FNAB Cytology

The reported sensitivity for percutaneous pancreatic FNAB, with the use of a variety of guidance techniques, ranges from 45% to 97%. The specificity is almost 100%. Diagnostic accuracy ranges from 75% to 100%.²³ The low sensitivity and low negative predictive values are cited as major limitations of pancreatic FNAB.²³ However, in our experience, the sensitivity for a diagnosis of adenocarcinoma is greater than 90% when a cytotechnologist is on site to ensure specimen adequacy.¹² The sensitivity for EUS-FNAB has ranged from 60% to 96% in large series, with a specificity of almost 100%.^24–28

Factors that affect the accuracy of the procedure include accurate localization of the mass by the operator, adequate sampling of the mass, correct sample preparation, and correct interpretation by the pathologist. Accurate localization and adequate sampling are highly dependent on the skill of the operator. The sample should be representative of the intended target and should contain a sufficient number of cells for proper interpretation. Certain lesions may not yield diagnostic material regardless of the skill of the operator. These include most cystic lesions and sclerotic, vascular, or necrotic tumors. Aspiration of the periphery of necrotic tumors may help obtain better-preserved material. Poor sample preparation also hinders accurate interpretation by the pathologist. Factors that affect interpretation include blood, air-drying artifact on smears intended for alcohol fixation, crush artifact, and smears that are spread too thickly.

Preparation of FNAB Specimens

Preparation methods used for FNAB samples include direct smears, cytospins (Shandon CytoSpin, Thermo Fisher Scientific, Waltham, Mass.), liquid-based preparations such as ThinPrep or SurePath Prep (TriPath, Burlington, N.C.), and cellblocks. Guidelines for preparation of the various sample types follow.

Aneuploidy and Chromosomal Analyses

Two advanced cytologic techniques for detecting aneuploidy—digital image analysis (DIA) and fluorescence in situ hybridization (FISH)—are reported to improve the sensitivity of brushing cytology to diagnose adenocarcinoma in pancreatobiliary strictures. In one study, the authors confirmed that conventional cytology has a low sensitivity (4% to 20%) but 100% specificity. When conventional cytology results were negative, FISH had an increased sensitivity (35% to 60%) in assessing for chromosomal gains (polysomy) while preserving the specificity of cytology. The sensitivity and specificity of DIA was intermediate compared with routine cytology and FISH but was additive to FISH values, demonstrating only trisomy of chromosome 7 or chromosome 3.⁴⁶ When the panel developed to detect urothelial carcinoma (UroVysion panel) was used, FISH alone increased the sensitivity of routine cytology.^47,48 In another study, FISH had a sensitivity of 94%, compared with 81% for routine cytomorphology.⁴⁹ A technique of intraductal ultrasound combined with standard cytology, DIA, and FISH enhances the accuracy of standard techniques in the evaluation of indeterminate bile duct strictures, allowing diagnosis of malignancy in a substantial number of patients with false-negative cytology.⁵⁰

The combination of KRAS mutation and FISH analyses appears to increase the cancer detection rate in patients with pancreatobiliary strictures⁵¹ beyond that of cytology and FISH alone. KRAS mutation analysis was performed on residual cytology specimens by using the quantitative, polymerase chain reaction (PCR)-based DxS KRAS Mutation Test Kit (DxS Ltd., Manchester, U.K.) in patients who also had cytologic evaluation and FISH analysis of their brushing specimens. KRAS mutations were identified in 24 (69%) of pancreatic adenocarcinoma specimens, and FISH results were positive in 22 (63%), with a combined sensitivity of 86% (30/35). KRAS mutations and polysomic FISH results were identified in fewer cholangiocarcinoma patients—in 12 (29%) and 17 (41%) of cholangiocarcinoma specimens, respectively—with a combined sensitivity of 54% (22/41).

Protein Analysis

Some proteins reportedly increase the sensitivity of brushing cytology for detection of adenocarcinoma. Insulin-like growth factor-II mRNA-binding protein 3 (IMP3 cytology), when used alone, showed a sensitivity of 64.1% for diagnosis of adenocarcinoma; the sensitivity rose to 74.1% when combined with routine cytology. Cytology alone showed a sensitivity of 33.3%.⁵² S100 protein P has been reported to be upregulated in cholangiocarcinoma.⁵³ Analysis of S100P expression by reverse transcriptase (RT)-PCR showed that this protein was frequently expressed in adenocarcinomas of the biliary tract but not in normal tissues. K-homology domain containing protein overexpressed in cancer (KOC) and S100A4 are two additional proteins evaluated in biliary brushing specimens. The authors of this study assessed protein expression on alcohol-fixed smears and included cases that were negative, indeterminate, and diagnostic for malignancy. In this study, the sensitivity and specificity were, respectively, 83% and 95% for cytology, 92% and 95% for KOC, and 79% and 95% for S100A4 protein. The combined use of KOC and S100A4 showed a sensitivity of 100% and a specificity of 95%.⁵⁴

Ductal Cells

The lining of the large pancreatic ducts is composed of columnar epithelium arranged in flat, honeycombed, two-dimensional sheets of cells with centrally located nuclei (Fig. 35.15). The ductal cells may also be present in a palisaded, “picket-fence” arrangement in which the nuclei are basally located. In contrast to the epithelium of the large ducts, intralobular duct epithelium is composed of cuboidal-shaped cells with scant basophilic cytoplasm and are usually present as flat sheets, small clusters, or tubular structures.

Acinar Cells

Aspirates of the normal pancreas consist predominantly of acinar-type epithelium, which is typically arranged either singly or in small acinar structures (Fig. 35.16). The cells are pyramidal or triangular in shape and contain abundant, granular cytoplasm with numerous intracytoplasmic zymogen granules. The nuclei are round, have a granular chromatin pattern, contain prominent nucleoli, and are either central or eccentrically located.

Islet Cells

Islet cells are only rarely detected in aspirates of the normal pancreas. More often, they are seen in aspirates from patients with chronic pancreatitis with islet cell hyperplasia. When present, they occur as loose aggregates of cells that contain wispy, ill-defined amphophilic cytoplasm and oval nuclei with a stippled chromatin pattern (Fig. 35.17).

Acute Pancreatitis

Typical smears from patients with acute pancreatitis show dirty, necrotic background cells, cellular debris, and fat necrosis with saponification. Acute inflammation may be prominent. The normal pancreatic elements, when present, may show evidence of necrosis and degeneration (Fig. 35.18). Pancreatic ductal epithelium may show various degrees of reactive atypia as well (see Box 35.8).

Chronic Pancreatitis

Aspirates of chronic pancreatitis are not typically very cellular; they consist of varying amounts of inflammatory cells, ductal cells, acinar cells, calcifications, fibrous tissue fragments (Fig. 35.19), and debris.⁵⁵ Islet cells are sparse or absent. The inflammatory component usually consists of mononuclear cells and histiocytes. Acinar cells may be associated with inflammation and fibrosis. The ductal cells show mild atypia but remain monolayered and organized (see Box 35.8).

Autoimmune Pancreatitis

FNAB smears show a paucity of ductal epithelium and are composed of inflammatory cells and stromal fragments. The key feature that helps distinguish this process from others is the presence of cellular stromal fragments with embedded lymphocytes.⁵⁶ A dense plasmacytic infiltrate and eosinophils are also common components of this process, but a lymphocytic background is rare.

Reactive Atypia

Reactive atypia induced by pancreatitis or other processes shows a spectrum of changes (Fig. 35.20). When severe, the atypia may mimic adenocarcinoma. A key feature is that the atypical groups are fewer in number than the number of groups associated with ductal adenocarcinoma. Another feature is that there is usually a range of atypia in the ductal groups, from benign to malignant. The differentiation of benign and reactive processes from adenocarcinoma is discussed later (see Differential Diagnosis and Pitfalls).

Diagnostic Criteria

Several studies have evaluated a number of cytomorphologic criteria in pancreatic FNAB specimens to identify a set of minimal cytologic criteria that can reliably separate benign processes from adenocarcinoma.^57–59 Although the criteria identified as most accurate vary and their accuracy has not been proven in a prospective study, it is clear that a systematic approach incorporating these criteria leads to improved diagnostic accuracy. The criteria can be categorized into cellular composition and cellularity, architectural features, background, cytoplasmic features, and nuclear features and mitotic activity (Box 35.9).

Cellular Composition

Benign pancreas is composed of a mixture of ductal cells and acinar cells, whereas PDA is usually composed of a relatively pure population of ductal-type cells (Fig. 35.21). This fact is most useful in the examination of aspirates obtained at laparotomy, when it is presumed that the surgeon has inserted the needle directly into the mass. Care must be taken in EUS-FNAB not to misinterpret gastrointestinal contamination as lesional.

Architectural Features

Architectural features are the most helpful aspect. Malignant groups lose the normal, honeycomb, two-dimensional patterns of ductal epithelium. Architectural atypia is manifested as three-dimensionality, crowding and overlapping of the nuclei (Fig. 35.22, A and B), or an exaggerated (drunken) honeycomb sheet arrangement (see Fig. 35.22, C). Cribriforming is also a feature of malignancy. Cells may be arranged in sheets or in small clusters or groups. Other helpful features include loss of polarity and nuclear molding (see Fig. 35.22, A and B). Loss of cohesion and single cells (see Fig. 35.22, D) are a feature of malignancy, but well-differentiated carcinomas may retain their cohesion and not have any malignant single cells.

Background

Coagulative necrosis is typical of malignancy but is more frequent in higher-grade carcinomas (Fig. 35.23, A). Well-differentiated carcinomas may have a clean background (see Fig. 35.22, A through C). Other features that can be assessed as part of the background are inflammation (see Fig. 35.23, B), secretory products, and cellular dyshesion.

Cytoplasmic Features

Because normal pancreatic ductal cells do not display visible cytoplasmic mucin, the presence of abundant cytoplasmic mucin (as in Fig. 35.22, C) is a feature of a neoplastic process. In the setting of a radiologically detected mass lesion, it is a feature that supports a malignant interpretation. The cells may appear as columnar cells seen on edge with columnar, mucin-containing cytoplasm (Fig. 35.24, A), or the cells may show abnormal cytoplasmic mucin vacuoles (see Fig. 35.24, B).

Undifferentiated (Anaplastic) Adenocarcinoma

The cytologic features (Box 35.10) of undifferentiated adenocarcinoma have been described. Smears are usually very cellular. The carcinoma is composed of mononucleated and multinucleated cells. The mononuclear cells appear singly or in small clusters and can range from medium-sized, polygonal epithelioid cells with clear cytoplasm to large, bizarre sarcomatoid cells with dense and/or spindled cytoplasm. The multinucleated cells form bizarre giant cells^60,61 (Fig. 35.25). Cytophagocytosis, tumor necrosis, and marked inflammation are additional common findings.

Undifferentiated carcinomas are usually easily recognizable as malignant. However, the differential diagnosis includes metastatic undifferentiated carcinomas, sarcomas, and melanomas. Immunohistochemistry will separate these various entities.

Undifferentiated Carcinoma with Osteoclast-Like Giant Cells

Undifferentiated carcinoma with osteoclast-like giant cells, a variant of PDA, is composed of malignant, usually mononuclear, epithelial cells admixed with benign osteoclast-type giant cells. Aspirate smears are typically hypercellular with two cell populations—an obviously malignant mononuclear epithelial proliferation, similar to that as described in undifferentiated carcinoma, and benign-appearing osteoclast-like giant cells (Fig. 35.26; see Box 35.10). The giant cells vary in number and often contain 10 or more bland appearing, centrally clustered and slightly overlapping nuclei with even chromatin and occasionally prominent nucleoli.^62–65

Adenosquamous carcinoma is the most common variant of PDA. These tumors are typically high grade; aspirate smears typically show moderate to high cellularity and necrosis. The squamous component may predominate, with focal intracellular mucin or glandular groups of cells as evidence of glandular differentiation. Atypical, keratinized squamous cells may represent the only evidence of squamous differentiation in a predominantly glandular tumor⁶⁶ (Fig. 35.27). Without keratin, the squamous cells show dense cytoplasm. The cell block may show classic components as well (Boxes 35.11 and 35.12). The differential diagnosis includes metastatic squamous cell carcinoma.

Colloid (Mucinous Noncystic) Carcinoma

The classic cytomorphologic appearance of colloid carcinoma is that of malignant glandular cells floating in thick, background mucin (Fig. 35.28; see Box 35.12). It may be difficult to distinguish this carcinoma from metastases of similar tumors from the lung, colon, or breast, particularly in patients with a history of carcinoma at these sites, or from intracystic or intraductal mucinous neoplasms. When the FNAB is obtained from a solid mass, the diagnosis of this invasive process can be made. Uncertainty may occur if a similar pattern is found in material aspirated from what is described as a cyst or dilated duct, in which case the differential includes MCN and IPMN. The background mucin and malignant features of the glandular epithelium distinguish this process from gastrointestinal contamination. In the absence of neoplastic epithelium, the diagnosis cannot be rendered.

Medullary Carcinoma

The FNAB features of medullary carcinoma have not been reported. One case encountered at the Moffitt Cancer Center revealed syncytial groups composed of neoplastic cells with rounded to oval nuclei and prominent nucleoli (Fig. 35.29). Lymphocytes and plasma cells were associated with the neoplastic cells (Box 35.14).

Box 35.14

Cytologic Features of Medullary Carcinoma

Syncytial group

Scant, ill-defined cytoplasm

Round to oval nuclei with prominent nucleoli

Lymphocytic infiltrate

Differential Diagnosis and Pitfalls

Key diagnostic problems include differentiating adenocarcinoma from benign or reactive epithelium (Table 35.4) and differentiating primary from metastatic disease. Identification of well-differentiated adenocarcinoma can be particularly problematic because the cytomorphologic features of well-differentiated carcinoma are subtle. Crowded, hypercellular groups are the most frequent finding. The cells show nuclear enlargement and significant loss of polarity. Moderately and poorly differentiated adenocarcinomas are easier to diagnose (see Table 35.4), although on an inflamed smear, the differential may include reactive ductal groups. Variants of PDA are usually recognized as malignant; the differential diagnosis is with metastatic disease from other sites that produce carcinomas with similar features. In this case, immunohistochemistry is indicated.

Ancillary Studies

Indications for the use of ancillary studies when dealing with ductal-type neoplasms are to differentiate benign from malignant disease or to differentiate primary from metastatic disease.

Studies have focused on assessment of samples for molecular alterations identified in PDA. KRAS, the first mutation analyzed in PDA and has been the most frequently assessed mutation in small biopsy samples. In two studies that evaluated fine needle aspiration, the addition of KRAS mutation analysis to standard cytomorphology increased the detection of adenocarcinoma.^67,68 KRAS lacks specificity, however, because it is expressed in early lesions before significant morphological alterations occur, leading to false-positive results. Other studies have focused on other proteins altered in pancreatic carcinogenesis. Loss of DPC4 protein expression can serve as a marker of malignancy in FNAB samples, because benign tissues retain expression of DPC4. However, only 52% of PDA cases show loss of expression, so it is not a reliable marker by itself.⁶⁹ An approach with a panel of markers, such as analysis of KRAS mutations and loss of protein expression for TP53 and DPC4, is more sensitive and more specific.⁷⁰

In recent years, there has been an explosion in the identification of genes and their related proteins involved in pancreatic carcinogenesis.⁷¹ Examples of these markers include prostate stem cell antigen (PSCA) and mesothelin,⁷² von Hippel-Lindau gene product (pVHL) and S100P,⁷³ KOC,^74,75 IMP3,^76–78 and the mucins MUC1 and MUC2.⁷⁹ A recent study analyzed a large number of markers that are reported as useful for diagnosing PDA. This study found that the combination of pVHL (which is not expressed in PDA), mammary serine protease inhibitor (maspin), S100P, and IMP3 was best for diagnosing PDA on samples obtained by fine-needle aspiration.⁸⁰ Although these results are promising, to date none have been validated for routine use.

Analysis of microRNA profiles is promising because this technology is easily applied to the small samples obtained from FNABs. PDA has been found to have restricted microRNA patterns that can differentiate it from benign conditions of the pancreas.⁸¹

Because the pancreas is a site of metastasis, the immunohistochemical work-up may need to be geared toward differentiating primary from metastatic disease. This may be more of an issue with the variants of PDA, although adenocarcinoma from other sites can also metastasize to the pancreas and create a diagnostic dilemma.

Well-Differentiated Pancreatic Neuroendocrine Tumors

Aspirates of well-differentiated PanNET (WDPanNET) are very cellular. They are composed of a monomorphic, dyshesive population of cells occurring most often as single cells but also in loosely cohesive clusters (Fig. 35.30, A) that may occasionally show pseudorosettes. These patterns are often accompanied by stripped nuclei. The nuclei are round to oval, and the chromatin pattern has a characteristic salt-and-pepper appearance. Nucleoli are variably present but are prominent in some tumors. The cytoplasm varies in quantity and quality. It may be scant and wispy,^77,82 plasmacytoid, or vacuolated, as in the clear cell variant.⁸³ Less common variants include oncocytic and rhabdoid variants. The vascularity of WDPanNETs may be seen on the corresponding cytology smears and is characterized by vascular cores with loosely attached neoplastic cells (Box 35.15; see Fig. 35.30, B).

The immunoprofile of WDPanNET is characterized by expression of cytokeratins and markers of neuroendocrine differentiation, including synaptophysin, chromogranin, and neural cell adhesion molecule 1 (CD56). These neoplasms also show expression of a variety of hormones, but immunohistochemical analysis for hormone expression does not add further value to the diagnosis or prognosis.

The key entities in the differential diagnosis of WDPanNET include islet cell hyperplasia and a number of neoplasms, specifically ACC, SPN, adenocarcinoma (i.e., PDA), lymphomas, and metastases to the pancreas (e.g., melanoma, renal cell carcinoma). WDPanNET, ACC, SPN, and PB have many characteristics in common; their differential diagnosis is summarized at the end of this section and in Table 35.5.

WDPanNET may occasionally be mistaken for adenocarcinoma, if the nuclei have prominent nucleoli and show significant size variation, and the groups are cohesive with crowding or some pseudorosettes. Features that distinguish WDPanNET from PDA are that the nuclei retain a rounded or oval appearance and lack the nuclear angulations, sharpness, and convolutions seen in PDA. PDA tends to be more cohesive, particularly when well differentiated, and shows evidence of glandular differentiation.

The stripped nuclei and dyshesive pattern of WDPanNET raises the differential diagnosis with non-Hodgkin lymphomas (NHL). Lymphomas are characterized by lymphoglandular bodies in the background. Flow cytometry identifies a monoclonal lymphoid population. Melanoma⁸⁴ and plasmacytoma⁸⁵ may also be considered in the differential diagnosis of WDPanNET because of their plasmacytoid morphology. Melanoma is typically much more pleomorphic and has intranculear inclusions and prominent nucleoli. The presence of melanin pigment is diagnostic. Plasmacytomas show a perinuclear hof (Golgi zone) and a clock-face nuclear chromatin pattern, distinguishing these cells from WDPanNET. The pancreas is a common site for metastasis from renal cell carcinoma, which is also a vascular tumor and may have stripped nuclei, similar to WDPanNET. Renal cell carcinoma has much more nuclear pleomorphism and clear cell cytoplasm. There should also be a history of a renal mass or of renal cell carcinoma. Immunohistochemistry is beneficial in sorting out these various neoplasms.

Poorly Differentiated Pancreatic Neuroendocrine Carcinoma

Poorly differentiated PanNETs (PDPanNETs) resemble poorly differentiated neuroendocrine carcinomas from other sites, such as small cell carcinoma (Box 35.16). The cytomorphologic features are those of fusiform, small cells with molding, abundant necrosis, and mitotic activity (Fig. 35.31). These neoplasms are as aggressive as pulmonary small cell carcinomas. Large cell neuroendocrine carcinomas show vesicular nuclei with more abundant cytoplasm and prominent mitotic activity.

Acinar Cell Carcinoma

ACC typically produces moderately cellular smears with cohesive clusters, single cells, and stripped nuclei.⁸⁶ The background is clean but may contain cytoplasmic granules from the stripped cytoplasm of tumor cells. The tumor cells, when cohesive, have a number of architectural patterns, including clusters and an organoid appearance (Fig. 35.32, A). The cohesive groups lose their normal architectural arrangements and may form crowded, three-dimensional groups. These neoplasms are vascular, and vessels may be prominent on the smears (see Fig. 35.32, B). The individual acinar carcinoma cells are polygonal and uniform in size and shape and have a low N : C ratio. The nuclear chromatin in the tumor cells is generally coarsely clumped, and usually one, or sometimes two, prominent nucleoli are seen. Cytoplasmic granularity resulting from the zymogen granules is seen on Diff-Quik smears as vacuoles or negative images. Poorly differentiated ACC shows more prominent nucleoli and more significant nuclear pleomorphism and nuclear membrane irregularity (Box 35.17). The immunoprofile of ACC is characterized by expression for cytokeratins, trypsin, chymotrypsin, Bcl-10, and CD10 and nuclear Beta-catenin expression due to mutations in the Beta-catenin pathways. They express other pancreatic enzyme markers, but these are not as frequently assessed.

The cytologic differential diagnosis includes PanNET, PB, SPN, and non-neoplastic acinar parenchyma. The differential diagnosis with PanNET, SPN, and PB is discussed at the end of this section. As for PanNET, the differential diagnosis for the stripped nuclei seen in ACC includes NHL, but in ACC the stripped nuclei are the same as the nuclei in the intact tumor cells and not of lymphocytic origin. Furthermore, the smear of the ACC lacks the background lymphoglandular bodies seen in NHL.

FNAB of benign pancreas may yield abundant benign acinar cells, and these may be misinterpreted as ACC. The organoid, cohesive, grapelike pattern of normal acinar cells hints at their benign nature. Normal acinar cells form organized, uniform, grapelike clusters, either singly or attached to a fibrovascular matrix; this pattern provides a more monomorphic appearance than ACC on low power scanning. Benign acinar cells retain a very low N : C ratio, and groups do not show any crowding.

Solid-Pseudopapillary Neoplasm

SPN of the pancreas has a characteristic cytologic picture. Smears are typically quite cellular and contain numerous branching, papillary-like groups of epithelial cells with a central fibrovascular core. The papillary fronds have three layers: a central vessel, a mucoid stromal layer, and an outer neoplastic layer (Fig. 35.33, A). The layer of neoplastic epithelium is composed of monomorphic epithelial cells with round to oval nuclei, finely dispersed chromatin, nuclear indentations or grooves, and small nucleoli. The cytoplasm of these cells is basophilic, varies in shape and quantity, may be vacuolated or may contain periodic acid–Schiff (PAS)-positive granules, and may demonstrate cytoplasmic tails (see Fig. 35.33, B). The pathognomonic feature is “balls” of mucoid stroma that may or may not contain a rim of epithelial cells⁸⁷ (Box 35.18; see Fig. 35.33, C). The characteristic immunohistochemical profile is strong, and diffuse expression of vimentin, neuron-specific enolase (NSE), α₁-antitrypsin, and alpha1-antichymotrypsin chymotrypsin, and CD10 and nuclear expression of β-catenin.^88,89 These neoplasms also express progesterone receptors. They are variably positive for cytokeratins and neuroendocrine markers and negative for e-cadherin.⁹⁰

PanNET, ACC, and PB are the key entities in the differential diagnosis. The differential diagnosis with these entities is discussed at the end of this section.

Pancreatoblastoma

PB produces cellular smears with both cell clusters and single cells. The appearance of the epithelial cells varies depending on the direction of differentiation.^91–93 Cells with acinar differentiation have an oval to cuboidal shape, round central to eccentric nuclei, one or more small nucleoli, and a moderate amount of granular cytoplasm closely resembling ACC (Fig. 35.34, A and B). This component cannot be reliably differentiated from ACC. Cells with endocrine differentiation can resemble acinar cells but demonstrate a more cuboidal shape; a higher N : C ratio; denser, less granular cytoplasm; and less conspicuous nucleoli. In general, however, the tumor cell differentiation is difficult to appreciate clearly on smears. The distinguishing feature of these neoplasms is the squamoid nest or corpuscle, which is best recognized in cell block preparations. Stromal fragments may be scant or prominent with traversing capillaries (Box 35.19; see Fig. 35.34, A).

The immunophenotype depends on the components present. These neoplasms may show histochemical and immunohistochemical features of acinar differentiation, neuroendocrine differentiation, ductal differentiation, or squamous differentiation. Markers of acinar and neuroendocrine differentiation have already been discussed. The squamous areas may express markers of squamous differentiation such as cytokeratins CK5/6 and protein p63. The ductal areas show cytoplasmic mucin demonstrated by special stains for mucin, such as mucicarmine.

Differential Diagnosis of PanNET, ACC, SPN, and PB

These nonductal pancreatic neoplasms can be differentiated based on assessment of their cytomorphologic features and immunohistochemical findings (see Table 35.5). The cytomorphologic features to consider are nuclear, cytoplasmic, and stromal. The nuclei of WDPanNET show a salt-and-pepper chromatin pattern; the nuclei of SPN are subtly more irregular and pale, with grooves and micronucleoli; and the nuclei of ACC and PB with acinar differentiation may show prominent nucleoli. The cytoplasm of WDPanNET is variable in amount and quantity; when it is plasmacytoid, this neoplasm may be more readily recognized. The cytoplasm of ACC shows the presence of granularity due to zymogen granules. The cytoplasm of PB may be similar to that of ACC. Only the cytoplasm of PB will show squamous differentiation in the form of squamous corpuscles of nests. The cytoplasm of SPN may show tails, vacuoles, or inclusions, which are unique to this neoplasm.

All of these neoplasms are vascular, and a vascular stromal component will be present on the smears to a variable extent. However, only SPN shows a layer of mucoid stroma surrounding the capillaries in the fibrovascular cores. This mucoid stroma, when identified, is pathognomonic for SPN.

When the cytomorphologic features do not provide a clear diagnosis, immunohistochemical studies are indicated. A panel consisting of cytokeratins, vimentin, neuroendocrine markers, pancreatic enzymes, CD10, progesterone receptor, e-cadherin, and β-catenin can differentiate among these tumors (see Table 35.5).

Mucicarmine stains and Alcian blue stain aid in identifying background material as mucinous when its nature is questionable. However, mucin from gastrointestinal contaminants may lead to a false positive interpretation.⁹⁷ The presence of abundant, thick background mucin or cells containing mucin lead in the direction of an MCN or IPMN and excludes serous cystadenoma and pseudocyst. If the sample is obviously mucinous, mucin stains are not indicated.

Ciliated Foregut Cyst

Aspirates from ciliated foregut cysts are characterized by ciliated columnar cells and detached ciliary tufts. The background fluid contains amorphous debris and rare macrophages.¹¹⁴ The differential diagnosis includes cystic neoplasms. The presence of ciliated cells is diagnostic of this entity.

Lymphoepithelial Cyst of the Pancreas

LECP is typically discovered incidentally. These lesions have a very typical appearance on computed tomography. They show a well-circumscribed, loculated or lobulated mass with low attenuation and with a density equal to water. TUS shows an inhomogeneous echogenic mass with posterior enhancement.¹¹⁵ EUS imaging shows a hypoechoic, unilocular or multilocular mass possibly with hyperechoic, sludgelike material in the cyst.¹¹⁶ Aspirates of LECP usually yield a variable amount of pasty, yellow-gray or yellow-white material.¹¹⁷ The smears show abundant anucleated squamous cells, keratinous debris , and few nucleated squamous cells. The background may also contain lymphocytes, histiocytes, background debris, and cholesterol clefts and crystals (Fig. 35.36, A).^118,119 The cell block may show squamous cells with a preserved granular layer (see Fig. 35.36, B). This cytologic finding is diagnostic (Box 35.21).

Measurement of cyst fluid levels may lead to misinterpretation of an LECP as a MCN or IPMN, because LECP may produce high levels of cyst fluid CEA, and the cancer antigen, CA 19-9. The reported range of CEA levels is 2700 to 35,028 ng/mL.^{104,119–121} The cyst fluid amylase levels range from normal to 480 U/L.^{104,119,122,123} The cyst fluid CA 19-9 level may be elevated,^121,122 reportedly as high as 5 × 10⁶ U/mL or greater.¹⁰⁴ The wide range of these test results helps to emphasize why cytology is considered the gold standard diagnostic method.

The differential diagnosis of LECP includes pseudocysts and cystic neoplasms. The presence of squamous material within the cytology sample helps exclude these other entities. The morphologic differential diagnosis includes any other entity with a squamous component, such as squamous metaplasia, squamous cyst of the pancreas, dermoid cyst, epidermoid cyst involving intrapancreatic accessory spleen, adenosquamous carcinoma, and metastatic squamous cell carcinoma. Squamous metaplasia occurs mainly in association with chronic pancreatitis and is unlikely to produce a cystic mass lesion. It may be difficult to differentiate dermoid cysts of the pancreas and splenic epidermoid cysts from LECP on the basis of cytologic features alone. Epidermoid cysts arising in intrapancreatic accessory spleen show mostly macrophages and debris.¹²⁴ Squamous cyst of the pancreatic ducts (SCOPD) is a recently described entity that is characterized by a benign-appearing squamous component and mucinous epithelium. The lack of malignant epithelium helps exclude adenosquamous and squamous cell carcinoma.

Squamoid Cyst of the Pancreatic Ducts

SCOPD is a recently described entity that manifests as a large cystic mass on imaging studies.¹²⁵ The cysts are lined by a layer of mucinous epithelium in the lumen and a layer of squamous epithelium at the base (Fig. 35.37). I have encountered one case on cytology. The patient had a large, multiloculated mass in the tail of the pancreas. The cytologic cyst fluid sample showed groups of benign-appearing squamous cells with benign mucinous columnar cells either surrounding the squamous epithelium or occurring separately. The background contained degenerated cellular debris. The cytologic features of SCOPD are listed in Box 35.22. The CEA fluid was elevated at more than 400 ng/mL.

The differential diagnosis of SCOPD may include MCN or IPMN, which should lack such a well-formed squamous component. However, in the absence of a squamous component, and because of the elevated CEA level, these cysts present a potential for a false positive diagnosis of a cystic mucinous neoplasm. The combination of mucinous epithelium with squamous epithelium, the lack of abundant anucleated squamous cells, and the location within the pancreas instead of outside the pancreas, should differentiate this entity from LECP.

Serous Cystic Neoplasm (Serous Cystadenoma/Serous Cystadenocarcinoma)

Aspiration of a serous cystadenoma is generally nondiagnostic because of scant cellularity.¹⁰⁵ Typically, a scant amount of clear, thin fluid is aspirated. The background is usually watery and may contain fibrovascular stromal fragments that are stripped of epithelial cells. The cells are usually arranged in flat, monolayered sheets or as dispersed single cells or stripped nuclei (Fig. 35.38). The nuclei are round and uniform in shape but may vary in size. The nuclei lack nucleoli and mitoses.¹²⁶ Aspirates may contain only a few cuboidal cells or histiocytes.¹²⁷ Because the stroma is typically quite vascular, these cysts may hemorrhage, in which case the aspirate may reveal blood and hemosiderin-laden macrophages. The cytologic features of serous cystic neoplasms are listed in Box 35.23.

Cytology lacks sensitivity for serous cystic neoplasms because of the difficulty in obtaining cells from these neoplasms.¹²⁶ Therefore, knowledge of the radiologic findings is essential. If imaging studies are interpreted as characteristic of serous cystadenoma, then the pathologist should evaluate the sample carefully for diagnostic cells. Smears of serous cystic neoplasms do not contain many diagnostic cells. Oligocystic¹²⁸ and solid variants^129,130 of serous cystic neoplasms occur as well.

A diagnosis of cystadenocarcinoma depends primarily on histologic identification of tissue invasion.^131,132 There are no reliable differences in the cytologic appearance between benign and malignant serous neoplasms because there are no reliable differences in histopathologic specimens.¹³³

The PAS stain, with and without diastase digestion, may be performed on cytology samples to demonstrate the presence of cytoplasmic glycogen, a characteristic feature of serous cystadenomas. CEA and amylase levels in the cyst fluid will be low.^31,99,105 These neoplasms are negative for mutations in KRAS and GNAS.¹⁰⁶

The differential diagnosis includes pseudocyst, MCN, and solid neoplasms manifesting as cysts. Because aspirates of these neoplasms frequently lack neoplastic epithelium, psuedocyst remains a diagnosis of exclusion on cytology alone.

Mucinous Cystic Neoplasm

MCN manifests as a solitary unilocular or multilocuar cyst in the distal pancreas in middle-aged women.¹³⁴ Histology is characterized by columnar, mucinous cyst-lining cells surrounded by ovarian-type stroma. The fluid is viscous and difficult to aspirate. Aspirates of MCN are characterized by thick background mucin and neoplastic epithelial cells containing cytoplasmic mucin. The cytoplasm and nuclear atypia can be variable in appearance. The cytology of MCN is similar to that of IPMN. Additional details about the cytologic features of both entities are discussed later.

These fluids have a very high viscosity.⁹⁸ Analysis of pancreatic cyst fluid shows low levels of amylase and elevated levels of CEA.¹⁰⁵

Acinar Cell Cystadenocarcinoma

A cystic variant of ACC has been reported.^135–137 The aspiration features have not been described, but they are assumed to be similar to those of the solid variant.

Intraductal Papillary Mucinous Neoplasm

The diagnosis of IPMN may be suggested on the imaging studies if they show a cyst connecting to the main pancreatic duct or multiple large cysts. However, a connection to the ductal system is not always identified.

Cytology for Mucinous Cysts

Aspiration of fluid from an MCN/IPMN may be difficult because the fluid is often extremely viscous. Gross examination often reveals highly viscous fluid or clear fluid with strands of mucus plugs. In fact, the material may be expressed from the needle as a “plug” of mucin. This feature is considered diagnostic, regardless of the cellularity of the aspirate.

The classic cytomorphologic appearance is the presence of thick background mucin combined with neoplastic mucinous epithelium. Assessment therefore includes a stepwise approach to the background and cellular component.

The key feature is the presence of background mucin, which can vary in quantity. The most diagnostic appearance is when the mucin covers the entire slide and forms a thick film, similar to colloid from thyroid aspirates. On Papanicolaou staining, the mucin has a characteristic pink hue (Fig. 35.39). When not diffuse, it may be clumped. Ferning may be seen. Psammomatous calcifications are a feature of IPMNs. The background may also contain acute inflammation and necrosis. The mucin is usually accompanied by degenerated cells, called “oncotic cells,”¹³⁸ and macrophages (Fig. 35.41, B). The presence of thick background mucin with these characteristics indicates MCN/IPMN.

The cellularity is variable. The cells may be arranged singly, on edge, in sheets, in papillary clusters, or in small papillary tufts. The sheets or groups do not form a honeycomb pattern; rather, they are subtly hypercellular and crowded (see Fig. 35.40, A and B).

The appearance of the cytoplasm varies. The characteristic cell type is a columnar cell with abundant cytoplasmic mucin and the appearance of mucinous metaplasia (see Fig. 35.39), or the cells may be rounder, with vacuolated cytoplasm.

The nuclei show varying degrees of abnormalities. The subtlest are mild nuclear enlargement and hypochromasia with subtle nuclear membrane foldings. The hypochromatic nuclei may have small, peripherally located nucleoli, similar to those seen in papillary thyroid carcinoma. Nuclear grooves and pseudoinclusions become more evident and are evidence of MCN/IPMN. The chromatin becomes coarser, with abnormal paranuclear clearing and micronucleoli (see Fig. 35.40). Mitotic figures will be evident in high-grade dysplasia. The single dysplastic cell, which is a small cell with a high N : C ratio and nuclear convolutions (see Fig. 35.41), is diagnostic of a neoplastic process; it is typically associated with higher-grade dysplasias. Because these cells may be obscured by the cystic debris, careful examination of the specimen, particularly in the areas of thick mucin, is needed (Box 35.24).

Invasive Carcinoma Associated with MCN or IPMN

Invasion typically cannot be determined on the basis of evaluation of cystic or ductal fluid only. Aspiration of a solid area of an MCN/IPMN, when visualized with radiologic guidance, may prove diagnostic of invasive adenocarcinoma.¹³⁹

The presence of coagulative tumor necrosis, cells with significant anisonucleosis and mitotic features, and other criteria of adenocarcinoma may indicate the presence of an invasive ductal carcinoma component. The intestinal type of IPMN is associated with noncystic mucinous carcinoma.

Intraductal Oncocytic Papillary Neoplasms and Invasive Oncocytic Carcinomas

IOPN is an intraductal neoplasm that has morphologic and clinical features overlapping those of IPMN.¹⁴⁰ Aspirates from these lesions may obtain viscous fluid. Cytology smears show a pattern similar to that of IPMN. The cells are usually arranged in papillary clusters with background mucin that contains macrophages and cell debris (Box 35.25). However, the main distinction is that most of the cells have dense oncocytic cytoplasm (Fig. 35.42). Invasive oncocytic carcinomas develop from IOPNs. Aspirate smears show features of malignancy. The cells have abundant oncocytic cytoplasm; others have glandular-type cytoplasm. The nuclei show grooves and inclusions.

These oncocytic neoplasms lack mutations in KRAS.¹⁴¹ The differential diagnosis includes ACC, PanNET with oncocytic cytoplasm, and hepatoid PDA. Immunohistochemistry will assist with these differential diagnoses. ACC expresses trypsin and Bcl-10.¹⁴² PanNET expresses neuroendocrine markers, and hepatoid carcinomas shows expression for HepPar and α-fetoprotein. Oncocytic carcinomas do not express either of these markers. Furthermore, the oncocytic epithelium is intermixed with glandular, mucinous-containing epithelium and shows papillary fronds and background mucin.

Role of Cytology in the Management of Mucinous Cysts

Analysis of cyst fluid for cytology and ancillary studies is accurate at classifying pancreatic cysts as mucinous or nonmucinous.^44,143,144 Current management strategies are to resect all mucinous cysts that appear to be MCNs based on correlation with imaging findings. A mucinous cyst is suspected to be an IPMN if it connects to the ductal system or if the disease is multicentric. Preoperative assessment of patients with suspected IPMN now focuses on identifying patients who are at high risk of harboring high-grade dysplasia or invasive carcinoma by using a combination of clinical history, gender, imaging characteristics, cytology, and cyst fluid biochemical and mutational analyses.

The 2012 international consensus guidelines have identified certain features as high-risk stigmata for the presence of malignancy and other features as worrisome for malignancy. If worrisome features are present, the patient is referred for EUS assessment with FNAB.¹⁴⁵ It is at this juncture that cytology becomes critical, because the patient will be referred for surgery if the aspirate is suspicious or positive for malignancy. In this context, cytology is considered a screening test for pancreatic cysts,¹⁴⁶ and it should be assessed with an eye toward identifying cells that indicate the presence of at least high-grade dysplasia, if not invasive carcinoma.

The cytologic features that are predictive of high-grade dysplasia or adenocarcinoma have been grouped together as “high-grade epithelial atypia,” because outright features of malignancy are infrequently present and there is some overlap in the features of high-grade dysplasia with moderate dysplasia. The specific cytologic features that warrant classifying a cyst as showing high-grade dysplasia are discussed earlier (see Cytology for Mucinous Cysts) (Box 35.26).

Cystic Pancreatic Neuroendocrine Tumors

Cystic PanNETs are uncommon, accounting for 3.4% of all cystic neoplasms and 4.3% of all PanNETs in one series.¹⁴⁷ However, cystic degeneration in large, nonfunctional tumors is a relatively common occurrence.¹⁴⁸ The cytologic features of these tumors are similar to those of their solid counterpart (see Fig. 35.30). A pitfall is that the aspirates may be scantly cellular. Therefore, a lower threshold is needed to diagnose these tumors. Cyst fluid analysis shows low values for CEA, CA 125, and CA 15.3; low viscosity; and variable amylase content.¹⁴⁹

Differential Diagnosis and Pitfalls: Neoplastic Mucinous Epithelium versus Gastrointestinal Contaminant

The main pitfall in the evaluation of pancreatic cysts is distinguishing benign gastric or duodenal epithelium from cystic mucinous neoplasms, MCN, or IPMN. Gastrointestinal epithelium mimics the epithelium derived from these neoplasms because it consists of glandular, mucin-containing epithelium and background mucin.

An approach to differentiating gastrointestinal epithelium from neoplastic mucinous epithelium incorporates assessment of (1) gross appearance of the fluid, (2) appearance of mucin on smears, and (3) cytomorphologic features of the cells.

It is also critical to know where the mass is located because aspirates of the pancreatic head and uncinate process traverse the duodenum, and aspirates of the neck, body, and tail traverse the stomach. Therefore, the location determines what type of contaminant one is trying to identify. In general, duodenal epithelium is easier to recognize because of the goblet cells and enterocytes.

Gastric and duodenal contents are usually watery and thin, in contrast to the mucin from MCN/IPMN, which is viscous. The mucin of gastrointestinal epithelium appears thinner and wispier on smears and is associated with the gastrointestinal epithelium. The mucin of neoplasms contains histiocytes and oncotic cells.¹³⁸ Mucin from gastrointestinal epithelium may contain degenerated cells.³²

Key to the diagnosis of a neoplasm is evaluation of the cytologic features. Gastric or duodenal epithelium retains a two-dimensional architecture in which the nuclei are evenly spaced within the sheets. Epithelium from MCN/IPMN shows crowding and nuclear overlap, which may be subtle. The nuclei are irregularly distributed.

Both gastric and duodenal epithelium may yield single cells. Single columnar cells with abundant columnar cytoplasm containing mucin that extends to the nucleus—metaplastic mucinous cells—are usually neoplastic. The mucin in foveolar cells typically does not fill the entire cytoplasm. Single cells with a brush border are enterocytes and are derived from the duodenum. Goblet cells are seen in IPMN and MCN, but these are less uniform and are unevenly distributed, in contrast to the goblet cells in the duodenum.

The nuclei from gastrointestinal epithelium are round and uniform. Neoplastic nuclei are subtly atypical, with nuclear membrane irregularities. Pseudoinclusions and grooves are typical of neoplastic epithelium, although gastric epithelium may also exhibit grooves and inclusions as a result of degeneration. Gastric epithelium frequently produces stripped nuclei with atypia and grooves in a background of mucin; this pattern should be recognized as part of the spectrum of gastric epithelium and not overinterpreted as being from gastric epithelium.

Even with these criteria, it is difficult to differentiate gastrointestinal epithelium, particularly gastric epithelium, from neoplastic epithelium. However, as discussed earlier (see Role of Cytology in the Management of Mucinous Cysts), it is not as crucial to differentiate gastric epithelium from low-grade, foveolar-type neoplastic epithelium. What is crucial is identifying the features that indicate the presence of high-grade dysplasia or invasive carcinoma.