Diagnostic Cytology of the Biliary Tract and Pancreas

Guidance Methods

Guidance methods for endobiliary brushing include endoscopy, endoscopic retrograde cholangiopancreatography (ERCP), and percutaneous transhepatic cholangiopancreatography (PTC). Guidance techniques for FNA include intraoperative palpation and direct visualization, transabdominal ultrasound (TUS), intraoperative ultrasound, computed tomography (CT), and endoscopic ultrasound (EUS). EUS has become 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 stomach, into the pancreatobiliary mass, under real-time imaging.

Indications for Cytological Sampling of the Pancreatobiliary Tract

Indications for sampling of the biliary and pancreatic ducts using brushing cytology include the presence of a stricture or obstruction. The biliary tree is also sampled using brushing cytology to monitor patients with primary sclerosing cholangitis (PSC) , who are at risk of developing cholangiocarcinoma. FNA is indicated for the sampling of solid or cystic masses of the pancreatobiliary tract.

Contraindications to Cytological Sampling of the Pancreatobiliary Tract

Contraindications to brushing of the biliary tract or pancreatic ducts include the contraindications to the techniques used to guide ERCP and PTC. Absolute contraindications to ERCP include patient refusal to undergo the procedure; unstable cardiopulmonary, neurological, 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 and significant ascites.

Contraindications to EUS-FNA include an uncorrectable bleeding disorder or lack of a safe needle access route. Gastrointestinal (GI) obstruction is an absolute contraindication for EUS-FNA 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 going to be prepared on-site, then it is preferable that they be prepared by an experienced cytotechnologist or pathologist.

ROSE, performed by either a cytotechnologist or pathologist, is probably the single greatest factor responsible for improving the diagnostic accuracy of FNA for solid masses of the pancreatobiliary tract. ROSE of selected smears ensures specimen adequacy and reduces the number of inadequate specimens due to poor localization of the lesion, scant cellularity, obscuring blood, as well as poor sample preparation or preservation. Thus ROSE produces significant cost savings and may spare the patient additional procedures. ROSE also allows the interpreter to determine the need for adjunctive studies, such as flow cytometry, immunohistochemistry, or microbiology. ROSE is not indicated for purely cystic masses but can be performed on the solid component of a cystic mass.

Sensitivity and Specificity

Specimen Preparation

Papanicolaou Society of Cytopathology Terminology and Reporting for Pancreatobiliary Cytopathology

The Papanicolaou Society of Cytopathology terminology system for reporting pancreatobiliary cytology specimens (PSCSRPBC) is a standardized terminology scheme for reporting pancreatobiliary specimens that includes diagnostic criteria. The PSCSRPBC created six reporting categories: Nondiagnostic, Negative (for malignancy), Atypical, Neoplastic, Suspicious (for malignancy), and Positive/Malignant. These categories are for use in laboratories where the information system requires a diagnostic category for reporting cytology specimens; otherwise samples may be reported with only a diagnosis line.

Correlation of cytology findings with clinical, imaging, and ancillary findings is required to determine sample adequacy. If a sample does not meet adequacy criteria, it is categorized as nondiagnostic. Adequacy criteria are summarized in Box 36.1 . The term nondiagnostic is preferred to unsatisfactory because unsatisfactor y implies that the sample could not be evaluated microscopically.

The negative category is used when the sample is representative of a nonneoplastic process, such as acute or chronic pancreatitis ( Table 36.1 ). The atypical and suspicious categories are both indeterminate categories, and criteria for these are summarized in Boxes 36.2 and 36.3 , respectively. In general terms, these categories are used when the sample shows abnormal cells, which cannot be classified as definitely benign or definitely malignant, because of either a scant number of representative cells or artifact precluding evaluation. These categories should be used sparingly. The positive/malignant category is used for samples with high-grade and aggressive neoplasms, such as ductal adenocarcinoma (see Table 36.1 ).

The neoplastic category is further subdivided into Neoplastic: Benign and Neoplastic: Other . Neoplastic: Benign includes benign neoplasms such as serous cystadenoma or lymphangioma. The Neoplastic: Other category includes neoplastic processes that are preinvasive precursor lesions for adenocarcinoma, such as mucinous cystic neoplasm (MCN) and intraductal papillary mucinous neoplasm (IPMN), with low- and high-grade dysplasia. Pancreatic neuroendocrine tumor (PanNET) is included under the Neoplastic: Other category but this decision remains controversial. It is recognized that PanNET > 0.5 cm have malignant potential. The decision to categorize them as Neoplastic: Other was the fact that some PanNET in elderly patients with comorbid conditions may be managed expectantly and to maintain the classification consistent with the term “tumor.” Solid pseudopapillary neoplasm is also categorized as Neoplastic: Other. The categories and corresponding diagnostic entities are listed in Table 36.1 .

Any cyst classified as mucinous, based on the presence of background mucin, CEA levels, or KRAS or GNAS mutations is categorized as Neoplastic: Other. A key caveat is that it is never acceptable to interpret the cytology of a pancreatic cyst as consistent with cyst contents.

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 FNA of either the biliary tract or pancreas ( Box 36.4 ).

Mesothelial Cells

Mesothelial cells are polygonal cells arranged in flat sheets and show round to oval nuclei and intercytoplasmic “windows” ( Fig. 36.1 ). They may be mistaken for benign ductal cells or squamous cells.

Hepatocytes

Normal hepatocytes are large polygonal cells with dense, sharply defined cytoplasmic borders and round, central, or eccentric nuclei with prominent nucleoli ( Fig. 36.2 ).

Gastric Epithelium

The surface mucus cells of the gastric epithelium are columnar cells that are typically arranged in large folded sheets, with 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. 36.3A,B ). The gastric pits may appear as rosettes in the center of a cellular sheet. The mucus glands of the cardia or pylorus are indistinguishable from the foveolar surface mucous cells. The cells derived from the foveolae and mucous glands display mucinous cytoplasm, often contained in the upper third of the cytoplasmic compartment ( Fig. 36.4 ), 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. 36.5 ). ^, Chief and parietal cells may be noted if the needle traverses the gastric fundus or body ( Fig. 36.6 ). Intact fragments may show attachment of the surface epithelium to the lamina propria. Gastric epithelium is associated with abundant watery mucin that may contain degenerated cells if there is an inflammatory process.

Small Intestinal Epithelium

Small intestinal epithelium has a similar architectural appearance to the surface mucous epithelium of the stomach, but the epithelial component is composed of a dual population of absorptive enterocytes with interspersed pale goblet cells and is also associated with background mucin ( Fig. 36.7A ). The brush border is visible when the cells are seen on edge ( Fig. 36.7B ). 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 cytoplasm.

Both the 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.

Normal Biliary Tract

Normal bile duct epithelial cells are tall columnar or cuboidal in appearance and are typically arranged in flat monolayer sheets ( Fig. 36.8A ), or in a picket fence arrangement with basally located nuclei ( Fig. 36.8B ). Nuclei are round to oval, and cytoplasmic borders are distinct ( Box 36.5 ).

Reactive Changes

Inflammation of the biliary system results from choledocholithiasis, primary sclerosing cholangitis, acute cholangitis, infections, calculi, stents, or instrumentation. The changes induced by inflammation include loss of surface structures, cytoplasmic vacuolization, nuclear enlargement, coarse hyperchromasia, and multinucleation ( Fig. 36.9 ). The epithelium remains monolayered and lacks pseudoacinar structures. Increased numbers of mitoses may be seen but these are normal in appearance. Reactive ductal cells may undergo degeneration including single-cell necrosis with resultant apoptotic and necrotic debris in the smear background. Necrosis alone is therefore insufficient for a diagnosis of malignancy. In inflammation or injury, the epithelial lining may undergo mucinous metaplasia or squamous metaplasia ( Box 36.6 ).

Dysplasia or Adenoma of the Biliary Tract and Ampulla

Adenomatous epithelium with low-grade dysplasia appears as slender, elongated columnar cells arranged singly or in small sheets and clusters. The long, thin, basally oriented nuclei occupy about one third of the cell. The chromatin is typically fine and granular with one or more small nucleoli . Brushings from these may be difficult to separate from adenocarcinoma or dysplasia (see Box 36.7 ). Premalignant lesions of the bile ducts have historically been called biliary dysplasia or atypical biliary epithelium. The original 3-tier classification of Biliary Intraepithelial Neoplasia (BilIN) that was published in 2007 has been replaced by a more reproducible 2-tier system of low- and high-grade dysplasia. Low-grade BilIN encompasses BilIN grade 1 and 2, while high-grade BilIN represents severe or grade 3 dysplasia. The diagnostic histopathological criteria are similar to those used for other intraepithelial lesions, but the diagnostic cytopathological criteria for these lesions have not been defined. However, it can be assumed that their cytological features will be similar to what has been described as dysplasia in the biliary tract, with low-grade (grade 1 and 2) 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 (grade 3).

Low-grade dysplasia or BilIN is characterized by sheets and clusters of cells that show nuclear crowding and overlapping, smooth nuclear membranes, and a moderate increase in nuclear-to-cytoplasmic (N/C) ratio ( Fig. 36.10A ). Chromatin is characteristically clear and granular, with mild clumping. Low-grade dysplastic cells may have one or two distinct nucleoli. More pronounced nuclear crowding and overlapping occurs in high-grade dysplasia/BilIN (grade 3). In these cases, nuclear membranes are irregular and the N/C ratio is significantly increased, with coarse or pale chromatin and distinct and/or prominent nucleoli ( Fig. 36.10B ; Box 36.8 ).

Intraductal Neoplasms of the Biliary Tract

Intraductal papillary neoplasms of the bile ducts (IPN-Bs) correspond to lesions previously referred to as biliary papillomatosis, or 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 has been previously described as atypical papillary or mucinous cells indicative of well-differentiated adenocarcinoma are probably derived from these lesions. Cytological features of papillary differentiation include cell groups with crowding and overlapping, papillae formation, and elongated nuclei. IPN-B may or may not contain intracytoplasmic mucin depending on their lining epithelium ( Fig. 36.11 ).

Adenocarcinoma

Cytology preparations of adenocarcinoma tend to be cellular and contain both cohesive groups and singly dispersed cells. In well-differentiated examples, groups of cells may be arranged in crowded overlapping “drunken honeycomb” sheets with minimal loss of polarity. However, 3-dimensional clusters of markedly atypical cell are more common in poorly differentiated carcinoma. Other characteristics that are frequent in adenocarcinoma include (from greatest to least common) changes in chromatin pattern (both hypochromasia and hyperchromasia), nuclear membrane irregularity, pleomorphism, two-cell populations, high N/C ratio, and cytoplasmic mucin vacuoles ( Figs. 36.12 and 36.13 ). Other nonspecific and less common changes that can be seen in malignancy include dyscohesion of cells with high N/C ratio, specimen hypercellularity, prominent nucleoli, and necrosis. In poorly differentiated or high-grade carcinomas nuclei are less rounded and become rectangular, pointed, or angulated with convolutions and notches and have more prominent nucleoli. Pseudoacinar formations are another feature of adenocarcinoma ( Fig. 36.14 ; Box 36.9 ). Some adenocarcinomas are rich in acute inflammatory cells, which may lead to misinterpretation as representing reactive atypia. Usually in reactive atypia there is no nuclear membrane folding.

Mucinous and papillary differentiation have been described as subtle findings in well-differentiated adenocarcinoma, and it was once recommended that pathologists 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 morphological patterns are derived from IPN-B if identified on a brushing as previously described in this chapter. If these findings are identified on an FNA of a solid mass they are more likely to correlate with an invasive process.

One major pitfall in the interpretation of biliary tract cytology samples is the presence of papillary pattern because normal bile epithelium may form pseudopapillary clusters. Papillary clusters in normal duct cells are 2-dimensional while the cell clusters in true papillary lesions are crowded, and nuclei are haphazardly arranged. The nuclei of true papillary lesions are also more angulated and elongated, in contrast to normal cells, which remain round to oval in shape. Occasionally, the nuclei of benign epithelium may also appear elongated, but again, the architectural arrangement is key. Carcinomas show a greater degree of architectural abnormalities and the presence of hyperchromatic or hypochromatic, transparent nuclei.

Secondary Tumors

Secondary tumors may also involve the biliary tree and lead to positive brushings. These may represent biliary tract extension of nearby nonductal cancers such as hepatocellular carcinoma or gallbladder carcinoma. They also include metastatic neoplasms such as neuroendocrine neoplasms, lymphoma, and metastases from lung, esophageal, and colonic adenocarcinoma. ^, In patients without a known history of prior malignancy these malignant cells may be misinterpreted as primary pancreatobiliary carcinomas. A high index of suspicion is therefore warranted, along with ancillary studies (immunohistochemistry and flow cytometry) whenever this is suspected.

Differential Diagnosis: Reactive Processes vs. 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 cytological criteria on smears found architectural abnormalities, such as nuclear enlargement, “cell-in-cell” arrangement, loss of polarity, the presence of flat nuclei, nuclear molding, chromatin clumping, as well as a bloody background, as indicative of adenocarcinoma, although a cytologist’s overall gestalt appeared to be just as good. Another description cites adenocarcinoma as showing architectural features of high-grade dysplasia but with more single cells.

In ThinPrep (Cytyc Corporation, Marlborough, MA) prepared specimens; features that were typical of carcinomas were 3-dimensionality (3D), anisonucleosis, high N/C ratio, nuclear contour irregularity, and prominent nucleoli. A recent study showed that the most frequent characteristics that were predictive of carcinoma were 3-dimensional clusters, pleomorphism, 2-cell population, change in chromatin pattern (hypo/hyperchromasia), high N/C ratio, cytoplasmic mucin vacuoles, nuclear irregularity, cellular dyscohesion, hypercellularity, nuclear molding, and prominent nucleoli. Most malignant brushings had ≥3 malignant characteristics, while 77% of benign brushings had none. Cytomorphological features that are not helpful in distinguishing malignant and benign cases are single naked nuclei, inflammation, and necrosis. ^{, ,} Table 36.2 summarizes these criteria.

Ancillary Studies

A number of technologies have been applied to indeterminate bile duct cytology specimens to improve sensitivity and specificity. The ancillary techniques employed include next-generation sequencing (NGS), fluorescent in situ hybridization (FISH), protein analysis, and immunocytochemistry.