Introduction
Endobiliary brushing is currently the preferred method to sample the pancreatobiliary system in patients with a stricture or obstruction without an associated mass. It is also used to screen patients with primary sclerosing cholangitis (PSC). Guidance methods include endoscopy, endoscopic retrograde cholangiopancreatography (ERCP), and percutaneous transhepatic cholangiopancreatography (PTC). Fine-needle aspiration biopsy (FNAB) is the most effective procedure to sample solid and cystic masses of the pancreatobiliary tract. Guidance techniques include intraoperative palpation and direct visualization, transabdominal ultrasound (TUS), intraoperative ultrasound, computed tomography (CT), and endoscopic ultrasound (EUS).
EUS is the preferred imaging technique at many academic centers and large community hospitals. Briefly, a biopsy needle is passed through the biopsy chamber of an echoendoscope, which consists of a linear array transducer mounted distal to the end of the viewing optical component of the endoscope. The needle is passed through either the duodenum or the stomach into the pancreatobiliary mass in real-time imaging.
Three manufacturers construct the needles used for EUS. They are made in 25-, 22-, and 19-gauge diameters and have a standard tip design. New sampling devices have been developed to improve the cellular collection from pancreatic masses. A through-the-needle cytologic brush system, the EchoBrush Endoscopic Ultrasound Cytology Brush (Cook Medical, Bloomington, Ind.), samples the interior lining of pancreatic cysts. Initial studies show that diagnostic yield and cellularity are better with the EchoBrush than with the standard EUS needle.
The EchoTip ProCore High-Definition Ultrasound Biopsy Needle (Cook Medical) is a single-use, disposable needle for EUS-FNAB. It is designed as a regular needle but has an added reverse bevel, which increases tissue collection. The larger-gauge needles obtain corelike samples, but the 25-gauge needle obtains fragmented, fluid-like samples, similar to those obtained with a regular EUS needle.
Cytologic Sampling of the Pancreatobiliary Tract
Indications
Indications for sampling of the biliary tree and pancreatic ducts using brushing cytology include the presence of a stricture or obstruction. Brushing cytology is also used to monitor patients with PSC who are at risk for cholangiocarcinoma. FNAB is indicated for sampling either solid or cystic masses of the pancreatobiliary tract.
Contraindications
Contraindications to brushing of the biliary tract or pancreatic ducts include contraindications to the techniques used to guide the procedures for ERCP and PTC. Absolute contraindications to ERCP include patient refusal to undergo the procedure; unstable cardiopulmonary, neurologic, or cardiovascular status; and existing bowel perforation. Relative contraindications include structural abnormalities of the esophagus, stomach, or small intestine. PTC is contraindicated in patients with bleeding diatheses or significant ascites.
Contraindications to EUS-guided FNAB include the presence of an uncorrectable bleeding disorder and lack of a safe needle access route. Gastrointestinal obstruction is an absolute contraindication for EUS-FNAB because of the risk of intestinal perforation.
Rapid On-Site Evaluation
The role of rapid on-site evaluation (ROSE) is not clearly established for brushing cytology. However, if smears are prepared on site, then it is preferable that they be prepared by an experienced cytotechnologist or pathologist.
ROSE, performed by a cytotechnologist or a pathologist, is probably the single greatest factor responsible for improvement in the diagnostic accuracy of FNAB for solid masses of the pancreatobiliary tract. ROSE of selected smears ensures adequacy of the specimen and reduces the number of inadequate specimens caused by poor localization of the lesion, scant cellularity, poor sample preparation, poor sample preservation, or obscuring blood. Thus, ROSE produces significant cost savings and may spare the patient from the need for another procedure. ROSE also allows the interpreter to determine the need for adjunctive studies such as flow cytometry, immunohistochemistry, or microbiology and prepare the sample accordingly. ROSE is not indicated for purely cystic masses but can be performed on the solid component of a cystic mass.
Sensitivity and Specificity
Biliary Brushing Cytology
Prospective and retrospective studies have documented a higher level of sensitivity for biliary brushings compared with exfoliative cytology in the detection of biliary carcinomas. A review of the literature shows that duct brushing cytology has an overall sensitivity of 26% to 88.9%, a specificity of 80% to 100%, and an overall accuracy of 48.1% to 96%. In PSC, the sensitivity was reported to be 60%, with a specificity of 89%. The sensitivity of bile duct brushings has been shown to increase after repeated attempts. In fact, after three negative brushings, the probability that a patient has a carcinoma is less than 6%. Predictors of positive yield include older patient age, mass size greater than 1 cm, and stricture length greater than 1 cm or the presence of a true stricture. The presence of stones correlates with benign cytology.
Most false-negative results are caused by sampling error, which can occur if the tumor does not invade biliary mucosa. In addition, tumors with sclerotic desmoplastic stroma do not exfoliate cells as readily as tumors without desmoplasia and therefore may not be detected with brushings. Poor visualization of the area by the endoscopist may negatively impact the sampling. Interpretation errors (17%) and technical errors (17%) are the second most frequent causes of false-negative results. Most interpretation errors result from underinterpretation of adenocarcinoma caused by the difficulty of distinguishing adenocarcinoma from reactive changes. The converse is also true: Reactive changes can mimic adenocarcinoma and lead to false-positive findings. Degeneration of malignant cells is another source of false-negative results.
False positive findings commonly result from overinterpretation of reactive and degenerative changes or of adenomatous epithelium from villous tumors of the ampulla or the bile duct. Intraductal neoplasms and dysplasia within the biliary ductal system may also lead to false-positive results.
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%. The low sensitivity and low negative predictive values are cited as major limitations of pancreatic FNAB. 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. The sensitivity for EUS-FNAB has ranged from 60% to 96% in large series, with a specificity of almost 100%.
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.
Specimen Preparation
Preparation of Pancreatobiliary Tract Brushing Specimens
The traditional method of specimen preparation is to prepare smears from the brushing—both air-dried smears for on site interpretation and alcohol-fixed smears for Papanicolaou stains. The brush may also be collected in Cytolyt solution (Hologic, Marlborough, Mass.) and the sample prepared as a ThinPrep (Hologic). The latter technique shows greater sensitivity. In another method, the brush is vortexed and then the sheath and guide wire are cut into 5-cm pieces and vortexed separately. The specimen is prepared as a cytospin. This procedure significantly increases the cellularity. Another published procedure entails processing the entire brush as an actual surgical pathology specimen.
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.
Samples from Cystic Lesions.
An aliquot of cyst fluid obtained by FNAB should be submitted for measurement of carcinoembryonic antigen (CEA) and amylase. It may be possible to send a sample of the supernatant after the fluid has been spun down. If the sample is insufficient, it may be diluted and then the final CEA and amylase values corrected by the dilution ratio. A sample of the fluid may also be sent for molecular analyses (e.g., KRAS mutational analysis). It is recommended that the remainder of the fluid be prepared by using cytospin and cell block techniques rather than smears.
The EchoBrush is used to sample the interior wall of cystic masses. The EchoBrush is passed through the biopsy chamber and used to brush the cyst wall. The endoscopy staff cuts off the brush tip and put it in saline. The brush tip is spun down with the saline. The sample is then used to make cytospins and cell blocks.
Samples from Solid Masses.
At the Moffitt Cancer Center, a cytotechnologist is present on site for adequacy interpretations of solid masses. Two smears are prepared from an aliquot of each pass; one is used to make an air-dried Diff-Quik–stained smear and the other to make an alcohol-fixed Papanicolaou-stained smear. The remainder of the sample is rinsed in saline for processing as a cell block. If lymphoma is suspected, the sample is rinsed in Roswell Park Memorial Institute (RPMI) medium.
The ProCore needle obtains a sample similar to a core biopsy when the larger-gauge needles are used. Touch imprints may be made from the larger-gauge needles for on-site evaluation. The 25-gauge needle most often yields fragmented, liquid-like material that can be smeared. The cores are placed in saline and prepared with the use of a cell block technique.
Contaminants
Contaminants from the duodenum may occasionally be seen with ERCP-guided brushing cytology of the pancreatobiliary tree, and contaminants from other sites may be obtained with FNAB of either the biliary tract or pancreas ( Box 35.1 ).
Mesothelial cells
Round nuclei, even chromatin
Flat, evenly spaced sheets, sometimes folded over
Intercytoplasmic windows
Hepatocytes
Polygonal cells with well-defined, granular cytoplasm
Cells may occur singly, in small clusters, or in jagged, irregular groups
Round nuclei with small nucleoli
Cytoplasmic inclusions including fat, lipofucsin, bile
Gastric epithelium
Flat, monolayered, honeycomb sheets or columnar cells on edge with basally located nuclei
Foveolar mucinous cells with mucin in upper two thirds of cytoplasm or involving the entire cytoplasm
Stripped nuclei in a background of mucin
Parietal cells and chief cells
Small-intestinal epithelium
Flat sheets in a honeycomb pattern with distinct cytoplasmic borders or picket-fence arrangement with basally located nuclei
Columnar enterocytes with microvillous brush border
Goblet cells
Paneth cells
Background mucin
Mesothelial Cells.
Mesothelial cells are polygonal cells arranged in flat sheets that show round to oval nuclei and intercytoplasmic “windows” ( Fig. 35.1 ). They may be mistaken for benign ductal cells or possibly squamous cells.
Hepatocytes.
Normal hepatocytes are large polygonal cells with dense, sharply defined cytoplasm and round, central or eccentric nuclei with prominent nucleoli ( Fig. 35.2 ).
Gastrointestinal Epithelium.
Gastrointestinal epithelium is a frequent contaminant in EUS-FNAB samples.
Gastric Epithelium.
The surface mucus cells of the gastric epithelium are columnar cells arranged in large folded sheets, palisaded rows, or single cells, usually associated with background mucin. Typically, a luminal border may be seen along one edge of the cellular aggregates. The sheets are typically monolayered but occasionally may be folded or thick ( Fig. 35.3 ). The gastric pits may appear in some groups of cells as rosettes present in the center of a cellular sheet. The mucus glands of the cardia or pylorus are indistinguishable from the foveolar surface mucus cells. The cells derived from the foveolae and mucus glands display mucinous cytoplasm, often contained in the upper third of the cytoplasmic compartment ( Fig. 35.4 ), although the mucin may extend to the nucleus.
Gastric epithelium may also appear as stripped, bland, slightly elongated nuclei within a background of mucin ( Fig. 35.5 ). Chief and parietal cells may be observed if the needle has traversed the fundic or body region of the stomach ( Fig. 35.6 ). Intact fragments may show attachment of surface epithelium to the lamina propria. Gastric epithelium is associated with abundant mucus that may contain degenerated cells if there is an inflammatory process.
Small-Intestinal Epithelium.
Small-intestinal epithelium has an architectural appearance similar to that of the surface mucus epithelium of the stomach, but the epithelial component is composed of absorptive enterocytes with interspersed goblet cells and is also associated with background mucin ( Fig. 35.7, A ). The brush border is visible when the cells are seen on edge (see Fig. 35.7, B ). The epithelium may contain lymphocytes, which appear as darker, small cells within the epithelium. Paneth cells are occasionally seen and are identified by the presence of coarse eosinophilic granules in the apical cytoplasm.
Both small-intestinal epithelium and gastric epithelium may produce abundant degenerated material that should not be confused with neoplastic cells. Both may also be associated with lamina propria that may resemble a stromal neoplasm.
Cytology of the Biliary Tract
Because brushing cytology is the technique most frequently used to sample the biliary tract, the cytologic descriptions in this section pertain to brushing cytology. The cytologic criteria for the FNAB diagnosis of benign and malignant entities in the biliary tract are similar to those in the pancreas.
Normal Biliary Tract
Normal bile duct epithelial cells are tall and columnar or cuboidal in appearance The cells are typically arranged in flat monolayered sheets ( Fig. 35.8, A ) or in a picket-fence arrangement with basally located nuclei (see Fig. 35.8, B ). The nuclei are round to oval, and the cytoplasmic borders are distinct ( Box 35.2 ).
Flat, monolayered, honeycomb sheets or picket-fence arrangement with basally located nuclei
Round to oval, uniformly sized and spaced nuclei
Reactive Changes
Inflammation of the biliary system results from choledocholithiasis, sclerosing cholangitis, acute cholangitis, infections, calculi, stents, or instrumentation. The changes induced by inflammation include loss of surface structures, vacuolization of the cytoplasm, nuclear enlargement, coarse hyperchromasia, and multinucleolation ( Fig. 35.9 ). The epithelium remains monolayered and lacks pseudoacinar structures. In situations of inflammation or injury, the epithelial lining may develop mucinous metaplasia or squamous metaplasia ( Box 35.3 ).
Less orderly groups
Loss of surface structures
Vacuolization of the cytoplasm
Minimal nuclear enlargement and anisonucleosis
Coarse hyperchromasia
Multinucleolation
Squamous or mucinous metaplasia
Dysplasia
Premalignant lesions of the bile ducts have historically been referred to as biliary dysplasia or atypical biliary epithelium . A new consensus classification of biliary intraepithelial neoplasia (BilIN) was published in 2007. This proposal classified BilIN into three grades, similar to the scheme used for other organs such as the pancreas and prostate. The histopathologic criteria are similar to those for other intraepithelial lesions. The cytopathologic criteria have not been defined but can be assumed to be similar to what has been described as dysplasia in the biliary tract, with grade 1 lesions causing atypia on bile duct brushings previously referred to as low-grade dysplasia and grade 3 lesions causing atypia previously referred to as high-grade dysplasia.
Lesions with grade 2 dysplasia may fall into either category on brushings. Low-grade dysplasia is characterized by sheets and clusters of cells that show nuclear crowding and overlapping, smooth nuclear membranes, and a moderate nucleus–to-cytoplasm (N : C) ratio ( Fig. 35.10, A ). The chromatin is characteristically clear in appearance and granular, with mild clumping. Low-grade dysplastic cells may have one or two distinct nucleoli. More pronounced nuclear crowding and overlapping occurs in BilIN grade 3, or high-grade dysplasia. In these cases, nuclear membranes are irregular and the N : C ratio is significantly increased; chromatin is coarse; and nucleoli are distinct and prominent ( Box 35.4 ; see Fig. 35.10, B ).
Low-grade dysplasia (BilIN 1 and 2)
Sheets and clusters with nuclear crowding and overlapping
Smooth nuclear membranes
Moderately increased N : C ratio
Minimal nuclear enlargement
High-grade dysplasia (BilIN 3)
Three-dimensional, crowded groups with nuclear overlapping
Nuclear enlargement
Nuclear membrane irregularities
Coarse chromatin
BilIN , Biliary intraepithelial neoplasia; N : C , nucleus-to-cytoplasm ratio.
Adenoma of the Biliary Tract and Ampulla
Adenomatous epithelium appears as slender, elongated, columnar cells arranged singly or in small sheets and clusters. The long, thin, basally oriented nuclei occupy approximately one third of the cell. The chromatin is typically fine and granular with one or more nucleoli ( Fig. 35.11 ). It may be difficult to separate these lesions from adenocarcinoma or dysplasia based on brushings ( Box 35.5 ).
Loosely cohesive sheets, clusters, or single cells
Columnar cells with elongated, basally located nuclei
Nuclei occupy approximately one third of the cell
Fine, granular chromatin
Prominent nucleoli
Intraductal Neoplasms of the Biliary Tract
Intraductal papillary mucinous neoplasms of the bile ducts (IPMN-B) correspond to lesions previously referred to as biliary papillomatosis or as cystic or mucinous lesions of the biliary tract, similar to intraductal papillary mucinous neoplasms of the pancreas (IPMN-P). The role of cytology in diagnosing these lesions is not established. What have been previously described as atypical papillary or mucinous cells indicative of well-differentiated adenocarcinoma are probably derived from these lesions. Cytologic features of papillary differentiation include groups with crowding and overlapping, papillary formations, and elongated nuclei. Features of mucinous differentiation are groups with mucinous cytoplasm ( Fig. 35.12 ).
Adenocarcinoma
Cytology preparations of adenocarcinoma tend to be cellular and contain both cohesive and single cells. Groups of cells are usually arranged in sheets with nuclear overlapping and crowding and loss of polarity. A very useful finding is the presence of two-cell populations ( Fig. 35.13 ). The nuclei lose their normal round shape and become rectangular, pointed, or angulated with convolutions and notches, particularly in high-grade carcinomas ( Fig. 35.14, A ). The N : C ratio is altered. The chromatin is typically coarse but may be pale in well-differentiated adenocarcinomas. Carcinomas with more abundant mucin in their cytoplasm show both nuclear and cellular enlargement and appear in an exaggerated honeycomb pattern. Pseudoacinar formations are features of adenocarcinoma ( Box 35.6 ; see Fig. 35.14, A ).
Cellular smears
Two-cell populations
Crowded groups of cells with nuclear overlapping and loss of polarity
Dyshesion
Excessive mucinous cytoplasm
Anisonucleosis
Nuclear membrane irregularities
Increased nuclear size
Increased nucleus–to-cytoplasm ratio
Mucinous and papillary differentiation have been described as subtle findings that need to be recognized because they may represent well-differentiated adenocarcinoma. The recommendation was to report any mucinous change as possibly representative of a mucinous neoplasm and any papillary change as suggestive of a papillary neoplasm. It now seems that these morphologic patterns are derived from IPMN-B if identified on a brushing (see earlier discussion). These findings identified on an FNAB of a solid mass are more likely to correlate with an invasive process.
A pitfall occurs with the papillary pattern because normal bile epithelium may form pseudopapillary clusters. Papillary clusters in normal duct cells are two dimensional. The cell clusters in true papillary lesions are crowded, and nuclei are usually arranged haphazardly. The nuclei of true papillary lesions are pointy and elongated, in contrast to their normal round to oval shape. Occasionally, the nuclei of benign epithelium may appear elongated, but again, the architectural arrangement is key. Carcinomas show a greater degree of architectural abnormalities and the presence of hypochromatic, transparent nuclei.
Differential Diagnosis
Reactive Processes versus Adenocarcinoma in the Biliary Tract
The key problem when assessing pancreatobiliary brushing cytology specimens is differentiating a reactive process from adenocarcinoma. A number of studies have focused on identifying sets of criteria that are most specific for adenocarcinoma. Initial studies assessing cytologic criteria on smears found architectural abnormalities, nuclear enlargement, “cell-in-cell” arrangement, loss of polarity, a bloody background, the presence of flat nuclei, nuclear molding, and chromatin clumping to be indicative of the presence of adenocarcinoma, although a cytologist’s overall gestalt assessment appeared to be just as good. Another report described adenocarcinoma as showing architectural features of high-grade dysplasia but characterized by the presence of a greater number of single cells.
In ThinPrep-prepared specimens, features typical of carcinomas were three-dimensional micropapillae, anisonucleosis, high N : C ratio, nuclear contour irregularity, and prominent nucleoli. Cytomorphologic features that were not helpful in distinguishing positive and negative cases were single naked nuclei, chromatin granularity, and necrosis.
A prospective study of brush smears obtained during ERCP found tightly cohesive, small, three-dimensional cell clusters that formed cell balls as the most consistent feature of malignancy. The cells in the clusters displayed features of malignancy. The authors concluded that the presence of single cells, cytoplasmic vacuoles, and prominent nucleoli are not essential for a diagnosis of malignancy.
In summary, cytologic features that are indicative of malignancy include loss of normal honeycomb pattern, loss of polarity, nuclear enlargement, anisonucleosis, nuclear membrane irregularities, and chromatin abnormalities. Identification of a two-cell population is also a very helpful feature. Single cells are not always identified in malignancy, and macronucleoli and necrosis may be seen in inflammatory processes ( Table 35.1 ).
| Criterion | Reactive Processes | Adenocarcinoma |
|---|---|---|
| Architecture | Retained honeycomb pattern, 2-dimensionality | Loss of honeycomb pattern, 3-dimensionality |
| Loss of polarity | Absent | Present |
| Nuclear molding | Absent | Present |
| Nucleus–to-cytoplasm ratio | Low | Increased |
| Nuclear membrane irregularities | Minimal | Loss of roundness, nuclei become angulated |
| Anisonucleosis | Minimal | >4 : 1 |
| Nuclear enlargement | Absent to minimal | Present |
| Chromatin | Finely dispersed | Coarse or hypochromatic |
| Cell population | Range of atypia | Two-cell population |
| Single, malignant cells | Absent | Present |
Ancillary Studies
Molecular Investigations
One study that assessed a panel of 12 polymorphic microsatellite markers linked to six tumor suppressor genes and KRAS codon 12 mutation detection showed that this panel was effective. A subsequent study assessing 20 bile duct brushing specimens for mutations in TP53 , KRAS , BRAF , and EGFR found these mutations too infrequent in biliary tract adenocarcinomas to be of added diagnostic benefit.
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. When the panel developed to detect urothelial carcinoma (UroVysion panel) was used, FISH alone increased the sensitivity of routine cytology. In another study, FISH had a sensitivity of 94%, compared with 81% for routine cytomorphology. 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.
The combination of KRAS mutation and FISH analyses appears to increase the cancer detection rate in patients with pancreatobiliary strictures 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%. S100 protein P has been reported to be upregulated in cholangiocarcinoma. 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%.
Cytology of the Pancreas
Assessment of the pancreatic FNAB sample needs to begin before the slides are reviewed. An integrative approach to the evaluation of pancreatic aspirates incorporating the clinical history, radiologic findings, cytologic findings, and ancillary studies yields the most clinically relevant interpretation of the aspirate material ( Box 35.7 ).
Evaluate clinical information
Evaluate radiologic information
Evaluate cytologic features
Evaluate ancillary studies
Correlate all of above
Age and gender are important because some neoplasms show specific gender and age predilections. Because metastases to the pancreas from known primaries may occur, it is important to obtain a history of any previous malignancies. A prolonged history of pancreatitis without an underlying cause may suggest IPMN. A cyst associated with a history of alcohol-induced pancreatitis may suggest pseudocyst.
Most crucial are the imaging findings indicating whether the mass is solid or cystic; this information will determine the cytopathologic algorithm. Different entities are considered, depending on whether the imaging studies show a mass that is solid, solid and cystic, purely cystic, or cystic with a connection to the ductal system (intraductal lesion). Chronic pancreatitis, lobular atrophy, ductal adenocarcinoma, pancreatic neuroendocrine tumor (PanNET), acinar cell carcinoma (ACC), and metastases are among the entities that are considered if the lesion is solid. Solid and cystic lesions include any solid tumor that has undergone cystic degeneration, such as PanNET, and also solid-pseudopapillary neoplasm (SPN). Purely cystic-appearing lesions include mucinous cystic neoplasms (MCN), serous cystadenoma, side branch IPMNs, and pseudocysts. More specific information, such as an impression of serous cystadenoma, may be provided by the person reporting the imaging findings.
A stepwise analysis of the aspirate sample begins with evaluation of the gross sample. This evaluation is particularly informative for cystic lesions. At low power, assessment of cellularity, architectural features, and background yields a great deal of information, which may in itself be diagnostic. At intermediate power, architectural details can be assessed in greater detail. At high power, the nuclear, cytoplasmic, and mitotic features are appreciated.
Ancillary studies include immunohistochemistry, flow cytometry for suspected lymphoma, cyst fluid analysis for CEA and amylase, and mutational analyses. Ancillary studies are most routinely used for the differential diagnosis of nonductal neoplasms and metastases and in the work-up of pancreatic cyst fluids.
Normal Pancreas and Contaminants
Table 35.2 summarizes the cytologic features of normal pancreatic epithelia.
| Epithelium | Cytoplasm | Nuclei | Architectural Features |
|---|---|---|---|
| Ductal epithelium | Tall columnar or cuboidal, with mucin | Round to oval | Flat, honeycomb sheets or palisade columns of cells |
| Acinar epithelium | Pyramidal, abundant, well defined, contains zymogen granules | Round, with prominent nucleoli | Acinar groups of cells or single cells |
| Islet cells | Scant and variable, amphophilic, contains neuroendocrine granules | Round to oval with salt-and-pepper chromatin | Loosely cohesive groups of cells |
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 ).
Solid Masses
Cytologic Approach to Aspirates of Solid Masses: Ductal Pattern versus Solid Cellular Pattern
Primary neoplasms of the pancreas manifesting as solid masses have predominantly two distinct morphologic patterns. The first is the typical histopathologic pattern of ductal neoplasms, which shows ductal-type groups with significant pleomorphism associated with desmoplastic stroma. Aspiration may be difficult because of the fibrous stroma, and sometimes the stroma results in samples that are scantly cellular. The differential diagnosis is usually with pancreatitis. The other pattern is that of the solid, cellular neoplasm. PanNET, ACC, SPN, and pancreatoblastoma (PB) fall in this category (see later discussion). Histopathology shows a neoplastic process growing as solid cell sheets, without a desmoplastic stroma. The cytology smears are diffusely cellular with numerous single cells and loose groups. These neoplasms may also be vascular.
Reactive Processes
Reactive processes of the pancreas that may be sampled by FNAB include acute and chronic pancreatitis and autoimmune pancreatitis. The role of FNAB is to rule out adenocarcinoma or other neoplasms. Occasionally, the FNAB may sample a reactive process adjacent to a neoplasm; clinical follow-up and further evaluation is always indicated for patients with clinical and imaging findings suspicious for adenocarcinoma or neoplasia ( Box 35.8 ).
Acute Pancreatitis
Acute inflammation
Necrotic debris composed of degenerating cells
Foamy histiocytes
Fat necrosis
Chronic Pancreatitis
Background mixed inflammation and histiocytes
Fat necrosis
Calcific debris
Epithelial groups with only slightly crowded nuclei
Absent or only rare isolated atypical cells
Enlarged cells <4 : 1 in a single sheet with low nucleus-to-cytoplasm ratio
Round to oval nucleus and smooth nuclear membranes
Prominent nucleoli common
Autoimmune Pancreatitis
Cellular stromal fragments
Lymphoplasmacytic infiltrate with eosinophils
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. 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. 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 ).
Ductal Adenocarcinoma
Diagnosis of pancreatic ductal adenocarcinoma (PDA), not otherwise specified (NOS), based on FNAB samples is usually straightforward. Occasionally, it is difficult to distinguish between benign lesions and adenocarcinoma or between reactive lesions and adenocarcinoma.
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. 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 ).
Cellularity varies with degree of sclerosis
Predominantly ductal population
Loss of honeycomb architecture with nuclear crowding, overlapping, loss of polarity, and uneven spacing (“drunken honeycomb”)
Irregular nuclear membranes; may be subtle in well-differentiated carcinoma
Hyperchromasia generally, but often hypochromasia with parachromatin clearing in well-differentiated carcinoma
Nuclear enlargement (1.5 × RBC on Diff-Quik stain, 2.5 × normal nucleus on Papanicolaou stain)
Anisonucleosis (4 : 1 or greater)
Single malignant cells
Mitoses
Necrosis
RBC , Red blood cells.
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.
Cellularity
Adenocarcinoma tends to produce cellular smears (see Fig. 35.21 ). However, tumors with a sclerotic stroma or necrosis may yield paucicellular samples. In this case, close attention to the atypia of particular groups is needed. Preparation of a cellblock from the collected material is helpful because it may contain diagnostic tissue, or even tissue fragments with invasive cells, when the smears are scant and indeterminate.
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.
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