Guidelines for Pancreaticobiliary Cytology from the Papanicolaou Society of Cytopathology: A Review

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1 Guidelines for Pancreaticobiliary Cytology from the Papanicolaou Society of Cytopathology: A Review Martha B. Pitman, MD 1 and Lester J. Layfield, MD 2 The newest installment on state-of-the-art standards of practice in cytopathology from the Papanicolaou Society of Cytopathology (PSC) focuses on the pancreaticobiliary system. Similar to the National Cancer Institute recommendations for aspiration cytology of the thyroid, the PSC guidelines for pancreaticobiliary cytology addresses indications, techniques, terminology and nomenclature, ancillary studies, and postprocedure management. Each committee was composed of a multidisciplinary group of experts in diagnosing, managing, and treating patients with pancreaticobiliary disease. Draft documents were posted on an interactive Web-based forum hosted by the PSC Web site ( and the topics of terminology, ancillary testing, and management were presented at national and international meetings over an 18-month period for discussion and feedback from practicing pathologists around the world. This review provides a synopsis of these guidelines. Cancer (Cancer Cytopathol) 2014;122: VC 2014 American Cancer Society. KEY WORDS: pancreas; guidelines; Papanicolaou Society of Cytopathology; review. INTRODUCTION The newest installment on state-of-the-art standards of practice in cytopathology from the Papanicolaou Society of Cytopathology (PSC) focuses on the pancreaticobiliary system. The PSC has a long history of producing guidelines for the interpretation and reporting of cytologic specimens derived from various body sites. 1-5 Similar to the National Cancer Institute recommendations for aspiration cytology of the thyroid, 6 the PSC guidelines for pancreaticobiliary cytology addresses indications, techniques, terminology and nomenclature, ancillary studies, and postprocedure management Each committee was composed of a multidisciplinary group of experts in diagnosing, managing, and treating patients with pancreaticobiliary disease. Draft documents were posted on an interactive Web-based forum hosted by the PSC Web site ( and the topics of terminology, ancillary testing, and management were presented at national and international meetings over an 18-month period for discussion and feedback from practicing pathologists around the world. Below is a synopsis of these guidelines. Clinical Evaluation, Imaging Studies, Indications for Cytologic Study, and Preprocedural Requirements for Duct Brushing Studies and Pancreatic Fine-Needle Aspiration The clinical evaluation of patients who present with a biliary stricture, pancreatic cyst, or solid mass begins when the patient comes to medical attention due to symptoms, or when a lesion is discovered incidentally during workup for another reason. Managing these patients is challenging due to the wide variety of options for Corresponding author: Martha Bishop Pitman, MD, Department of Pathology, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 01907; Fax: (617) ; mpitman@partners.org 1 Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts; 2 Department of Pathology and Anatomical Sciences, University of Missouri, Columbia, Missouri Received: March 24, 2014; Accepted: March 25, 2014 Published online April 28, 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: /cncy.21427, wileyonlinelibrary.com Cancer Cytopathology June

2 Table 1. Imaging Modalities for Evaluation of the Pancreas and Biliary Tract Modality ERCP TAU MDCT MRI, MRCP EUS Primary Utility Relief of biliary or pancreatic duct obstruction; sampling of stricture with brushing Evaluation of liver and bile duct dilatation; insensitive for pancreatic masses and small lesions; least invasive; lowest cost First line evaluation of PB lesions, eg pancreas protocol CT ; high sensitivity and specificity Improved determination of cystic components and connection of BD-IPMN to MPD; best resolution for small lesions; used selectively due to low marginal improvement over MDCT and higher cost, but preferred for surveillance due to lack of radiation FNA of pancreatic and periductal masses and cysts for diagnosis; better resolution for small lesions compared to CT; shortest trajectory to the lesion; multiple targets during single procedure (stage the patient); operator dependent Abbreviations: BD-IPMN, branch-duct intraductal papillary mucinous neoplasms; ERCP, endoscopic retrograde cholangopancreatography; EUS, endoscopic ultrasound; FNA, fine-needle aspirate; MDCT, multidetector computed tomography; MRI, magnetic resonance imaging; MRCP, magnetic resonance cholangopancreatography; PB, pancreaticobiliary; TAU, transabdominal ultrasound. treatment ranging from observation to surgery. 7 Carcinoma is a clinical concern for patients with newly diagnosed late-onset diabetes mellitus or unexplained acute pancreatitis in an older individual. 12 Development of jaundice, pruritus, or cholestasis not explained by underlying liver or gallbladder disease or choledocholithiasis should initiate an evaluation for neoplasia of the hepaticobiliary tract. 13,14 Abdominal pain that radiates to the back, anorexia, weight loss, steatorrhea, and abnormal liver enzymes are also common presenting symptoms of pancreaticobiliary disease. There are many imaging modalities that can be used in evaluating the pancreas and biliary tract (Table 1). The general standard pancreas protocol begins with a computed tomography (CT) scan, usually multiphase or multidetector CT (MDCT) with contrast. Magnetic resonance imaging (MRI) techniques including MR cholangiopancreatography (MRCP) and MR angiography (MRA) are used in selected cases such as small lesions, unclear solid versus cystic composition, or in search of a connection to the main pancreatic duct in suspected branch-duct intraductal papillary mucinous neoplasms (IPMN). 15,16 Endoscopic retrograde cholangiopancreatography (ERCP) is most useful clinically for providing relief of obstructive jaundice by either plastic or metal biliary stent placement. Brush cytology from biliary strictures or, to a lesser extent, pancreatic duct strictures, is used to sample duct strictures. It is only rarely used as a primary tool for establishing the diagnosis of pancreatic adenocarcinoma or in the workup of pancreatic cysts, because these lesions are best evaluated with endoscopic ultrasound and fine-needle aspiration biopsy (EUS-FNA). For pancreatic cysts, the most important clinical determination is whether a pancreatic cyst is mucinous or nonmucinous on the one hand, and benign or malignant (high-grade or high-risk cyst) on the other. 17 This distinction by imaging alone is quite challenging, but imaging classification systems have been proposed. 18 Most patients with inflammatory pseudocysts can be managed medically, and serous cysts are benign neoplasms that do not warrant surgical resection unless the patient is symptomatic or the cyst is large (>4 cm). 19 Management guidelines have been proposed for suspected mucinous cysts 20 (Fig. 1). These guidelines base management primarily on clinical and imaging findings with conservative management of small cysts (<3 cm) occurring in patients without high-risk clinical or imaging findings. Patients with high-risk imaging features or suspicious/malignant cytology are triaged to surgery. Patients with worrisome imaging features should be evaluated by endoscopic ultrasound and FNA biopsy for further assessment of high-risk features. For solid masses, EUS is used for preoperative staging in patients with suspected pancreatic or biliary tract carcinoma. EUS is particularly useful when CT or MR findings are equivocal, in CT-resectable disease to screen for subtle vascular involvement (especially in borderline surgical candidates), or when unresectable disease is suspected and a tissue diagnosis is needed. 21 EUS is better than CT and MR in establishing the presence of vascular invasion, and for detecting small masses, evaluating for tumor involvement of the superior mesenteric and portal veins and for detecting lymph node metastases. 15,22,23 A key advantage of EUS is that EUS-FNA can be performed as an integral part of EUS examination. This allows definitive tissue acquisition and rapid diagnosis. When EUS suggests resectability, EUS-guided biopsy may not be necessary prior to definitive resection. EUS- FNA can confirm the presence of metastatic disease in image-detected lymphadenopathy. Patients considered candidates for preoperative neoadjuvant therapy require a definitive tissue diagnosis. Aspiration of pancreatic cysts 400 Cancer Cytopathology June 2014

3 PSC Pancreaticobiliary Guidelines Review/Pitman and Layfield Figure 1. Management algorithm for suspected branch-duct IPMN. (Reproduced with permission from Tanaka et al. 20 ) provides fluid for biochemical, cytological, and molecular analysis that help to determine the nature of the cyst and if the patient requires surgical or conservative management. All specimens submitted to the pathology laboratory are accompanied by a specimen requisition form. This form is a means of communication, not only to the pathologist interpreting the specimen, but also to the laboratory personnel who process the specimen. Often, tissue management protocols are in place for certain specimen types and clinical diseases, so this information must be clearly delineated on the requisition form. Additional helpful information specific to the pancreaticobiliary tract includes precise site of the lesion being sampled, the tissue traversed during FNA (stomach or duodenum), the solid or cystic nature of the lesion, the presence or absence of bile duct or pancreatic duct stricture, the size of the lesion and the presence of high-risk or worrisome features. For pancreatic cysts, the configuration of the cyst is vital, eg, borders, loculations, wall thickness, mural nodules or masses, and so forth. Clinical suspicion of neuroendocrine tumor or lymphoma should be indicated, because tissue for the proper evaluation of these neoplasms requires special handling. Techniques for Cytologic Sampling of Pancreatic and Bile Duct Lesions Major developments in the diagnosis and management of patients with pancreaticobiliary diseases have stemmed from improvements in sampling of the pancreas and biliary system. 8 EUS-FNA is now the procedure of choice for establishing a diagnosis of pancreatic malignancy, and the recent development of core needles that can be used with EUS will further improve diagnostic accuracy. 24 ERCP is the traditional and simplest method of obtaining a cytology specimen of a biliary stricture. 25 Brush cytology has a much better yield than bile aspiration alone, and brushing cytology provides a more accurate diagnosis than directed focal biopsy because the entire surface area of the stricture is sampled by the brush. 26 Tissue can be smeared on a slide or released from the brush in preservative solutions for liquid-based processing. 27 Biopsies are often used to supplement brush Cancer Cytopathology June

4 cytology. When brushing, standard forceps, and miniforceps methods of sampling are compared, mini-forceps biopsy provides significantly better sensitivity and overall accuracy compared with cytology brushing and standard forceps biopsy. 28 Needle biopsy of biliary strictures and masses can also be performed with fluoroscopic guidance. 29 Endoscopic cholangioscopy is another technique performed using a dedicated, small-diameter endoscope that is placed through the instrument channel of a duodenoscope. Finally, EUS-FNA of a bile duct mass may also be used as a technique to establish the diagnosis of malignancy. EUS-FNA of pancreatic masses requires linear endosonographic instruments. 30 The appropriate gauge EUS needle should be selected for the procedure, on the basis of the vascularity of the target lesion, the difficulty in accessing the lesion, and the type of tissue needed for a diagnosis. Simple aspiration needles (usually 22- or 25- gauge) are used in the vast majority of biopsies and provide similar cytologic yield. 31 Highly vascular lesions such as neuroendocrine tumors and metastatic renal cell carcinoma, as well as lesions of the uncinate process, should be aspirated with a 25-gauge needle. Smaller gauge needles are easier to use and are generally safer. Mucinous cysts are aspirated with 22-gauge needles because of the high viscosity of the cyst fluid. Core biopsy and Trucut needles (19-gauge) are used for fibrotic and stromal-rich lesions such as are found in autoimmune pancreatitis. Small-gauge core biopsy needles have recently been made available and are often used when standard aspiration techniques do not provide a diagnostic tissue. During FNA of cystic lesions, one passage of the needle should be used to evaluate a cyst and high suction will aid in the rapid emptying of the cyst. Mural nodules or adjacent masses should be targeted or aspirated separately. Whenever possible, rapid on-site evaluation (ROSE) of cytology should be used in FNAs of solid masses because it reduces the frequency of false-negative FNAs, particularly in the evaluation of pancreatic masses. 32 Without ROSE, approximately 5 passes of a pancreatic mass are needed to ensure adequate sampling. ROSE is not advised for FNAs of cysts unless there is a solid component that provides tissue for direct smears. Aspirated cyst fluid should be carefully triaged for cytology, biochemical testing (carcinoembryonic antigen [CEA] and amylase), and molecular testing. Standardized Terminology and Nomenclature The proposed terminology scheme has 6 categories (Table 2). 11 New and somewhat controversial is the category Neoplastic that is divided into clearly benign neoplasms and other neoplasms. Using a standardized terminology and nomenclature system provides intra- and interdepartmental guidance for diagnosis and attempts to correlate the diagnosis with our current understanding of the lesion s biological behavior and management recommendations. Interpretation categories do not have to be used, but some pathology laboratory information systems require them, and such categorization may aid in clinical and translational research. Table 2. Proposed Pancreatobiliary Terminology Classification Scheme I. Nondiagnostic II. Negative (for malignancy) III. Atypical IV. Neoplastic: Benign Other V. Suspicious (for malignancy) VI. Positive/Malignant Category I. Nondiagnostic A nondiagnostic cytology specimen is one that provides no diagnostic or useful information about the solid or cystic lesion sampled: for example, an acellular aspirate of a cyst without evidence of a mucinous etiology, as determined by cytology and ancillary testing. 11 Cellular atypia precludes a nondiagnostic report regardless of the cellularity. The clinical and imaging context should be taken into consideration when assessing whether a sample is adequate. The absence of epithelial cells in the sample does not necessarily make a specimen nondiagnostic. A pseudocyst, for example, lacks an epithelial cyst lining by definition, and thick colloid-like mucin without epithelial cells may be aspirated from mucinous cysts, but this finding or an elevated CEA level in the cyst fluid is sufficient to support an interpretation of a neoplastic mucinous cyst Category II. Negative (for malignancy) A negative cytology sample is one that contains adequate cellular and/or extracellular tissue to evaluate or define a lesion that is identified on imaging. When using the negative category, one should give a specific diagnosis when practical; for example, pancreatitis (acute, chronic, and 402 Cancer Cytopathology June 2014

5 PSC Pancreaticobiliary Guidelines Review/Pitman and Layfield autoimmune), pseudocyst and lymphoepithelial cyst. 11 Benign pancreaticobiliary tissue in the setting of vague fullness and no discrete mass also qualifies as a negative interpretation. A negative interpretation with a descriptive diagnosis implies that the sample is adequately cellular and that no cytological atypia is present. A negative report with a descriptive diagnosis such as mucinous debris of uncertain origin (lesional versus gastrointestinal contamination) is reasonable. Category III. Atypical The atypical category is appropriate when cells display cytoplasmic, nuclear, or architectural features inconsistent with normal or reactive cellular changes, and that are insufficient to classify the cells as a neoplasm or suspicious for a high-grade malignancy. The category is heterogeneous and includes cases with reactive changes, low cellularity specimens, premalignant changes (dysplasia), and cases assigned to this category due to pathologist caution in diagnosis. In addition, the cytological findings may be suggestive but not diagnostic of a low-grade neoplasm due to insufficient tissue for confirmation of a specific diagnosis. Brushing cytology yielding atypical biliary epithelium remains in this category, because premalignant lesions of the biliary tract have not been as well defined with correlative management algorithms. Category IV. Neoplastic IV Neoplastic: Benign. This interpretation category connotes the presence of a cytological specimen sufficiently cellular and representative, with or without the context of clinical, imaging, and ancillary studies, to be diagnostic of a benign neoplasm. The most commonly encountered benign neoplasm of the pancreas is serous cystadenoma. Other benign neoplasms of the pancreas such as cystic teratoma and schwannoma are extremely rare and are also placed in this category. IV B. Neoplastic: Other. This interpretation category defines a neoplasm that is either premalignant such as IPMN or mucinous cystic neoplasm (MCN) with low, intermediate or high-grade dysplasia using cytological criteria, or a low-grade malignant neoplasm such as welldifferentiated pancreatic neuroendocrine tumors (Pan- NET) or solid-pseudopapillary neoplasms (SPN). This category represents the most controversial aspect of this terminology proposal. The rationale for this proposed category relates the desire to standardize and correlate the cytological nomenclature with the 2010 World Health Organization terminology classification that maintains the nomenclature for both PanNET and SPN as neoplasms rather than carcinomas, and to take into consideration the increasingly conservative management approaches for many of the lesions. These other neoplasms are either preinvasive, premalignant neoplasms (IPMN and MCN with low-, intermediate-, or high-grade dysplasia) or demonstrate lowgrade malignant behavior (PanNET and SPN), and, as such, warrant distinction from aggressive, high-grade malignancies (most notably ductal adenocarcinoma). All of the tumors in this category are clearly neoplastic, and even though some are low-grade malignant, the heading Neoplastic: Other is an accurate and reasonable generic term that accurately reflects the preoperative cytological terminology and does not define the neoplasm as benign or malignant. The cytological categories of atypical and suspicious for malignancy connote an indeterminate interpretation and do not relate the detection of a neoplasm, which could lead to unnecessary repeat biopsy. The cytological interpretation of PanNET indicates a well-differentiated neoplasm. Although it is now widely accepted that well-differentiated PanNETs all have malignant potential, 36 many PanNET are very slow growing, and even curable if caught at an early stage, and some are detected incidentally in asymptomatic, elderly patients. To distinguish PanNETs from highly aggressive malignant neoplasms and to offer management flexibility in elderly patients with small, asymptomatic tumors where the risk-to-benefit ratio of surgery is high compared to conservative management, PanNETs are placed in this category rather than the malignant category. Convincing a patient that conservative management of their incidental 1-cm PanNET is the best option for them is virtually impossible when cytological analysis produces a diagnosis of malignant. SPN is a low-grade malignancy but with a small local recurrence rate and low metastatic potential. 37 For these reasons coupled with the fact that the tumor is called a neoplasm and not carcinoma, it is included in this Neoplastic: Other category. The two primary neoplastic mucinous cysts of the pancreas include IPMN and MCN. Premalignant cysts are lined by low-, intermediate-, or high-grade dysplasia; Cancer Cytopathology June

6 Table 3. Useful Ancillary Tests in the Diagnosis of Pancreaticobiliary Lesions Ancillary Test Target Diagnostic Utility Histochemical Stains Periodic acid Schiff (PAS) PAS with diastase Stains cytoplasmic glycogen; extracellular or intracytoplasmic mucin Intracytoplasmic zymogen granules Removes PAS-stained cytoplasmic glycogen; does not remove PAS-stained mucin, or zymogen granules Serous cystadenoma Adenocarcinoma Mucinous cysts Acinar cell proliferations Serous cystadenoma Adenocarcinoma Mucinous cysts Acinar cell proliferations Mucicarmine Neutral mucin Adenocarcinoma Mucinous cysts Alcian Blue ph 2.5 Acid mucin Adenocarcinoma Mucinous cysts Immunohistochemical Stains Smad4 Loss of cytoplasmic staining supports adenocarcinoma Adenocarcinoma Mesothelin Positive cytoplasmic staining supports adenocarcinoma Adenocarcinoma Synaptophysin Strong, diffuse cytoplasmic staining Neuroendocrine tumor Chromogranin A Strong to patchy cytoplasmic staining Neuroendocrine tumor Trypsin Strong cytoplasmic staining (despite high background) Acinar cell carcinoma and other acinar proliferations Beta-catenin Strong nuclear staining Solid-pseudopapillary neoplasm Biochemical Tests Carcinoembryonic Antigen Amylase Cyst fluid: elevated >192 ng/ml; Low CEA (<5 ng/ml) Low CEA (<100 ng/ml) Cyst fluid: elevated (usually 1000 s); Low amylase (usually 100 s) Mucinous cyst Serous cystadenoma Cystic neuroendocrine tumor Pseudocyst Pseudocyst Serous cystadenoma Cystic neuroendocrine tumor Molecular Tests KRAS Cyst fluid: mutant Mucinous cyst GNAS Cyst fluid: mutant Intraductal papillary mucinous tumor Fluorescence In Situ Hybridization (FISH) Bile duct epithelial cells: copy number abnormalities in CEP3, CEP7, CEP17, and band 9p21 Adenocarcinoma malignant counterparts require invasive carcinoma. Atypia less than overtly malignant is included in this category of Neoplastic: Other. Distinguishing the atypia in these cysts is challenging using a 4-tiered system, and it is not always possible to distinguish high-grade dysplasia and carcinoma, or intermediate-grade dysplasia and high-grade dysplasia. A 2-tiered system of low-grade (low-grade and intermediate-grade dysplasia) and highgrade (high-grade dysplasia or adenocarcinoma) epithelial atypia provides the best information for clinical management A diagnosis of adenocarcinoma (positive or malignant category) requires unequivocal features of adenocarcinoma. Category IV. Suspicious (for Malignancy) A specimen is suspicious for malignancy when some but an insufficient number of the typical features of a specific malignant neoplasm, mainly pancreatic adenocarcinoma, are present. The cytological features raise a strong suspicion for malignancy, but the findings are qualitatively and/or quantitatively insufficient for a conclusive diagnosis, or tissue is not present for ancillary studies to define a specific neoplasm. The morphologic features must be sufficiently atypical that malignancy is considered more probable than not. 11 This category of suspicious for malignancy generally refers to pancreatic adenocarcinoma because most malignancies in the pancreas are ductal adenocarcinoma, but this category is used for all high-grade, aggressive malignancies. The suspicious category is an appropriate classification for aspirates that produce a solid-cellular clearly neoplastic epithelial proliferation which includes PanNET, acinar cell carcinoma, pancreatoblastoma, and SPN in the differential diagnosis, but which has insufficient tissue for confirmatory ancillary studies necessary to make a specific diagnosis. This category has a very high positive predictive value for malignancy The risk of malignancy for brushing specimens designated 404 Cancer Cytopathology June 2014

7 PSC Pancreaticobiliary Guidelines Review/Pitman and Layfield suspicious for malignancy is approximately 80% to 96%. 45 Category VI. Positive or Malignant This category includes a group of neoplasms that unequivocally display malignant cytological characteristics including pancreatic ductal adenocarcinoma and its variants, cholangiocarcinoma, acinar cell carcinoma, high-grade neuroendocrine carcinoma (small cell and large cell), pancreatoblastoma, lymphomas, sarcomas, and metastases to the pancreas. Because 9 of 10 malignancies in the pancreas are conventional ductal adenocarcinoma, the positive or malignant category is often related to this neoplasm. The specificity of a positive or malignant interpretation for both pancreatic FNA and biliary brushing is very high, > 90% to 95% in most studies. 42,46-53 Relying on strict criteria contributes to this high specificity at the expense of sensitivity. Rapid on-site evaluation of solid mass lesion FNAs contributes to diagnostic yield Use of Ancillary Studies in the Cytologic Diagnosis of Biliary and Pancreatic Lesions Ancillary tests are invaluable tools that assist in refining our cytological diagnoses. There is a wide range of biochemical and molecular tests available with variable diagnostic and prognostic utility 10 (Table 3). Providing the most complete and accurate information from the biopsy is necessary for optimal patient management, which may not be in the form of a specific diagnostic entity, but rather a general category such as a mucinous cyst, not otherwise specified The most clinically useful test today for evaluation of indeterminate atypia in bile duct brushing specimens is fluorescent in situ hybridization (FISH). A commercially available DNA probe set (UroVysion; Abbott Molecular, Abbott Park, Ill) analyzes individual indeterminate cells for genetic abnormalities. 57,59-64 Probes target the pericentromeric regions of chromosomes 3 (CEP3), 7 (CEP7), and 17 (CEP17) as well as chromosomal bands 9p21 (LSI 9p21). The method can be automated using the Bioview Imaging Duet system (Bioview, Ltd., Nes- Zionu, Israel). FISH analysis has been shown to outperform routine cytology with very high specificity and much higher sensitivity than routine cytology for the identification of carcinoma. 60,62,63 Tissue processed as a cell block from FNAs of solid and cystic lesions provides readily available tissue for ancillary testing, but cytospins and liquid-based processed slides can also be used if proper controls are evaluated. The histochemical stains periodic acid Schiff (PAS) and PAS with diastase (dpas) can be used to highlight the glycogen in the cytoplasm of the clear cuboidal cells of serous cystadenoma, or the zymogen granules in the granular cytoplasm of acinar cell lesions. These stains also detect intracytoplasmic mucin. Other mucin stains include mucicarmine stain for neutral mucin and alcian blue ph 2.5 for acid mucin, which can be used to assess extra- and intracellular mucin in cystic lesions. Thick, colloid-like extracellular mucin does not need such stains, or any other ancillary tests for that matter, because this quality and quantity of mucin is consistent with cyst mucin and not consistent with gastrointestinal contamination. 41 Immunocytochemical testing is the primary ancillary testing mechanism for FNA samples of solid masses given the ease of obtaining tissue for cell block preparations. Immunolabeling for Smad4, the protein product of the SMAD4 gene, is a good surrogate for SMAD4 genetic alterations and is useful in establishing a diagnosis of adenocarcinoma, because it is retained in benign pancreatic ducts and lost in more than half of pancreatic ductal adenocarcinomas. 65,66 Smad4 loss also appears to be associated with a poor prognosis for pancreatic ductal adenocarcinoma. 67 Immunolabeling for mesothelin may also serve as a useful marker for supporting the diagnosis of pancreatic adenocarcinoma. 68,69 The most commonly used markers to establish endocrine differentiation include chromogranin and synaptophysin. The proliferation marker Ki-67 is of importance in the histologic grading of PanNETs, but its utility in cytologic preparations including cell block material remains to be determined. 36 Functional pancreatic endocrine neoplasms produce a number of hormones which can be demonstrated by immunocytochemical techniques including insulin, glucagon, somatostatin, gastrin, vasoactive intestinal protein, and pancreatic polypeptide Strong cytoplasmic staining for trypsin is the most specific marker for the diagnosis of acinar cell carcinoma. 75,76 Expression of cytokeratin-7 may also aid in separation of acinar cell neoplasms (positive) from normal acinar cells (negative). 77 Strong nuclear staining for beta-catenin distinguishes SPNs from the other solid-cellular neoplasms of the pancreas. 78 Because most cyst FNAs produce so little fluid (and thus cells) for evaluation, immunohistochemical analysis of pancreatic cyst fluid is rarely an Cancer Cytopathology June

8 option, but it is usually critical for establishing a diagnosis of cystic neuroendocrine tumors. 79,80 Biochemical testing of cyst fluid is invaluable in assessing cyst fluids. Cyst fluid CEA level is the best test of mucinous differentiation. 34 Suggested cutoff values for distinguishing between nonmucinous and mucinous cysts vary, but the generally accepted level is 192 ng/ml as a useful cut-point. 33 Increasing the cutoff point for support of a mucinous cyst increases specificity but at the expense of sensitivity. 35 CEA is not a reliable test for malignancy, however, and a low CEA level does not exclude a mucinous etiology. Amylase testing is important for the diagnosis of pseudocysts and serous cystadenoma. Pseudocysts have very high amylase levels, typically in the thousands of U/L; an amylase level below 250 U/L is very unlikely to be a pseudocyst. 35 Tumors without connection to the pancreatic ductal system such as serous cystadenoma and cystic neuroendocrine tumors have consistently low levels of amylase with rare exception. 80,81 Although MCNs also do not communicate with the pancreatic ductal system, amylase levels can be quite elevated in MCNs, making the specificity of this test for distinguishing IPMN and MCN of little value. Molecular analysis of the 4 most common cystic neoplasms of the pancreas (serous cystadenoma, SPN, MCN, and IPMN) has identified a specific mutational profile in each cyst type. VHL mutations are seen in serous cystic neoplasms, CTNNB1 (beta-catenin) in solidpseudopapillary neoplasms, RNF43, KRAS, TP53, and SMAD4 in MCN, and KRAS, RNF43, GNAS, P53, and SMAD4 in IPMN. 82,83 It has therefore been suggested that molecular analysis for these mutant genes may aid in the differential diagnosis of cystic lesions of the pancreas. GNAS mutations appear to be specific for IPMN, 84,85 whereas KRAS and RNF43 mutations can be seen in both MCNs and IPMNs. The von Hippel-Lindau gene (3p25) is a tumor suppressor gene somatically mutated in serous cyst adenomas and is not seen in other cystic lesions of the pancreas. 82,86 Virtually all SPNs harbor a CTNNB1 gene mutation, which appears to be relatively specific for SPNs in the pancreas. Molecular testing for solid pancreatic tumors is not currently of clinical value in routine clinical care, given that most of the tumors are readily recognized upon morphological analysis or the mutations that are helpful in establishing the diagnosis have protein products that can be exploited with immunohistochemical stains, eg, Smad4 (SMAD4) and beta-catenin (CTNNB1). Postbrushing and FNA Biopsy Follow-Up and Treatment Options for Patients With Pancreatobiliary Lesions Optimally, patient treatment is based on a specific diagnosis, which is not always possible with only cytology due to limitations of sampling and the availability of tissue for ancillary tests, as well as the less than 100% specificity of these tests. 9 General categorization of a lesion, however, may be sufficient to guide treatment. For example, classifying a tumor as a neoplasm, most likely endocrine rather than ductal adenocarcinoma, or identifying a cyst as mucinous rather than nonmucinous, or recognizing high-grade epithelial cells in an otherwise benign-appearing cyst by imaging can alter patient management. With a nondiagnostic FNA or brushing, clinical management becomes solely dependent on the clinical and imaging findings. A nondiagnostic brushing of a distal hepatic, mid/proximal bile duct, or intrapancreatic common bile duct lesion should be followed by an EUS-FNA of any mass lesion. A nondiagnostic percutaneous FNA should be rebiopsied using EUS-FNA, even if this means moving the patient to an institution better suited to performing EUS-FNA. A nondiagnostic EUS-FNA warrants reassessment of the EUS findings, and other imaging should be undertaken, followed by a review of the FNA line of approach. A second opinion of the cytology specimen by an experienced pancreatic cytopathologist may yield valuable information as well and may preclude a repeat biopsy. Repeat EUS-FNA of cystic lesions of the pancreas should be considered carefully, because it has been recommended that only one draining pass be made because of risks as high as 14% of infection even if there is no cellular cytological material. Correlation with imaging is mandatory. 87 Repeat EUS-FNA is a costly procedure, but it is still a less expensive and less invasive option than biopsy via laparoscopy or laparotomy. 88,89 For negative interpretations, a multidisciplinary team or individual in charge of the patient s care must perform the triple test, ie, an assessment that includes the clinical presentation, radiologic findings, and cytology diagnosis. If any one of the elements of the triple test is discrepant, it is mandatory to reassess that component, and if found on review to be sound, then the overall diagnosis and other elements have to be reassessed. 87,90-93 Pancreatic EUS-FNA has a very high specificity with very few false-positive interpretations; however, false-negative interpretations are not 406 Cancer Cytopathology June 2014

9 PSC Pancreaticobiliary Guidelines Review/Pitman and Layfield uncommon due to sampling and interpretive errors, which affect the sensitivity of the test. Patients with acute pancreatitis are usually managed by an institutional-specific protocol, which commonly consists of cessation of oral intake, intravenous fluid hydration, and narcotic analgesia. Autoimmune pancreatitis is difficult to diagnose, but the radiologic pattern of autoimmune pancreatitis can be distinctive and highly suggestive of the disease, which would be supported by cytological findings of inflamed stromal fragments and the absence of malignancy. Elevated serum immunoglobulin G4 in this clinical context and response to steroids help to confirm the diagnosis. Steroids are the main treatment modality Pseudocyst management is dependent on the size of the cyst, patient symptoms, and presence or absence of ductal communication. Treatment options include endoscopic stenting, internal drainage (endoscopic or operative), or external drainage (percutaneous). 97,98 Lymphoepithelial cysts and splenules are managed conservatively if the patient is asymptomatic and the diagnosis is confidently established by cytology. 99 In other organ systems, the usual response to an atypical cytologic interpretation is a repeat procedure. Although the logistics of repeat EUS, ERCP, or percutaneous FNA are not trivial and use expensive equipment and scores of hospital personnel, the costs and resource utilization of operative biopsy are even greater. The appropriate course of action is dependent on a multidisciplinary review, the functional status of the patient, and the wishes of the patient after clinical consultation. When imaging, cytology, and/or cyst fluid biochemistry (CEA and amylase) support an interpretation of a serous cystadenoma, the patient can be conservatively managed with observation, with the proviso that the patient is asymptomatic and that there is no evidence of significant growth, which raises the risk of hemorrhage and rupture. 19 The generic classification of the tumors in the Neoplastic: Other category as neoplasms provides maximum flexibility for patient management, which is becoming increasingly conservative. Pancreatic neuroendocrine tumors present two problems that require management: tumor growth and spread and hormonal activity. Functioning PanNET may have elevated serum pancreatic polypeptide, insulin, C-peptide, proinsulin, gastrin, vasoactive intestinal peptide, glucagon, calcitonin, or somatostatin. Levels for these hormones should be drawn and monitored during the therapeutic process. For patients with nonfunctioning PanNET, serum chromogranin A, is useful for following treatment response. Pancreatic polypeptide may also be elevated in apparently nonfunctional PanNET and can serve as a useful posttreatment marker. 100 Whether to perform surgery and the type of surgery is dependent on patient age, fitness, and symptoms as well as lesion location, size, grade, and stage. With the increasing use of cross-sectional imaging, very small (1 cm) tumors are being discovered incidentally in a large number of patients, many of whom are elderly with comorbid conditions increasing surgical risk. PanNETs may grow very slowly for prolonged periods, and although the majority (50%-60%) eventually exhibit malignant behavior, surgical intervention may not be the best option for all patients. Local and or hepatic resection is done for functioning and nonfunctioning tumors with the aim of curative resection or debulking/palliation dependent on size and location. If resection fails or is impossible, a number of molecularly based modern treatments are available including sunitinib (tyrosine kinase inhibitor), rapamycin (mtor inhibitor), and PRRt (peptide receptor radionuclide therapy). 90,101 The National Comprehensive Cancer Network (NCCN) provides guidelines and algorithms for management of functioning and nonfunctioning Pan- NETs that are available online. Patients with a diagnosis of SPN are treated by surgical resection. Most patients are cured by resection, with 95% or better survival at 5 years. Even with hepatic and lymph node metastases, the tumor shows low aggressiveness. 102,103 Surgical resection is the treatment of choice for all MCN regardless of grade. Although most MCN are benign, nearly 18% will undergo malignant transformation, and because the typical patient is a 50-year-old female, the usual distal pancreatectomy is a better option than the expense and anxiety associated with lifelong surveillance for features suggestive of malignant transformation. 20,98,102 For patients with IPMN, surgical intervention versus observation depends on the type of IPMN. Main-duct IPMNs are treated by resection due to the high-risk of malignancy. Resection is usually a pancreatoduodenectomy because most occur in the pancreatic head. 20 Management options for patients with branch-duct IPMN, on the other hand, are more controversial (Fig. 1). Branchduct IPMNs are most often benign and occur in asymptomatic patients. Because the time to malignant transformation is estimated to be approximately 10 years, many Cancer Cytopathology June

10 patients, especially those with comorbid conditions, benefit more by conservative observation than surgery. The decision to operate is determined by the risk of malignancy, which in many centers is solely based on imaging features, but it is cytological evaluation of the cyst fluid that is the best test for the detection of malignancy. 20 Although the typical branch-duct IPMN is lined by lowgrade dysplastic gastric-type epithelium, when invasive carcinoma occurs, it is of the tubular type with the same dismal prognosis as conventional ductal adenocarcinoma. 104 As such, the patient has the best prognosis when premalignant (eg, preinvasive) carcinoma is identified, preferably at the level of high-grade dysplasia. The cytological detection of high-grade epithelial atypia (eg, highgrade dysplasia or invasive carcinoma) is a high-risk feature warranting surgical resection. 40 Demographics (age/ sex), family history of pancreatic cancer, social history of tobacco usage, obesity, and serum tumor markers (CA19-9, Ha1c) may also influence the patient s risk of pancreatic cancer, and thus, the decision to operate. As with the benign and atypical interpretations, when there are discrepant imaging findings suggesting malignancy, and an atypical interpretation, management of the patient with a suspicious cytological interpretation requires a stringent multidisciplinary review of the clinical and imaging findings. If the cytological findings of suspicious for malignancy do correlate with the imaging findings suggestive of malignancy, then the patient can be worked-up and staged. This could include laparoscopy with biopsy, further EUS-FNA of lymph nodes, and PET and CT scans to exclude distant metastases. Biochemical and molecular analytical markers may increase the sensitivity and specificity of the interpretation. The exact workup is dependent on the availability of these modalities, personnel, and local established protocols. Because of the low false-positive rate of pancreatic cytology, patient management is generally determined from cytological analysis alone. Management relates to the specific type of malignancy present. Surgical resection offers the best patient prognosis, but, unfortunately, most patients who present with pancreatic cancer are unresectable. The determination of operability of the patient requires an extensive multidisciplinary evaluation of the patient. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) outline in detail the workup of patients with operable and nonoperable ductal adenocarcinoma and other pancreatic malignancies. 105 Conclusions The PSC Guidelines for Pancreatobiliary Cytology provides general recommendations by multidisciplinary leaders in the field of pancreatology regarding the indications for biopsy, the techniques of sampling, a new proposed terminology scheme, ancillary techniques and postprocedure treatment, and management recommendations for each of the proposed diagnostic categories Although controversial in part, the proposed terminology scheme attempts to standardize the highly varied nomenclature of the pancreaticobiliary tract used in the interpretation of cytology specimens around the world. 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