;35:869-874.

412. Shibahara H, Tamada S, Goto M, et al. Pathologic fea-tures of mucin-producing bile duct tumors: two histopatho-logic categories as counterparts of pancreatic intraductal papillary-mucinous neoplasms. Am J Surg Pathol. 2004;28:327-338.

413. Nagata S, Ajioka Y, Nishikura K, et al. Co-expression of gas-tric and biliary phenotype in pyloric-gland type adenoma of the gallbladder: immunohistochemical analysis of mucin profile and CD10. Oncol Rep. 2007;17:721-729.

414. Yamamoto M, Nakajo S, Tahara E. Immunohistochemical analysis of estrogen receptors in human gallbladder. Acta Pathol Jpn. 1990;40:14-21.


415. Wistuba II , Miquel JF, Gazdar AF, Albores-Saavedra J. Gall-bladder adenomas have molecular abnormalities different from those present in gallbladder carcinomas. Hum Pathol. 1999;30:21-25.

416. Tian Y, Ding RY, Zhi YH, et al. Analysis of p53 and vascu-lar endothelial growth factor expression in human gallbladder carcinoma for the determination of tumor vascularity. World J Gastroenterol. 2006;12:415-419.

417. Chang HJ, Jee CD, Kim WH. Mutation and altered expression of beta-catenin during gallbladder carcinogenesis. Am J Surg Pathol. 2002;26:758-766.

418. Devaney K, Goodman ZD, Ishak KG. Hepatobiliary cystad-enoma and cystadenocarcinoma. A light microscopic and im-munohistochemical study of 70 patients. Am J Surg Pathol. 1994;18:1078-1091.

419. Ojeda VJ, Shilkin KB, Walters MN. Premalignant epithelial le-sions of the gall bladder: a prospective study of 120 cholecystec-tomy specimens. Pathology. 1985;17:451-454.

420. Chan KW. Review of 253 cases of significant pathology in 7,910 cholecystectomies in Hong Kong. Pathology. 1988;20:20-23.

421. Wistuba II , Albores-Saavedra J. Genetic abnormalities involved in the pathogenesis of gallbladder carcinoma. J Hepatobiliary Pancreat Surg. 1999;6:237-244.

422. Stolzenberg-Solomon R, Fraumeni JF, Wideroff L. New Ma-lignancies Following Cancer of the Digestive Tract, Excluding Colorectal Cancer (Chapter 4) New Malignancies Among Can-cer Survivors: SEER. Cancer Registries. 2000:1973-2000.

423. Roa I, Ibacache G, Roa J, et al. Gallstones and gallbladder cancer-volume and weight of gallstones are associated with gall-bladder cancer: a case-control study. J Surg Oncol. 2006;93:624-628.

424. Kumar S. Infection as a risk factor for gallbladder cancer. J Surg Oncol. 2006;93:633-639.

425. Esterly JR, Spicer SS. Mucin histochemistry of human gallblad-der: changes in adenocarcinoma cystic fibrosis, and cholecysti-tis. J Natl Cancer Inst. 1968;40:1-10.

426. Lack EE. Pathology of the pancreas, gallbladder, extrahepatic biliary tract and ampullary region. New York: Oxford Univer-sity Press; 2003.

427. Rashid A, Ueki T, Gao YT, et al. K-ras mutation, p53 over-expression, and microsatellite instability in biliary tract can-cers: a population-based study in China. Clin Cancer Res. 2002;8:3156-3163.

428. Argani P, Shaukat A, Kaushal M, et al. Differing rates of loss of DPC4 expression and of p53 overexpression among carcinomas of the proximal and distal bile ducts. Cancer. 2001;91:1332-1341.

429. Parwani AV, Geradts J, Caspers E, et al. Immunohistochemi-cal and genetic analysis of non-small cell and small cell gall-bladder carcinoma and their precursor lesions. Mod Pathol. 2003;16:299-308.

430. Kim YT, Kim J, Jang YH, et al. Genetic alterations in gallbladder adenoma, dysplasia and carcinoma. Cancer Lett. 2001;169:59-68.

431. Shi YZ, Hui AM, Li X, et al. Overexpression of retinoblastoma protein predicts decreased survival and correlates with loss of p16INK4 protein in gallbladder carcinomas. Clin Cancer Res. 2000;6:4096-4100.

432. Quan ZW, Wu K, Wang J, et al. Association of p53, p16, and vascular endothelial growth factor protein expressions with the prognosis and metastasis of gallbladder cancer. J Am Coll Surg. 2001;193:380-383.

433. Rashid A. Cellular and molecular biology of biliary tract can-cers. Surg Oncol Clin N Am. 2002;11:995-1009.

434. Masuhara S, Kasuya K, Aoki T, et al. Relation between K-ras codon 12 mutation and p53 protein overexpression in gallblad-der cancer and biliary ductal epithelia in patients with pancre-aticobiliary maljunction. Pancreat Surg. 2000;7:198-205.

435. Imai M, Hoshi T, Ogawa K. K-ras codon 12 mutations in biliary tract tumors detected by polymerase chain reaction denaturing gradient gel electrophoresis. Cancer. 1994;73:2727-2733.

436. Roa JC, Roa I, de Aretxabala X, et al. [K-ras gene mutation in gallbladder carcinoma]. Rev Med Chil. 2004;132:955-960.

437. Ajiki T, Fujimori T, Onoyama H, et al. K-ras gene mutation in gall bladder carcinomas and dysplasia. Gut. 1996;38:426-429.

438. Kim YW, Huh SH, Park YK, et al. Expression of the c-erb-B2 and p53 protein in gallbladder carcinomas. Oncol Rep. 2001;8:1127-1132.

439. Jan YY, Yeh TS, Yeh JN, et al. Expression of epidermal growth factor receptor, apomucins, matrix metalloproteinases, and p53 in rat and human cholangiocarcinoma: appraisal of an animal model of cholangiocarcinoma. Ann Surg. 2004;240:89-94.

440. Albores-Saavedra J, Molberg K, Henson DE. Unusual malig-nant epithelial tumors of the gallbladder. Semin Diagn Pathol. 1996;13:326-338.

441. Watanabe M, Hori Y, Nojima T, et al. Alpha-fetoprotein-pro-ducing carcinoma of the gallbladder. Dig Dis Sci. 1993;38:561-564.

442. Gakiopoulou H, Givalos N, Liapis G, et al. Hepatoid adenocar-cinoma of the gallbladder. Dig Dis Sci. 2007;52:3358-3362.

443. Terracciano LM, Glatz K, Mhawech P, et al. Hepatoid adeno-carcinoma with liver metastasis mimicking hepatocellular car-cinoma: an immunohistochemical and molecular study of eight cases. Am J Surg Pathol. 2003;27:1302-1312.

444. Kim KW, Kim SH, Kim MA, et al. Adenosquamous carcinoma of the extrahepatic bile duct: clinicopathologic and radiologic features. Abdom Imaging. 2008.

445. Oohashi Y, Shirai Y, Wakai T, et al. Adenosquamous carcino-ma of the gallbladder warrants resection only if curative resec-tion is feasible. Cancer. 2002;94:3000-3005.

446. Chan S, Currie J, Malik AI, Mahomed AA. Paediatric cholecys-tectomy: shifting goalposts in the laparoscopic era. Surg Endosc. 2008;22:1392-1395.

447. Nishihara K, Tsuneyoshi M. Undifferentiated spindle cell car-cinoma of the gallbladder: a clinicopathologic, immunohisto-chemical, and flow cytometric study of 11 cases. Hum Pathol. 1993;24:1298-1305.

448. Modlin IM, Sandor A. An analysis of 8305 cases of carcinoid tumors. Cancer. 1997;79:813-829.

449. Barron-Rodriguez LP, Manivel JC, Mendez-Sanchez N, Jessu-run J. Carcinoid tumor of the common bile duct: evidence for its origin in metaplastic endocrine cells. Am J Gastroenterol. 1991;86:1073-1076.

450. Anjaneyulu V, Shankar-Swarnalatha G, Rao SC. Carcinoid tu-mor of the gall bladder. Ann Diagn Pathol. 2007;11:113-116.

451. Jun SR, Lee JM, Han JK, Choi BI. High-grade neuroendocrine carcinomas of the gallbladder and bile duct: report of four cases with pathological correlation. J Comput Assist Tomogr. 2006;30:604-609.

452. Papotti M, Cassoni P, Sapino A, et al. Large cell neuroendocrine carcinoma of the gallbladder: report of two cases. Am J Surg Pathol. 2000;24:1424-1428.

453. Kimura W, Futakawa N, Yamagata S, et al. Different clinico-pathologic findings in two histologic types of carcinoma of pa-pilla of Vater. Jpn J Cancer Res. 1994;85:161-166.

454. Fathallah L, Vyas P, Klimstra D, et al. MUC1 and MUC2 ex-pression in carcinomas of ampullary region and their diagnostic and clinical significance. Mod Pathol. 2002;15:284A.

455. Chu PG, Schwarz RE, Lau SK, et al. Immunohistochemical staining in the diagnosis of pancreatobiliary and ampulla of Vater adenocarcinoma: application of CDX2, CK17, MUC1, and MUC2. Am J Surg Pathol. 2005;29:359-367.

456. Hansel DE, Maitra A, Lin JW, et al. Expression of the caudal-type homeodomain transcription factors CDX 1/2 and out-come in carcinomas of the ampulla of Vater. J Clin Oncol. 2005;23:1811-1818.

457. Paulsen FP, Varoga D, Paulsen AR, et al. Prognostic value of mucins in the classification of ampullary carcinomas. Hum Pathol. 2006;37:160-167.

458. Zhou H, Schaefer N, Wolff M, Fischer HP. Carcinoma of the ampulla of Vater: comparative histologic/immunohistochemical classification and follow-up. Am J Surg Pathol. 2004;28:875-882.

459. Sessa F, Furlan D, Zampatti C, et al. Prognostic factors for am-pullary adenocarcinomas: tumor stage, tumor histology, tumor location, immunohistochemistry and microsatellite instability. Virch Arch. 2007;451:649-657.

460. Kamisawa T, Fukayama M, Koike M, et al. Carcinoma of the ampulla of Vater: expression of cancer-associated anti-gens inversely correlated with prognosis. Am J Gastroenterol. 1988;83:1118-1123.

587REFERENCES

u-for 25:

tins as.

a: mi-

mi-the

tra-a.

ids dy

ers,

re-

ra-ing to 51-

ro-

ult

us:

ing em.

ro-or

tol.

gen scle Res

a-ion ical tol.

al is.

a-is.

ide.

est.

ter-

ex-ells he-

idic in

vel ol.

of ced

J

461. Scarpa A, Capelli P, Zamboni G, et al. Neoplasia of the am-pulla of Vater. Ki-ras and p53 mutations. Am J Pathol. 1993;142:1163-1172.

462. Takashima M, Ueki T, Nagai E, et al. Carcinoma of the am-pulla of Vater associated with or without adenoma: a clinico-pathologic analysis of 198 cases with reference to p53 and Ki-67 immunohistochemical expressions. Mod Pathol. 2000;13:1300-1307.

463. McCarthy DM, Hruban RH, Argani P, et al. Role of the DPC4 tumor suppressor gene in adenocarcinoma of the ampulla of Vater: analysis of 140 cases. Mod Pathol. 2003;16:272-278.

464. Howe JR, Klimstra DS, Cordon-Cardo C, et al. K-ras mutation in adenomas and carcinomas of the ampulla of vater. Clin Can-cer Res. 1997;3:129-133.

465. Thirabanjasak D, Tang L, Klimstra DS, et al. Glandular carcinoids of the ampulla with psammoma bodies (so-called ampullary somatostatinomas): analysis of 12 cases of an under-recognized entity. Modern Pathology. 2008;20:623A.

466. Nassar H, Albores-Saavedra J, Klimstra DS. High-grade neu-roendocrine carcinoma of the ampulla of vater: a clinicopatho-logic and immunohistochemical analysis of 14 cases. Am J Surg Pathol. 2005;29:588-594.

467. Rindi G, Kloppel G, Alhman H, et al. TNM staging of fore-gut (neuro)endocrine tumors: a consensus proposal including a grading system. Virchows Arch. 2006;449:395-401.

468. Van Eyken P, Sciot R, Desmet VJ. Immunocytochemistry of cyto-keratins in primary human liver tumors. APMIS. 1991;23(Sup-pl):77-85.

469. Fischer HP, Altmannsberger M, Weber K, Osborn M. Keratin polypeptides in malignant epithelial liver tumors. Differential di-agnostic and histogenetic aspects. Am J Pathol. 1987;127:530-537.

470. van Eyken P, Sciot R, van Damme B, et al. Keratin immuno-histochemistry in normal human liver. Cytokeratin pattern of hepatocytes, bile ducts and acinar gradient. Virch Arch A Pathol Anat Histopathol. 1987;412:63-72.

471. Moll R, Franke WW, Schiller DL, et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982;31:11-24.

472. Zatloukal K, Stumptner C, Fuchsbichler A, et al. The keratin cy-toskeleton in liver diseases. The diagnostic value of hepatocyte paraffin antibody 1 in differentiating hepatocellular neoplasms from nonhepatic tumors: a review. J Pathol. 2004;204:367-376.

473. Suriawinata AA, Thung SN. Liver. In: Mills SE, ed. Histology for Pathologists. 3rd ed. Philadelphia: Lippincott Williams & Wilkins; 2007:685-703.

474. Theise ND, Fiel IM, Hytiroglou P, et al. Macroregenerative nod-ules in cirrhosis are not associated with elevated serum or stain-able tissue alpha-fetoprotein. Liver. 1995;15:30-34.

475. Dodson A, Campbell F. Biotin inclusions: a potential pitfall in immunohistochemistry avoided. Histopathology. 1999;34:178-179.

476. Iezzoni JC, Mills SE, Pelkey TJ, Stoler MH. Inhibin is not an immunohistochemical marker for hepatocellular carcinoma. An example of the potential pitfall in diagnostic immunohis-tochemistry caused by endogenous biotin. Am J Clin Pathol. 1999;111:229-234.

477. Bussolati G, Gugliotta P, Volante M, et al. Retrieved endog-enous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry. Histopathology. 1997;31:400-407.

478. Wennerberg AE, Nalesnik MA, Coleman WB. Hepatocyte par-affin 1: a monoclonal antibody that reacts with hepatocytes and can be used for differential diagnosis of hepatic tumors. Am J Pathol. 1993;143:1050-1054.

479. Leong AS, Sormunen RT, Tsui WM, et al. Hep Par 1 and se-lected antibodies in the immunohistological distinction of hepatocellular carcinoma from cholangiocarcinoma, com-bined tumours and metastatic carcinoma. Histopathology. 1998;33:318-324.

480. Wieczorek TJ, Pinkus JL, Glickman JN, Pinkus GS. Compari-son of thyroid transcription factor-1 and hepatocyte antigen immunohistochemical analysis in the differential diagnosis of hepatocellular carcinoma, metastatic adenocarcinoma, renal cell carcinoma, and adrenal cortical carcinoma. Am J Clin Pathol. 2002;118:911-921.

481. Borscheri N, Roessner A, Rocken C. Canalicular immnostaining of neprilysin (CD10) as a diagnostic marker hepatocellular carcinomas. Am J Surg Pathol. 2001;1297-1303.

482. Lai YS, Thung SN, Gerber MA, et al. Expression of cytokerain normal and diseased livers and in primary liver carcinomArch Pathol Lab Med. 1989;113:134-138.

483. Nakajima T, Kondo Y. Well-differentiated cholangiocarcinomdiagnostic significance of morphologic and immunohistochecal parameters. Am J Surg Pathol. 1989;13:569-573.

484. Terayama N, Terada T, Nakanuma Y. An immunohistochecal study of tumour vessels in metastatic liver cancers and surrounding liver tissue. Histopathology. 1996;29:37-43.

485. Haratake J, Scheuer PJ. An immunohistochemical and ulstructural study of the sinusoids of hepatocellular carcinomCancer. 1990;65:1985-1993.

486. Ruck P, Xiao JC, Kaiserling E. Immunoreactivity of sinusoin hepatocellular carcinoma. An immunohistochemical stuusing lectin UEA-1 and antibodies against endothelial markincluding CD34. Arch Pathol Lab Med. 1995;119:173-178.

487. Afdhal NH, Nunes D. Evaluation of liver fibrosis: a conciseview. Am J Gastroenterol. 2004;99:1160-1174.

488. James J, Lygidakis NJ, van Eyken P, et al. Application of ketin immunocytochemistry and sirius red staining in evaluatintrahepatic changes with acute extrahepatic cholestasis duehepatic duct carcinoma. Hepatogastroenterology. 1989;36:1155.

489. Manning DS, Afdhal NH. Diagnosis and quantitation of fibsis. Gastroenterology. 2008;134:1670-1681.

490. Desmet VJ. Histopathology of chronic cholestasis and adductopenic syndrome. Clin Liver Dis. 1998;2:249-264, viii.

491. Halfon P, Cacoub P. [Viral quantitation of hepatitis C virpresent and future]. Rev Med Interne. 2000;21:174-181.

492. Mardini H, Record C. Detection assessment and monitorof hepatic fibrosis: biochemistry or biopsy? Ann Clin Bioch2005;42:441-447.

493. Lockwood DS, Yeadon TM, Clouston AD, et al. Tumor pgression in hepatocellular carcinoma: relationship with tumstroma and parenchymal disease. J Gastroenterol Hepa2003;18:666-672.

494. Gulubova M. Immunohistochemical localization of collatype III and type IV, laminin, tenascin and alpha-smooth muactin (alphaSMA) in the human liver in peliosis. Pathol Pract. 2002;198:803-812.

495. Torimura T, Ueno T, Inuzuka S, et al. The extracellular mtrix in hepatocellular carcinoma shows different localizatpatterns depending on the differentiation and the histologpattern of tumors: immunohistochemical analysis. J Hepa1994;21:37-46.

496. Schuppan D. Structure of the extracellular matrix in normand fibrotic liver: collagens and glycoproteins. Semin Liver D1990;10:1-10.

497. Schuppan D, Ruehl M, Somasundaram R, Hahn EG. Mtrix as a modulator of hepatic fibrogenesis. Semin Liver D2001;21:351-372.

498. Schuppan D, Porov Y. Hepatic fibrosis: from bench to bedsJ Gastroenterol Hepatol. 2002;17(Suppl 3):S300-305.

499. Bataller R, Brenner DA. Liver fibrosis. J Clin Inv2005;115:209-218.

500. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenology. 2008;134:1655-1669.

501. Knittel T, Aurisch S, Neubauer K, et al. Cell-type-specific pression of neural cell adhesion molecule (N-CAM) in Ito cof rat liver. Up-regulation during in vitro activation and in patic tissue repair. Am J Pathol. 1996;149:449-462.

502. Neubauer K, Knittel T, Aurisch S, et al. Glial fibrillary acprotein—a cell type specific marker for Ito cells in vivo andvitro. J Hepatol. 1996;24:719-730.

503. Cassiman D, van Pelt J, De Vos R, et al. Synaptophysin: A nomarker for human and rat hepatic stellate cells. Am J Path1999;155:1831-1839.

504. Ogawa K, Suzuki J, Mukai H, Mori M. Sequential changesextracellular matrix and proliferation of Ito cells with enhanexpression of desmin and actin in focal hepatic injury. AmPathol. 1986;125:611-619.


505. Zatloukal K, French SW, Stumptner C, et al. From Mallory to Mallory-Denk bodies: what, how and why? Exp Cell Res. 2007;313:2033-2049.

506. Van Eyken P, Sciot R, Desmet VJ. A cytokeratin immunohisto-chemical study of alcoholic liver disease: evidence that hepato-cytes can express ‘bile duct-type’ cytokeratins. Histopathology. 1988;13:605-617.

507. Hazan R, Denk H, Franke WW, et al. Change of cytokeratin organization during development of Mallory bodies as revealed by a monoclonal antibody. Lab Invest. 1986;54:543-553.

508. Callea F. Immunohistochemical techniques for the demonstra-tion of viral antigens in liver tissue. Ric Clin Lab. 1988;18:223-231.

509. Roskams T. The role of immunohistochemistry in diagnosis. Clin Liver Dis. 2002;6:571-589:x.

510. Booth JC, Goldin RD, Brown JL, et al. Fibrosing cholestatic hepatitis in a renal transplant recipient associated with the hepa-titis B virus precore mutant. J Hepatol. 1995;22:500-503.

511. Chen CH, Chen PJ, Chu JS, et al. Fibrosing cholestatic hepatitis in a hepatitis B surface antigen carrier after renal transplanta-tion. Gastroenterology. 1994;107:1514-1518.

512. Fang JW, Wright TL, Lau JY. Fibrosing cholestatic hepatitis in patient with HIV and hepatitis B. Lancet. 1993;342:1175.

513. Davies SE, Portmann BC, O’Grady JG, et al. Hepatic histologi-cal findings after transplantation for chronic hepatitis B virus infection, including a unique pattern of fibrosing cholestatic hepatitis. Hepatology. 1991;13:150-157.

514. Fischer HP, Willsch E, Bierhoff E, Pfeifer U. Histopathologic findings in chronic hepatitis C. J Hepatol. 1996;24:35-42.

515. Kasprzak A, Biczysko W, Adamek A, Zabel M. Morphological lesions detected by light and electron microscopies in chronic type B hepatitis. Pol J Pathol. 2002;53:103-115.

516. Sansonno D, Iacobelli AR, Cornacchiulo V, et al. Immunohisto-chemical detection of hepatitis C virus-related proteins in liver tissue. Clin Exp Rheumatol. 1995;13(Suppl 13):S29-32.

517. Lau JY, Krawczynski K, Negro F, Gonzalez-Peralta RP. In situ detection of hepatitis C virus—a critical appraisal. J Hepatol. 1996;24:43-51.

518. Scheuer PJ, Krawczynski K, Dhillon AP. Histopathology and detection of hepatitis C virus in liver. Springer Semin Immuno-pathol. 1997;19:27-45.

519. Gowans EJ. Distribution of markers of hepatitis C virus infec-tion throughout the body. Semin Liver Dis. 2000;20:85-102.

520. Brody RI, Eng S, Melamed J, et al. Immunohistochemical detec-tion of hepatitis C antigen by monoclonal antibody TORDJI-22 compared with PCR viral detection. Am J Clin Pathol. 1998;110:32-37.

521. Uchida T, Shikata T, Tanaka E, Kiyosawa K. Immunoperoxi-dase staining of hepatitis C virus in formalin-fixed, paraffin-embedded needle liver biopsies. Virchows Arch. 1994;424:465-469.

522. Negro F, Pacchioni D, Mondardini A, et al. In situ hybridization in viral hepatitis. Liver. 1992;12:217-226.

523. Negro F, Papotti M, Taraglio S, et al. Relationship between hepatocyte proliferation and hepatitis delta virus replication in neoplastic and non-neoplastic liver tissues. J Viral Hepat. 1997;4:93-98.

524. Raymond E, Tricottet V, Samuel D, et al. Epstein-Barr virus- related localized hepatic lymphoproliferative disorders after liver transplantation. Cancer. 1995;76:1344-1351.

525. Chen CJ, Jeng LB, Huang SF. Lymphoepithelioma-like hepato-cellular carcinoma. Chang Gung Med J. 2007;30:172-177.

526. Chen TC, Ng KF, Kuo T. Intrahepatic cholangiocarcinoma with lymphoepithelioma-like component. Mod Pathol. 2001;14:527-532.

527. Vortmeyer AO, Kingma DW, Fenton RG, et al. Hepatobiliary lymphoepithelioma-like carcinoma associated with Epstein-Barr virus. Am J Clin Pathol. 1998;109:90-95.

528. Vanstapel MJ, Desmet VJ. Cytomegalovirus hepatitis: a his-tological and immunohistochemical study. Appl Pathol. 1983;1:41-49.

529. Lautenschlager I, Halme L, Hockerstedt K, et al. Cytomegalo-virus infection of the liver transplant: virological, histological, immunological, and clinical observations. Transpl Infect Dis. 2006;8:21-30.

530. Humphrey DM, Weiner MH. Mycobacterial antigen detection by immunohistochemistry in pulmonary tuberculosis. Hum Pathol. 1987;18:701-708.

531. Lau SK, Prakash S, Geller SA, Alsabeh R. Comparative immu-nohistochemical profile of hepatocellular carcinoma, cholangio-carcinoma, and metastatic adenocarcinoma. Human pathology. 2002;33:1175-1181.

532. Hurlimann J, Gardiol D. Immunohistochemistry in the dif-ferential diagnosis of liver carcinomas. Am J Surg Pathol. 1991;15:280-288.

533. Van Eyken P, Sciot R, Desmet VJ. A cytokeratin immunohisto-chemical study of cholestatic liver disease: evidence that hepato-cytes can express ‘bile duct-type’ cytokeratins. Histopathology. 1989;15:125-135.

534. van Eyken P, Sciot R, Callea F, Desmet VJ. A cytokeratin- immunohistochemical study of focal nodular hyperplasia of the liver: further evidence that ductular metaplasia of hepato-cytes contributes to ductular “proliferation.” Liver. 1989;9:372-377.

535. Sell S, Leffert HL. Liver cancer stem cells. J Clin Oncol. 2008;26:2800-2805.

536. Kamiya A, Gonzalez FJ, Nakauchi H. Identification and differ-entiation of hepatic stem cells during liver development. Front Biosci. 2006;11:1302-1310.

537. Gerber MA, Thung SN. Liver stem cells and development. Lab Invest. 1993;68:253-254.

538. Sell S. Is there a liver stem cell? Cancer Res. 1990;50:3811-3815.

539. Goldstein NS, Blue DE, Hankin R, et al. Serum alpha- fetoprotein levels in patients with chronic hepatitis C. Relationships with serum alanine aminotransferase values, histologic activity index, and hepatocyte MIB-1 scores. Am J Clin Pathol. 1999;111:811-816.

540. Cabibi D, Licata A, Barresi E, et al. Expression of cytokeratin 7 and 20 in pathological conditions of the bile tract. Pathol Res Pract. 2003;199:65-70.

541. Nakanuma Y, Ohta G. Immunohistochemical study on bile ductular proliferation in various hepatobiliary diseases. Liver. 1986;6:205-211.

542. Washington K, Clavien PA, Killenberg P. Peribiliary vascular plexus in primary sclerosing cholangitis and primary biliary cir-rhosis. Hum Pathol. 1997;28:791-795.

543. Vertemati M, Minola E, Goffredi M, et al. Computerized mor-phometry of the cirrhotic liver: comparative analysis in primary biliary cirrhosis, alcoholic cirrhosis, and posthepatitic cirrhosis. Microsc Res Tech. 2004;65:113-121.

544. Kobayashi S, Nakanuma Y, Matsui O. Intrahepatic peribiliary vascular plexus in various hepatobiliary diseases: a histological survey. Hum Pathol. 1994;25:940-946.

545. Daniels JA, Torbenson M, Anders RA, Boitnott JK. Immunos-taining of plasma cells in primary biliary cirrhosis. Am J Clin Pathol. 2009;131:243-249.

546. Deshpande V, Mino-Kenudson M, Brugge W, Lauwers GY. Autoimmune pancreatitis: more than just a pancreatic disease? A contemporary review of its pathology. Arch Pathol Lab Med. 2005;129:1148-1154.

547. Umemura T, Zen Y, Hamano H, et al. Immunoglobin G4-hepatopathy: association of immunoglobin G4-bearing plas-ma cells in liver with autoimmune pancreatitis. Hepatology. 2007;46:463-471.

548. Umemura T, Zen Y, Hamano H, et al. IgG4 associated autoim-mune hepatitis: a differential diagnosis for classical autoimmune hepatitis. Gut. 2007;56:1471-1472.

549. Zen Y, Fujii T, Sato Y, et al. Pathological classification of he-patic inflammatory pseudotumor with respect to IgG4-related disease. Mod Pathol. 2007;20:884-894.

550. Zen Y, Fujii T, Harada K, et al. Th2 and regulatory immune reactions are increased in immunoglobin G4-related sclerosing pancreatitis and cholangitis. Hepatology. 2007;45:1538-1546.

551. Lefkowitch JH. Hepatobiliary pathology. Current opinion in gastroenterology. 2008;24:269-277.

552. Bjornsson E, Chari ST, Smyrk TC, Lindor K. Immunoglobu-lin G4 associated cholangitis: description of an emerging clinical entity based on review of the literature. Hepatology. 2007;45:1547-1554.

589REFERENCES

553. Park DH, Kim MHD, Chari ST. Recent advances in autoim-mune pancreatitis. Gut. 2009.

554. Chari ST. Diagnosis of autoimmune pancreatitis using its five cardinal features: introducing the Mayo Clinic’s HISORt crite-ria. J Gastroenterol. 2007;42(Suppl 18):39-41.

555. Kwon S, Kim MH, Choi EK. The diagnostic criteria for auto-immune chronic pancreatitis: it is time to make a consensus. Pancreas. 2007;34:279-286.

556. Kojima M, Sipos B, Klapper W, et al. Autoimmune pancreatitis: frequency, IgG4 expression, and clonality of T and B cells. Am J Surg Pathol. 2007;31:521-528.

557. Deshpande V, Chicano S, Finkelberg D, et al. Autoimmune pan-creatitis: a systemic immune complex mediated disease. Am J Surg Pathol. 2006;30:1537-1545.

558. Zhang L, Notohara K, Levy MJ, et al. IgG4-positive plasma cell infiltration in the diagnosis of autoimmune pancreatitis. Mod Pathol. 2007;20:23-28.

559. Makhlouf HR, Abdul-Al HM, Goodman ZD. Diagnosis of focal nodular hyperplasia of the liver by needle biopsy. Hum Pathol. 2005;36:1210-1216.

560. Nguyen BN, Flejou JF, Terris B, et al. Focal nodular hyperplasia of the liver: a comprehensive pathologic study of 305 lesions and recognition of new histologic forms. Am J Surg Pathol. 1999;23:1441-1454.

561. Malone M, Mieli-Vergani G, Mowat AP, Portmann B. The fe-tal liver in PiZZ alpha-1-antitrypsin deficiency: a report of five cases. Pediatr Pathol. 1989;9:623-631.

562. Fairbanks KD, Tavill AS. Liver disease in alpha 1-antitrypsin deficiency: a review. Am J Gastroenterol. 2008;103:2136-2141:quiz 2142.

563. Portmann BC, Thompson RJ, Roberts EA, Paterson AC. Ge-netic and metabolic liver disease. In: Burt AD, Portmann BC, Ferrell LD, eds. MacSween’s Pathology of the Liver. 5th ed. Philadelphia: Churchill Livingstone Elsevier; 2007:199-326.

564. Medicina D, Fabbretti G, Brennan SO, et al. Genetic and im-munological characterization of fibrinogen inclusion bodies in patients with hepatic fibrinogen storage and liver disease. Ann N Y Acad Sci. 2001;936:522-525.

565. Demetris AJ, Adeyi O, Bellamy CO, et al. Liver biopsy interpre-tation for causes of late liver allograft dysfunction. Hepatology. 2006;44:489-501.

566. Haga H, Egawa H, Fujimoto Y, et al. Acute humoral rejection and C4d immunostaining in ABO blood type-incompatible liver transplantation. Liver Transpl. 2006;12:457-464.

567. Colvin RB. C4d in liver allografts: a sign of antibody-mediated rejection? Am J Transplant. 2006;6:447-448.

568. Schmeding M, Dankof A, Krenn V, et al. C4d in acute rejection after liver transplantation—a valuable tool in differential diag-nosis to hepatitis C recurrence. Am J Transplant. 2006;6:523-530.

569. Bu X, Zheng Z, Yu Y, Zeng L, Jiang Y. Significance of C4d de-position in the diagnosis of rejection after liver transplantation. Transplant Proc. 2006;38:1418-1421.

570. Lorho R, Turlin B, Aqodad N, et al. C4d: a marker for hepatic transplant rejection. Transplant Proc. 2006;38:2333-2334.

571. Watson R, Kozlowski T, Nickeleit V, et al. Isolated donor spe-cific alloantibody-mediated rejection after ABO compatible liver transplantation. Am J Transplant. 2006;6:3022-3029.

572. Troxell ML, Higgins JP, Kambham N. Evaluation of C4d stain-ing in liver and small intestine allografts. Arch Pathol Lab Med. 2006;130:1489-1496.

573. Jain A, Ryan C, Mohanka R, et al. Characterization of CD4, CD8, CD56 positive lymphocytes and C4d deposits to distin-guish acute cellular rejection from recurrent hepatitis C in post-liver transplant biopsies. Clin Transplant. 2006;20:624-633.

574. Bellamy CO, Herriot MM, Harrison DJ, Bathgate AJ. C4d immunopositivity is uncommon in ABO-compatible liver al-lografts, but correlates partially with lymphocytotoxic antibody status. Histopathology. 2007;50:739-749.

575. Sakashita H, Haga H, Ashihara E, et al. Significance of C4d staining in ABO-identical/compatible liver transplantation. Mod Pathol. 2007;20:676-684.

576. Ponticelli C. Renal transplantation 2004: where do we stand

577. Krukemeyer MG, Moeller J, Morawietz L, et al. Description of B lymphocytes and plasma cells, complement, and chemokines/receptors in acute liver allograft rejection. Transplantation. 2004;78:65-70.

578. Terasaki PI. Humoral theory of transplantation. Am J Trans-plant. 2003;3:665-673.

579. Dankof A, Schmeding M, Morawietz L, et al. Portal capillary C4d deposits and increased infiltration by macrophages indicate humorally mediated mechanisms in acute cellular liver allograft rejection. Virchows Arch. 2005;447:87-93.

580. Sawada T, Shimizu A, Kubota K, et al. Lobular damage caused by cellular and humoral immunity in liver allograft rejection. Clin Transplant. 2005;19:110-114.

581. Michaels PJ, Fishbein MC, Colvin RB. Humoral rejection of human organ transplants. Springer Semin Immunopathol. 2003;25:119-140.

582. Hirohashi S, Ishak KG, Kojiro M, et al. Hepatocellular carci-noma. In: Hamilton SR, Aaltonen LA, eds. Tumours of the Di-gestive System. Lyon, France: International Agency for Research on Cancer (IARC); 2000:159-172.

583. Torbenson M, Lee JH, Choti M, et al. Hepatic adenomas: analy-sis of sex steroid receptor status and the Wnt signaling pathway. Mod Pathol. 2002;15:189-196.

584. Zucman-Rossi J, Jeannot E, Nhieu JT, et al. Genotype- phenotype correlation in hepatocellular adenoma: new classification and relationship with HCC. Hepatology. 2006;43:515-524.

585. Bioulac-Sage P, Balabaud C, Bedossa P, et al. Pathological diag-nosis of liver cell adenoma and focal nodular hyperplasia: Bor-deaux update. J Hepatol. 2007;46:521-527.

586. Geller SA, Dhall D, Alsabeh R. Application of immunohisto-chemistry to liver and gastrointestinal neoplasms: liver, stom-ach, colon, and pancreas. Archives of Pathology & Laboratory Medicine. 2008;132:490-499.

587. Huang H, Fujii H, Sankila A, et al. Beta-catenin mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus infection. Am J Pathol. 1999;155:1795-1801.

588. Coston WM, Loera S, Lau SK, et al. Distinction of hepatocellular carcinoma from benign hepatic mimickers using Glypican-3 and CD34 immunohistochemistry. Am J Surg Pathol. 2008;32:433-444.

589. Shafizadeh N, Ferrell LD, Kakar S. Utility and limitations of glypican-3 expression for the diagnosis of hepatocellular carci-noma at both ends of the differentiation spectrum. Mod Pathol. 2008;21:1011-1018.

590. Fasano M, Theise ND, Nalesnik M, et al. Immunohistochemical evaluation of hepatoblastomas with use of the hepatocyte-specific marker, hepatocyte paraffin 1, and the polyclonal anti- carcinoembryonic antigen. Mod Pathol. 1998;11:934-938.

591. Stocker JT, Schmidt D. Hepatoblastoma. In: Hamilton SR, Aal-tonen LA, eds. Tumours of the Digestive System. Lyon, France: International Agency for Research on Cancer (IARC); 2000: 184-189.

592. Ruck P, Xiao JC, Pietsch T, et al. Hepatic stem-like cells in hepa-toblastoma: expression of cytokeratin 7, albumin and oval cell associated antigens detected by OV-1 and OV-6. Histopathol-ogy. 1997;31:324-329.

593. Ruck P, Xiao JC, Kaiserling E. Immunoreactivity of sinusoids in hepatoblastoma: an immunohistochemical study using lectin UEA-1 and antibodies against endothelium-associated antigens, including CD34. Histopathology. 1995;26:451-455.

594. Moll R, Divo M, Langbein L. The human keratins: biology and pathology. Histochem Cell Biol. 2008;129:705-733.

595. Strnad P, Stumptner C, Zatloukal K, Denk H. Intermediate fila-ment cytoskeleton of the liver in health and disease. Histochem Cell Biol. 2008;129:735-749.

596. Chu PG, Weiss LM. Keratin expression in human tissues and neoplasms. Histopathology. 2002;40:403-439.

597. Durnez A, Verslype C, Nevens F, et al. The clinicopathological and prognostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinoma. A possible progenitor cell origin. His-topathology. 2006;49:138-151.

598. Maeda T, Kajiyama K, Adachi E, et al. The expression of cyto-keratins 7, 19, and 20 in primary and metastatic carcinomas of

today? Nephrol Dial Transplant. 2004;19:2937-2947. the liver. Mod Pathol. 1996;9:901-909.


599. Kakar S, Gown AM, Goodman ZD, Ferrell LD. Best prac-tices in diagnostic immunohistochemistry: hepatocellular car-cinoma versus metastatic neoplasms. Arch Pathol Lab Med. 2007;131:1648-1654.

600. Morrison C, Marsh Jr W, Frankel WL. A comparison of CD10 to pCEA, MOC-31, and hepatocyte for the distinction of malig-nant tumors in the liver. Mod Pathol. 2002;15:1279-1287.

601. Goodman ZD, Ishak KG, Langloss JM, et al. Combined hepatocellular-cholangiocarcinoma. A histologic and immuno-histochemical study. Cancer. 1985;55:124-135.

602. Varma V, Cohen C. Immunohistochemical and molecular markers in the diagnosis of hepatocellular carcinoma. Adv Anat Pathol. 2004;11:239-249.

603. Wang L, Vuolo M, Suhrland MJ, Schlesinger K. HepPar1, MOC-31, pCEA, mCEA and CD10 for distinguishing hepato-cellular carcinoma vs. metastatic adenocarcinoma in liver fine needle aspirates. Acta Cytol. 2006;50:257-262.

604. Libbrecht L, Severi T, Cassiman D, et al. Glypican-3 expression distinguishes small hepatocellular carcinomas from cirrhosis, dysplastic nodules, and focal nodular hyperplasia-like nodules. Am J Surg Pathol. 2006;30:1405-1411.

605. Llovet JM, Chen Y, Wurmbach E, et al. A molecular signature to discriminate dysplastic nodules from early hepatocellular car-cinoma in HCV cirrhosis. Gastroenterology. 2006;131:1758-1767.

606. Wang XY, Degos F, Dubois S, et al. Glypican-3 expression in hepatocellular tumors: diagnostic value for preneoplastic lesions and hepatocellular carcinomas. Hum Pathol. 2006;37:1435-1441.

607. Yamauchi N, Watanabe A, Hishinuma M, et al. The glypican 3 oncofetal protein is a promising diagnostic marker for hepato-cellular carcinoma. Mod Pathol. 2005;18:1591-1598.

608. Sakamoto M, Mori T, Masugi Y, et al. Candidate molecular markers for histological diagnosis of early hepatocellular carci-noma. Intervirology. 2008;51(Suppl 1):42-45.

609. Krishna M, Lloyd RV, Batts KP. Detection of albumin messen-ger RNA in hepatic and extrahepatic neoplasms. A marker of hepatocellular differentiation. Am J Surg Pathol. 1997;21:147-152.

610. D’Errico A, Deleonardi G, Fiorentino M, et al. Diagnostic im-plications of albumin messenger RNA detection and cytokeratin pattern in benign hepatic lesions and biliary cystadenocarcino-ma. Diagn Mol Pathol. 1998;7:289-294.

611. Papotti M, Pacchioni D, Negro F, et al. Albumin gene expres-sion in liver tumors: diagnostic interest in fine needle aspiration biopsies. Mod Pathol. 1994;7:271-275.

612. Papotti M, Sambataro D, Marchesa P, Negro F. A combined hepatocellular/cholangiocellular carcinoma with sarcomatoid features. Liver. 1997;17:47-52.

613. Kakar S, Muir T, Murphy LM, et al. Immunoreactivity of Hep Par 1 in hepatic and extrahepatic tumors and its correlation with albumin in situ hybridization in hepatocellular carcinoma. Am J Clin Pathol. 2003;119:361-366.

614. Oliveira AM, Erickson LA, Burgart LJ, Lloyd RV. Differentia-tion of primary and metastatic clear cell tumors in the liver by in situ hybridization for albumin messenger RNA. Am J Surg Pathol. 2000;24:177-182.

615. Supriatna Y, Kishimoto T, Uno T, et al. Evidence for hepato-cellular differentiation in alpha-fetoprotein-negative gastric adenocarcinoma with hepatoid morphology: a study with in situ hybridisation for albumin mRNA. Pathology. 2005;37:211-215.

616. Lopez-Beltran A, Luque RJ, Quintero A, et al. Hepatoid adeno-carcinoma of the urinary bladder. Virch Arch. 2003;442:381-387.

617. Tanigawa H, Kida Y, Kuwao S, et al. Hepatoid adenocarcinoma in Barrett’s esophagus associated with achalasia: first case re-port. Pathol Int. 2002;52:141-146.

618. Roberts CC, Colby TV, Batts KP. Carcinoma of the stomach with hepatocyte differentiation (hepatoid adenocarcinoma). Mayo Clin Proc. 1997;72:1154-1160.

619. Minervini MI, Demetris AJ, Lee RG, et al. Utilization of hepatocyte-specific antibody in the immunocytochemical evaluation of liver tumors. Mod Pathol. 1997;10:686-692.

620. Lazaro J, Rubio D, Repolles M, Capote L. Hepatoid carcinoma of the ovary and management. Acta Obstet Gynecol Scand. 2007;86:498-499.

621. Hameed O, Xu H, Saddeghi S, Maluf H. Hepatoid carci-noma of the pancreas: a case report and literature review of a heterogeneous group of tumors. Am J Surg Pathol. 2007;31:146-152.

622. Yigit S, Uyaroglu MA, Kus Z, et al. Hepatoid carcinoma of the ovary: immunohistochemical finding of one case and literature review. Int J Gynecol Cancer. 2006;16:1439-1441.

623. Goodman ZD. Neoplasms of the liver. Mod Pathol. 2007;20(Suppl 1):S49-60.

624. Pan CC, Chen PC, Tsay SH, Chiang H. Cytoplasmic immunore-activity for thyroid transcription factor-1 in hepatocellular car-cinoma: a comparative immunohistochemical analysis of four commercial antibodies using a tissue array technique. Am J Clin Pathol. 2004;121:343-349.

625. Lei JY, Bourne PA, diSant’Agnese PA, Huang J. Cytoplasmic staining of TTF-1 in the differential diagnosis of hepatocellular carcinoma vs cholangiocarcinoma and metastatic carcinoma of the liver. Am J Clin Pathol. 2006;125:519-525.

626. Gaffey MJ, Traweek ST, Mills SE, et al. Cytokeratin expression in adrenocortical neoplasia: an immunohistochemical and bio-chemical study with implications for the differential diagnosis of adrenocortical, hepatocellular, and renal cell carcinoma. Hum Pathol. 1992;23:144-153.

627. Tsuji M, Kashihara T, Terada N, Mori H. An immunohisto-chemical study of hepatic atypical adenomatous hyperpla-sia, hepatocellular carcinoma, and cholangiocarcinoma with alpha-fetoprotein, carcinoembryonic antigen, CA19-9, epithe-lial membrane antigen, and cytokeratins 18 and 19. Pathol Int. 1999;49:310-317.

628. Pinkus GS, Kurtin PJ. Epithelial membrane antigen—a diag-nostic discriminant in surgical pathology: immunohistochemi-cal profile in epithelial, mesenchymal, and hematopoietic neo-plasms using paraffin sections and monoclonal antibodies. Hum Pathol. 1985;16:929-940.

629. Christensen WN, Boitnott JK, Kuhajda FP. Immunoperoxidase staining as a diagnostic aid for hepatocellular carcinoma. Mod Pathol. 1989;2:8-12.

630. Proca DM, Niemann TH, Porcell AI, DeYoung BR. MOC31 immunoreactivity in primary and metastatic carcinoma of the liver. Report of findings and review of other utilized markers. Appl Immunohistochem Mol Morphol. 2000;8:120-125.

631. Niemann TH, Hughes JH, De Young BR. MOC-31 aids in the differentiation of metastatic adenocarcinoma from hepatocellu-lar carcinoma. Cancer. 1999;87:295-298.

632. Porcell AI, De Young BR, Proca DM, Frankel WL. Immuno-histochemical analysis of hepatocellular and adenocarcinoma in the liver: MOC31 compares favorably with other putative markers. Mod Pathol. 2000;13:773-778.

633. Fucich LF, Cheles MK, Thung SN, et al. Primary vs metastatic hepatic carcinoma. An immunohistochemical study of 34 cases. Arch Pathol Lab Med. 1994;118:927-930.

634. Gottschalk-Sabag S, Ron N, Glick T. Use of CD34 and factor VIII to diagnose hepatocellular carcinoma on fine needle aspi-rates. Acta Cytol. 1998;42:691-696.

635. Tanigawa N, Lu C, Mitsui T, Miura S. Quantitation of sinusoid-like vessels in hepatocellular carcinoma: its clinical and prognostic significance. Hepatology. 1997;26:1216-1223.

636. Terada T, Nakanuma Y. Expression of ABH blood group antigens, receptors of Ulex europaeus agglutinin I, and fac-tor VIII-related antigen on sinusoidal endothelial cells in ad-enomatous hyperplasia in human cirrhotic livers. Hum Pathol. 1991;22:486-493.

637. Terada T, Nakanuma Y. Expression of ABH blood group an-tigens, Ulex europaeus agglutinin I, and type IV collagen in the sinusoids of hepatocellular carcinoma. Arch Pathol Lab Med. 1991;115:50-55.

638. Dhillon AP, Colombari R, Savage K, Scheuer PJ. An immuno-histochemical study of the blood vessels within primary hepato-cellular tumours. Liver. 1992;12:311-318.

591REFERENCES

639. Togni R, Bagla N, Muiesan P, et al. Microsatellite instability in hepatocellular carcinoma in non-cirrhotic liver in patients older than 60 years. Hepatol Res. 2009;39:266-273.

640. Chiappini F, Gross-Goupil M, Saffroy R, et al. Microsatellite in-stability mutator phenotype in hepatocellular carcinoma in non-alcoholic and non-virally infected normal livers. Carcinogenesis. 2004;25:541-547.

641. Wang L, Bani-Hani A, Montoya DP, et al. hMLH1 and hMSH2 expression in human hepatocellular carcinoma. Int J Oncol. 2001;19:567-570.

642. Llovet JM, Bruix J. Molecular targeted therapies in hepatocel-lular carcinoma. Hepatology. 2008;48:1312-1327.

643. Villanueva A, Chiang DY, Newell P, et al. Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology. 2008;135:1972-1983, 1983 e1971–e1911.

644. Guzman G, Alagiozian-Angelova V, Layden-Almer JE, et al. p53, Ki-67, and serum alpha feto-protein as predictors of he-patocellular carcinoma recurrence in liver transplant patients. Mod Pathol. 2005;18:1498-1503.

645. Jing Z, Nan KJ, Hu ML. Cell proliferation, apoptosis and the related regulators p27, p53 expression in hepatocellular carci-noma. World J Gastroenterol. 2005;11:1910-1916.

646. Sugo H, Takamori S, Kojima K, et al. The significance of p53 mutations as an indicator of the biological behavior of recurrent hepatocellular carcinomas. Surg Today. 1999;29:849-855.

647. Zhang T, Sun HC, Xu Y, et al. Overexpression of platelet-de-rived growth factor receptor alpha in endothelial cells of hepa-tocellular carcinoma associated with high metastatic potential. Clin Cancer Res Official J Am Assoc Cancer Res. 2005;11:8557-8563.

648. Berman MA, Burnham JA, Sheahan DG. Fibrolamellar carcino-ma of the liver: an immunohistochemical study of nineteen cases and a review of the literature. Hum Pathol. 1988;19:784-794.

649. Van Eyken P, Sciot R, Brock P, Casteels-Van Daele M, et al. Abundant expression of cytokeratin 7 in fibrolamellar carcino-ma of the liver. Histopathology. 1990;17:101-107.

650. Gornicka B, Ziarkiewicz-Wroblewska B, Wroblewski T, et al. Carcinoma, a fibrolamellar variant—immunohistochemical analysis of 4 cases. Hepatogastroenterology. 2005;52:519-523.

651. Garcia de Davila MT, Gonzalez-Crussi F, Mangkornkanok M. Fibrolamellar carcinoma of the liver in a child: ultrastructural and immunohistologic aspects. Pediatr Pathol. 1987;7:319-331.

652. Buckley AF, Burgart LJ, Kakar S. Epidermal growth factor re-ceptor expression and gene copy number in fibrolamellar hepa-tocellular carcinoma. Hum Pathol. 2006;37:410-414.

653. Thomas MB, Chadha R, Glover K, et al. Phase 2 study of er-lotinib in patients with unresectable hepatocellular carcinoma. Cancer. 2007;110:1059-1067.

654. Ikebe T, Wakasa K, Sasaki M, et al. Hepatocellular carcinoma with chondrosarcomatous variation: case report with immuno-histochemical findings, and review of the literature. J Hepatobi-liary Pancreat Surg. 1998;5:217-220.

655. Sasaki A, Yokoyama S, Nakayama I, et al. Sarcomatoid he-patocellular carcinoma with osteoclast-like giant cells: case report and immunohistochemical observations. Pathol Int. 1997;47:318-324.

656. Pan CC, Chen PC, Tsay SH, Ho DM. Differential immuno-profiles of hepatocellular carcinoma, renal cell carcinoma, and adrenocortical carcinoma: a systemic immunohistochemical survey using tissue array technique. Appl Immunohistochem Mol Morphol. 2005;13:347-352.

657. Van Eyken P, Sciot R, Paterson A, et al. Cytokeratin expression in hepatocellular carcinoma: an immunohistochemical study. Hum Pathol. 1988;19:562-568.

658. Wu PC, Fang JW, Lau VK, et al. Classification of hepatocellular carcinoma according to hepatocellular and biliary differentia-tion markers. Clinical and biological implications. Am J Pathol. 1996;149:1167-1175.

659. Hruban RH, Sturm PD, Slebos RJ, et al. Can K-ras codon 12 mutations be used to distinguish benign bile duct proliferations from metastases in the liver? A molecular analysis of 101 liver lesions from 93 patients. Am J Pathol. 1997;151:943-949.

660. Nakanuma Y, Sripa B, Vatanasapt V, et al. Intrahepatic cholan-giocarcinoma. In: Hamilton SR, Aaltonen LA, eds. Tumours of the Digestive System. Lyon: International Agency for Research on Cancer (IARC), 2000:173-180.

661. Haglund C, Lindgren J, Roberts PJ, Nordling S. Difference in tissue expression of tumour markers CA 19-9 and CA 50 in hepatocellular carcinoma and cholangiocarcinoma. Br J Cancer. 1991;63:386-389.

662. Ohshio G, Ogawa K, Kudo H, et al. Immunohistochemical stud-ies on the localization of cancer associated antigens DU-PAN-2 and CA19-9 in carcinomas of the digestive tract. J Gastroenterol Hepatol. 1990;5:25-31.

663. Loy TS, Sharp SC, Andershock CJ, Craig SB. Distribution of CA 19-9 in adenocarcinomas and transitional cell carcinomas. An immunohistochemical study of 527 cases. Am J Clin Pathol. 1993;99:726-728.

664. Aishima S, Kuroda Y, Nishihara Y, et al. Gastric mucin pheno-type defines tumour progression and prognosis of intrahepatic cholangiocarcinoma: gastric foveolar type is associated with ag-gressive tumour behaviour. Histopathology. 2006;49:35-44.

665. Cazals-Hatem D, Rebouissou S, Bioulac-Sage P, et al. Clinical and molecular analysis of combined hepatocellular- cholangiocarcinomas. J Hepatol. 2004;41:292-298.

666. Gutgemann I, Haas S, Berg JP, Zhou H, Buttner R, Fischer HP. CD56 expression aids in the differential diagnosis of cholan-giocarcinomas and benign cholangiocellular lesions. Virchows Arch. 2006;448:407-411.

667. Kozaka K, Sasaki M, Fujii T, et al. A subgroup of intrahepatic cholangiocarcinoma with an infiltrating replacement growth pattern and a resemblance to reactive proliferating bile ductules: ‘bile ductular carcinoma’. Histopathology. 2007;51:390-400.

668. Stroescu C, Herlea V, Dragnea A, Popescu I. The diagnostic val-ue of cytokeratins and carcinoembryonic antigen immunostain-ing in differentiating hepatocellular carcinomas from intrahe-patic cholangiocarcinomas. J Gastrointest Liver Dis. 2006;15:9-14.

669. Lodi C, Szabo E, Holczbauer A, et al. Claudin-4 differenti-ates biliary tract cancers from hepatocellular carcinomas. Mod Pathol. 2006;19:460-469.

670. Loy TS, Quesenberry JT, Sharp SC. Distribution of CA 125 in adenocarcinomas. An immunohistochemical study of 481 cases. Am J Clin Pathol. 1992;98:175-179.

671. Nakajima T, Tajima Y, Sugano I, et al. Intrahepatic cholan-giocarcinoma with sarcomatous change. Clinicopathologic and immunohistochemical evaluation of seven cases. Cancer. 1993;72:1872-1877.

672. Imazu H, Ochiai M, Funabiki T. Intrahepatic sarcomatous chol-angiocarcinoma. J Gastroenterol. 1995;30:677-682.

673. Sumiyoshi S, Kikuyama M, Matsubayashi Y, et al. Carcinosar-coma of the liver with mesenchymal differentiation. World J Gastroenterol. 2007;13:809-812.

674. Honda M, Enjoji M, Sakai H, et al. Case report: intrahepatic cholangiocarcinoma with rhabdoid transformation. J Gastroen-terol Hepatol. 1996;11:771-774.

675. Nomura K, Aizawa S, Ushigome S. Carcinosarcoma of the liver. Arch Pathol Lab Med. 2000;124:888-890.

676. Lim BJ, Kim KS, Lim JS, et al. Rhabdoid cholangiocarcinoma: a variant of cholangiocarcinoma with aggressive behavior. Yonsei Med J. 2004;45:543-546.

677. Kanamoto M, Yoshizumi T, Ikegami T, et al. Cholangiolocel-lular carcinoma containing hepatocellular carcinoma and chol-angiocellular carcinoma, extremely rare tumor of the liver:a case report. J Med Invest. 2008;55:161-165.

678. Wakasa T, Wakasa K, Shutou T, et al. A histopathological study on combined hepatocellular and cholangiocarcinoma: cholan-giocarcinoma component is originated from hepatocellular car-cinoma. Hepatogastroenterology. 2007;54:508-513.

679. Taguchi J, Nakashima O, Tanaka M, et al. A clinicopathologi-cal study on combined hepatocellular and cholangiocarcinoma. J Gastroenterol Hepatol. 1996;11:758-764.

680. Tickoo SK, Zee SY, Obiekwe S, et al. Combined hepatocellular-cholangiocarcinoma: a histopathologic, immunohistochemical, and in situ hybridization study. Am J Surg Pathol. 2002;26:989-997.

681. Jarnagin WR, Weber S, Tickoo SK, et al. Combined hepatocel-lular and cholangiocarcinoma: demographic, clinical, and prog-nostic factors. Cancer. 2002;94:2040-2046.

682. Siren J, Karkkainen P, Luukkonen P, et al. A case report of bili-ary cystadenoma and cystadenocarcinoma. Hepatogastroenter-ology. 1998;45:83-89.


683. Yanase M, Ikeda H, Ogata I, et al. Primary smooth muscle tu-mor of the liver encasing hepatobiliary cystadenoma without mesenchymal stroma. Am J Surg Pathol. 1999;23:854-859.

684. Terada T, Nakanuma Y, Ohta T, et al. Mucin-histochemical and immunohistochemical profiles of epithelial cells of several types of hepatic cysts. Virchows Arch A Pathol Anat Histo-pathol. 1991;419:499-504.

685. Maruyama S, Hirayama C, Yamamoto S, et al. Hepatobiliary cystadenoma with mesenchymal stroma in a patient with chronic hep-atitis C. J Gastroenterol. 2003;38:593-597.

686. Scott FR, More L, Dhillon AP. Hepatobiliary cystadenoma with mesenchymal stroma: expression of oestrogen receptors in for-malin-fixed tissue. Histopathology. 1995;26:555-558.

687. Grayson W, Teare J, Myburgh JA, Paterson AC. Immuno-histochemical demonstration of progesterone receptor in hepatobiliary cystadenoma with mesenchymal stroma. Histopa-thology. 1996;29:461-463.

688. Leger-Ravet MB, Borgonovo G, Amato A, et al. Carcinosarco-ma of the liver with mesenchymal differentiation: a case report. Hepatogastroenterology. 1996;43:255-259.

689. Lao XM, Chen DY, Zhang YQ, et al. Primary carcinosarcoma of the liver: clinicopathologic features of 5 cases and a review of the literature. Am J Surg Pathol. 2007;31:817-826.

690. Balta Z, Sauerbruch T, Hirner A, et al. [Primary neuroendo-crine carcinoma of the liver: From carcinoid tumor to small-cell hepatic carcinoma: case reports and review of the literature.]. Pathologe. 2008;29:53-60.

691. Cho MS, Lee SN, Sung SH, Han WS. Sarcomatoid hepatocel-lular carcinoma with hepatoblastoma-like features in an adult. Pathol Int. 2004;54:446-450.

692. Wang JH, Dhillon AP, Sankey EA, et al. ‘Neuroendocrine’ differentiation in primary neoplasms of the liver. J Pathol. 1991;163:61-67.

693. Lerut JP, Orlando G, Adam R, et al. The place of liver trans-plantation in the treatment of hepatic epitheloid hemangioendo-thelioma: report of the European liver transplant registry. Ann Surg. 2007;246:949-957, discussion 957.

694. Ishak KG, Anthony PP, Niederau C, Nakanuma Y. Mesenchy-mal tumours of the liver. In: Hamilton SR, Aaltonen LA, eds. Tumours of the Digestive System. Lyon, France: International Agency for Research on Cancer (IARC); 2000:191-198.

695. Zamboni G, Pea M, Martignoni G, et al. Clear cell “sugar” tu-mor of the pancreas. A novel member of the family of lesions characterized by the presence of perivascular epithelioid cells. Am J Surg Pathol. 1996;20:722-730.

696. Nonomura A, Minato H, Kurumaya H. Angiomyolipoma pre-dominantly composed of smooth muscle cells: problems in histologi-cal diagnosis. Histopathology. 1998;33:20-27.

697. Nishio J, Iwasaki H, Sakashita N, et al. Undifferentiated (em-bryonal) sarcoma of the liver in middle-aged adults: smooth muscle differentiation determined by immunohistochemistry and electron microscopy. Hum Pathol. 2003;34:246-252.

698. Stocker JT, Ishak KG. Undifferentiated (embryonal) sarcoma of the liver: report of 31 cases. Cancer. 1978;42:336-348.

699. Miettinen M, Kahlos T. Undifferentiated (embryonal) sarcoma of the liver. Epithelial features as shown by immunohistochemi-cal analysis and electron microscopic examination. Cancer. 1989;64:2096-2103.

700. Aoyama C, Hachitanda Y, Sato JK, et al. Undifferentiated (embryonal) sarcoma of the liver. A tumor of uncertain histo-genesis showing divergent differentiation. Am J Surg Pathol. 1991;15:615-624.

diagnostic immunohistochemistry || immunohistology of the pancreas, biliary tract, and liver

Documents