Review Practical Applications of Molecular Testing in the Cytologic Diagnosis of Pancreatic Cysts Mingjuan Lisa Zhang * and Martha B. Pitman * Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA * Correspondence: [email protected] (M.L.Z.); [email protected] (M.B.P.) Abstract: Mucinous pancreatic cysts are precursor lesions of ductal adenocarcinoma. Discoveries of the molecular alterations detectable in pancreatic cyst fluid (PCF) that help to define a mucinous cyst and its risk for malignancy have led to more routine molecular testing in the preoperative evaluation of these cysts. The differential diagnosis of pancreatic cysts is broad and ranges from non-neoplastic to premalignant to malignant cysts. Not all pancreatic cysts—including mucinous cysts—require surgical intervention, and it is the preoperative evaluation with imaging and PCF analysis that determines patient management. PCF analysis includes biochemical and molecular analysis, both of which are ancillary studies that add significant value to the final cytological diagnosis. While testing PCF for carcinoembryonic antigen (CEA) is a very specific test for a mucinous etiology, many mucinous cysts do not have an elevated CEA. In these cases, detection of a KRAS and/or GNAS mutation is highly specific for a mucinous etiology, with GNAS mutations supporting an intraductal papillary mucinous neoplasm. Late mutations in the progression to malignancy such as those found in TP53, p16/CDKN2A, and/or SMAD4 support a high-risk lesion. This review highlights PCF triage and analysis of pancreatic cysts for optimal cytological diagnosis. Keywords: pancreatic cytology; pancreatic cyst fluid; cyst fluid triage; molecular testing; mucinous cyst; intraductal papillary mucinous neoplasm; mucinous cystic neoplasm Citation: Zhang, M.L.; Pitman, M.B. Practical Applications of Molecular Testing in the Cytologic Diagnosis of Pancreatic Cysts. J. Mol. Pathol. 2021, 2, 11–22. https://doi.org/ 1. Introduction doi:10.3390/jmp2010002 Radiologic detection of pancreatic cysts in asymptomatic patients has increased in recent years, and distinguishing benign cystic lesions versus those with malignant potential Academic Editor: Giancarlo Troncone is essential for patient management. Currently, endoscopic ultrasound-guided fine needle Received: 15 January 2021 aspiration (EUS-FNA) with pancreatic cyst fluid (PCF) analysis is the standard of care for Accepted: 2 February 2021 the preoperative diagnosis of pancreatic cysts [1–4]. Published: 7 February 2021 There are many different types of cysts in the pancreas, but the most common differ- ential diagnosis of cysts that provide PCF includes non-neoplastic pseudocysts, benign Publisher’s Note: MDPI stays neutral neoplastic serous cysts, premalignant mucinous cysts, malignant mucinous cysts (such as with regard to jurisdictional claims in those with invasive carcinoma), and secondarily cystic solid malignancies such as cystic published maps and institutional affil- pancreatic neuroendocrine tumors. iations. The Papanicolaou Society for Cytopathology (PSC) System for Reporting Pancreati- cobiliary Cytology [5], published in 2015, promotes using a multimodal approach for the diagnosis of pancreatic cysts, whereby all the clinical, radiologic, cytologic, biochemical, and molecular information available are incorporated to provide the optimal preoperative Copyright: © 2021 by the authors. risk assessment. Licensee MDPI, Basel, Switzerland. PCF combines cytology and biochemical analysis for carcinoembryonic antigen (CEA) This article is an open access article and amylase to distinguish mucinous from non-mucinous cysts and to assess for high-risk distributed under the terms and features [6,7]. Cytologic evaluation provides high specificity but lower sensitivity for conditions of the Creative Commons the detection of a mucinous cyst. The interpretation of CEA levels is also not without Attribution (CC BY) license (https:// limitations: in one study, using a cutoff of 192 ng/mL resulted in the misdiagnosis of 39% of creativecommons.org/licenses/by/ cystic mucinous neoplasms [8]. Furthermore, CEA levels have been shown to fluctuate in 4.0/). J. Mol. Pathol. 2021, 2, 11–22. https://doi.org/10.3390/jmp2010002 https://www.mdpi.com/journal/jmp J. Mol. Pathol. 2021, 2, FOR PEER REVIEW 2 J. Mol. Pathol. 2021, 2 12 39% of cystic mucinous neoplasms [8]. Furthermore, CEA levels have been shown to fluc- repeatedtuate in FNArepeated samples FNA [9 ].samples At some [9] institutions,. At some institutions, molecular testing molecular is routinely testing performedis routinely onperformed all PCF with on sufficientall PCF with cyst suf fluidficient volumes cyst fluid to improve volumes the to detection improve of the mucinous detection cysts of mu- as wellcinous as those cysts at as high well risk as forthose malignancy. at high risk With for the malignancy. addition of With routine themolecular addition of analysis, routine wheremolecularKRAS/GNAS analysis,mutations where KRAS/GNAS are highly mutations specific for are a mucinoushighly specific etiology, for a the mucinous detection eti- ofology, mucinous the detection cysts by of PCF muc hasinous been cysts shown by PCF to has have been a sensitivity shown to have of 90% a sensitivity and specificity of 90% >90%and specificity [10–13]. >90% [10–13]. TheThe aim aim of of this this review review is is toto summarizesummarize howhow cytopathologists approach the the diagnosis diagno- sisof ofpancreatic pancreatic cysts cysts and and how how molecular molecular testing testing impacts impacts this this diagnosis diagnosis and and ultimat ultimatelyely pa- patienttient care. care. 2.2. Tissue Tissue Management Management Fresh,Fresh, unfixed, unfixed, and and undiluted undiluted PCF PCF is is required required for for accurate accurate evaluation evaluation and and analysis. analysis. FixingFixing and and diluting diluting PCF PCF in in liquid-based liquid-based preservatives preservatives or or saline saline precludes precludes the the ability ability to to obtainobtain biochemical biochemical information. information. Rapid Rapid on-site on-site evaluation evaluation does does not not aid aid in in patient patient manage- manage- ment,ment, and and all all drained drained cyst cyst fluid fluid should should instead instead be be saved saved and and appropriately appropriately triaged. triaged. If If the the lesionlesion has has a a solid solid component, component, that that area area should should be be separately separately sampled sampled and and made made into into direct direct smears.smears. EvenEven with with minimal minimal amounts amount ofs cystof cyst fluid, fluid, PCF PCF can becan triaged be triaged for cytology, for cytology, CEA/amylas CEA/am-e analysis,ylase analysis, and molecular and molecular testing testing (Figure (Figure1)[ 6]. One1) [6] study. One showed study showed that using that supernatant using super- fluidnatant for fluid CEA/amylase for CEA/amylase testing is testing comparable is comparable to using neatto using fluid/cell neat fluid/cell block preparation block prepara- [14]. Iftion the [14] PCF. If is the very PCF thick, is very a direct thick, smear a direct should smear be should made, be as made, the chemistry as the chemistry lab will lab reject will thereject specimen the specimen for CEA for analysisCEA analysis due to due high to high viscosity. viscosity. In these In these cases, cases, the the CEA CEA level level is is notnot necessary necessary because because the the thick, thick, viscous viscous nature nature of of the the cyst cyst fluid fluid indicates indicates that that the the cyst cyst is is mucinousmucinous (and (and therefore therefore CEA CEA does does not not contribute contribute additional additional information). information). Furthermore, Furthermore, KRAS thethe added added value value ofof testingtesting for KRAS maymay be be small small if ifcytology cytology and/or and/or CEA CEA levels levels are arecon- conclusive, as KRAS mutations do not add to the determination of a mucinous cyst and clusive, as KRAS mutations do not add to the determination of a mucinous cyst and does does not stratify the lesion by grade. not stratify the lesion by grade. Figure 1. Pancreatic cyst fluid triage. Figure 1. Pancreatic cyst fluid triage. J.J. Mol. Mol. Pathol. Pathol.2021 2021, 2, 2, FOR PEER REVIEW 13 3 3.3. Benign Benign Cystic Cystic Lesions Lesions TheThe benign benign pancreatic pancreatic cysts cysts have have no no malignant malignant potential potential and and can can be be conservatively conservatively managed.managed. Grossly, Grossly, the the cyst cyst fluid fluid appears appears thin thin and and brown brown or or clear, clear, and and on on cytology, cytology, these these lesionslesions tend tend to to be be sparsely sparsely cellular. cellular. 3.1.3.1. Pseudocyst Pseudocyst PseudocystsPseudocysts areare typicallytypically seen in the the setting setting of of acute acute pancreatitis pancreatitis and and result result from from au- autodigestivetodigestive damage damage of of pancreatic pancreatic parenchyma. parenchyma. These These cysts cysts lack lack a true a true epithelial epithelial lining lining and andare arecollections collections of necrotic of necrotic material material surrounded surrounded by a by thick, a thick, inflammatory inflammatory fibrous fibrous capsule. cap- sule.PCF PCFalmost almost always always shows shows a very a very high high amylase amylase level level (≥250 (≥ U/L)250 U/L)and low and CEA low CEAlevel level