Objectives. Pancreatic Cysts. Benefits and Limitations of the Cytologic Assessment of Cystic Pancreatic Lesions and Masses

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1 Benefits and Limitations of the Cytologic Assessment of Cystic Pancreatic Lesions and Masses Michelle D. Reid, MD, MSc Associate 1/24/15 Professor Director of Cytology - EUHM Michelle D Reid MD, MS Emory University Atlanta, GA Objectives Discuss: Endoscopic ultrasound (EUS)-guided FNA, its benefits and pitfalls New terminology for the sign-out of cystic pancreatic lesions Review: The most common cystic pancreatic lesions and neoplasms Use and limitations of clinical information, radiology and ancillary studies in diagnosing cystic neoplasms and distinction from common mimics Questions Pancreatic Cysts Prevalence of incidental pancreatic cysts is ~ 14% Incidence and size of the cysts increase with age Lee et al. Am J Gastroenterol Sep;105(9):

2 FNA Diagnosis of Pancreatic Cysts Requires correlation of: Clinical findings Age, sex, symptoms Radiologic findings Location of the cyst (head, body or tail) Configuration (cystic, mixed solid and cystic) Ancillary studies» Cyst fluid analysis (enzymes, CEA, viscosity)» Immunohistochemistry Cytologic findings Standardized Terminology & Nomenclature for Pancreatobiliary Cytology: Papanicolaou Society of Cytopathology Guidelines Classification Scheme: I) Non-diagnostic II) Negative (for malignancy) III) Atypical IV) Neoplastic: benign and other V) Suspicious (for malignancy) VI) Positive/malignant Diagnostic Cytopathology. 2014;42: Classification Scheme I) Non-diagnostic Low cellularity Technical/sampling issues Clinical/imaging considerations Provides no diagnostic or useful information about the lesion II) Negative for malignancy Adequate cellularity for evaluation A specific diagnosis should be given when practical Benign pancreatobiliary tissue Pancreatitis Pseudocyst Lymphoepithelial cyst Splenule 2

3 III) Atypical Classification Scheme Cytoplasmic/nuclear/ architectural atypia not consistent with normal/reactive changes Insufficient cells/features to classify as neoplastic/suspicious for malignancy Follow-up evaluation warranted IV) Neoplastic IV A) Neoplastic: Benign Specimen is cellular enough for diagnosis of a benign neoplasm Serous cystadenoma Cystic teratoma Schwannoma IV B) Neoplastic: Other Includes pre-malignant neoplasms Neoplastic mucinous cysts (IPMN, MCN) Solid-pseudopapillary neoplasm Well differentiated neuroendocrine tumor EUS-Guided Pancreatic FNA A major pitfall is specimen contamination by gastrointestinal (GI) tract epithelium Not seen in percutaneous pancreatic FNAB GI tract contaminants include: 1. Duodenal epithelium 2. Gastric epithelium 3. GI tract mucin CONTAMINANTS IN PANCREATIC FNA Must know location of lesion to determine the most likely contaminant Lesions in pancreatic head and uncinate process duodenal epithelial contaminants Lesions in body/tail gastric epithelial contaminants ww.learningradiology.com/archi ves

4 1. Duodenal Epithelial Contaminants Duodenum epithelium forms flat honeycomb sheets with goblet cells Tissue edges have distinct brush border best seen at very high power Distinction from a mucinous neoplasm can be difficult Distinct brush border on surface Duodenal Contaminants vs Mucinous Neoplasm Goblet cells Duodenal epithelium Mucinous neoplasm Duodenal epithelium has 2-dimensional honeycomb sheets with isolated goblet cells interspersed between benign columnar cells. This helps to distinguish duodenal epithelium from a mucinous neoplasm which has a pure population of mucin-filled cells. 2. Gastric Epithelial Contaminants Gastric epithelium on ThinPrep Gastric epithelial mucin does not typically fill the entire cell but is usually confined to the superficial 1/3rd of cell and forms a mucin cup. Distinction from a neoplastic mucinous cyst (especially gastric type IPMN) can be very difficult. 4

5 3. GI Tract Mucin GI tract mucin may also be seen in EUS-FNAB Usually scant, thin and watery in quality Not abundant and thick like mucin in mucinous neoplasms Neoplastic mucin may contain admixed tumor cells Thin watery GI tract mucin Thick colloid-like mucin in mucinous neoplasm Use of Ancillary Studies in the Diagnosis of Cystic Lesions Pancreatic Cyst Fluid Analysis 1. Enzymes Pseudocyst/Non-Neoplastic Cyst Neoplastic Cyst Amylase High Variable** 2. Viscosity Non-Mucinous Cyst Mucinous Cyst viscosity < serum viscosity > serum 3. Tumor Markers Non-Mucinous Cyst Mucinous Cyst CEA Not elevated (<5ng/mL) Elevated (>200ng/mL) CA19-9 not elevated elevated 4. Molecular Markers Non-Mucinous Cyst Mucinous Cyst GNAS mutation Negative IPMN RNF43 mutation Negative Mucinous cyst KRAS mutation Negative Mucinous cyst Loss of heterozygosity Negative Mucinous cyst CEA, carcinoembryonic antigen 5

6 Viscosity Drop of fluid placed between thumb and index finger Maximal length of stretch is measured (> 3.5mm if mucinous) = string sign Cyst fluid viscosity greater than that of serum Suggests mucinous cyst Limitation: False-positive results may occur with non-mucinous cysts Lymphoepithelial cysts Cyst Fluid CEA Each lab should establish its own cutoff values Values > 192 ng/ml are highly suggestive of a mucinous cyst Limitations: Low levels do not exclude a mucinous cyst CEA does not distinguish between benign & malignant mucinous cysts May be elevated in non-mucinous cysts Lymphoepithelial cyst, squamoid cyst of pancreatic duct Van der Vaaij. GastrointestEndos Accuracy of the Three Primary Tests in Differentiating Mucinous from Non-Mucinous Cysts EUS Cytology CEA Sensitivity 56.1% 34.5% 75% Specificity 45.4% 83.3% 83.6% Accuracy 50.9% 58.7% 79.2% Brugge WR et al. Diagnosis of pancreatic cystic neoplasms; A report of the cooperative pancreatic cyst study.gastroenterology

7 Molecular Markers in Cyst Fluid Analysis Commercially available test called PathFinderTG 3 test components 1. KRAS gene point mutation 2. Loss of heterozygosity (LOH) analysis 15 preselected loci associated with tumor suppressor genes 3. DNA quantity/quality Benign Non-Mucinous Cyst 3 Types of Cyst are described Mucinous Cyst Malignant Cyst 0/3 abnormalities 1/3 Abnormalities High amplitude KRAS mutation or LOH (>75% of DNA content) Shen et al. Molecular analysis of pancreatic cyst fluid: a comparative analysis with current practice of diagnosis. Cancer Jun 25;117(3): Mutation Type of Cyst Limitations KRAS oncogene Mucinous cysts IPMN MCN Doesnot distinguish between the 2 VHL Serous cystadenoma 50% of sporadic cases GNAS Oncogene (codon 201) Pancreatic NET IPMN 25% of sporadic cases > 60%of cases RNF43 tumor suppressor gene MEN1 tumor suppressor gene Mucinous cysts IPMN (75%) MCN Does not distinguish between the 2 Pancreatic NET 44% of sporadic cases CTNNB1 (β-catenin) Solid-pseudopapillary neoplasm 95% of cases MicroRNA alterations (mir-21,-221, -17-3p) IPMN Also seen in pancreatic adenocarcinoma CYSTIC PANCREATIC LESIONS 7

8 Cystic Pancreatic Lesions Non-neoplastic cysts Pseudocysts account for 75% of all cystic lesions Lymphoepithelial cyst Squamoid cyst of pancreatic duct (SCOPD) Paraduodenal pancreatitis, epidermoidcysts in heterotopic spleen Neoplastic cysts Serous cystadenoma Mucinous cystic neoplasm (MCN) Intraductal papillary mucinous neoplasm (IPMN) Solid tumors that undergo cystic degeneration» Neuroendocrine tumors» Solid-pseudopapillary neoplasm» Acinar cell carcinoma» Ductal adenocarcinoma The primary goal of FNAB of cystic lesions is to distinguish low-risk from high-risk pancreatic cysts Low-risk pancreatic cysts Have a low risk of harboring malignancy Resected if: Symptomatic When definitive diagnosis impossible Low-risk pancreatic cysts Pseudocyst Serous cystadenoma Lymphoepithelial cyst (LEC) High-risk pancreatic cysts Have a high risk of high-grade dysplasia or invasion Resection depends on: Worrisome radiologic features Cyst size, mural nodules High-grade atypia Carcinoma, high-grade dysplasia High-risk pancreatic cysts Intraductalpapillary mucinous neoplasm (IPMN) 30% harbor invasive carcinoma Mucinous cystic neoplasm (MCN) 15-20% harbor invasive carcinoma 8

9 NON-NEOPLASTIC PANCREATIC CYSTS Case # 1 68 year old male History of alcohol abuse, pancreatitis Presented with abdominal pain Abdominal CT scan revealed pancreatic tail cyst EUS-guided FNA was performed Pseudocyst Turbid fluid (18 ml) was aspirated from the cyst Fluid was NOT mucinous or gelatinous ThinPrephad granular debris, histiocytes, lymphocytes, cholesterol crystals Pigment Cyst fluid amylase 18,000 U/L 9

10 Pancreatic Pseudocyst - FNA Fluid collection of amylase-rich secretions, debris and blood Lacks a true epithelial lining Smears are paucicellular with amorphous granular debris, macrophages, fat necrosis and pigment Pigment is helpful in diagnosing pseudocyst Accurate diagnosis prevents unnecessary surgery Case # 2 56 year old male Incidental 3cm pancreatic cystic mass on abdominal CT scan EUS-FNAB was performed Anucleated squamous cells 10

11 Small mature lymphocytes and background debris Keratin debris Cell block with anucleated granular layer, keratotic debris and GI tract contaminants. 11

12 Case # 2 Cytologic diagnosis: Negative for malignant cells Lymphoepithelial cyst No further treatment was given Lymphoepithelial Cyst Non-neoplastic pancreatic cyst Occurs predominantly in males (80%), M:F 16:3 Etiology unknown Cyst is lined by mature squamous epithelium and contains keratin Surrounded by dense lymphoid infiltrate +/- germinal centers Lymphoepithelial Cyst FNA findings are similar to histologic findings Aspirated fluid is thick, white and cheesy Smears show: Nucleated and anucleated squamous cells, keratin, very few lymphocytes FNA is typically diagnostic Major benefit of FNA is that accurate cytologic diagnosis obviates the need for surgery Cyst lining consists of squamous epithelium surrounded by dense lymphoid infiltrate. 12

13 Case # 3 51 year old female with increased abdominal pressure CT -3cm pancreatic tail cyst Cyst fluid analysis: Amylase 38,161 U/L CEA ng/ml 13

14 Cell block with strips of eosinophilic cells in a syncytial arrangement p63 (brown)/cytokeratin 5 (red) cocktail +/+, mucicarmine negative p63 positive cells 14

15 Case # 3 Cytology diagnosis: Squamoid cyst of pancreatic duct(scopd) p63 and CK5 both focally positive Mucicarmine negative Squamoid Cyst of Pancreatic Duct (SCOPD) Rare non-neoplastic pancreatic cyst Typically unilocular cystically dilated duct Often misdiagnosed as mucinous cyst on imaging Cyst is lined by 2 types of epithelium Glandular epithelium (luminal surface) Squamous/ transitional epithelium beneath (basal location) Lacks granular layer and keratinization SCOPD 15

16 Squamoid Cyst of Pancreatic Duct FNA often paucicellular/nondiagnostic Smears show mixed squamous and glandular mucin-containing cells +/- mucoid background CEA and amylase are elevated Immunohistochemistry Positive for cytokeratin 5/6, p63in SQUAMOUS layer Positive for mucicarminein GLANDULAR layer MUC1 and MUC6 + (in glandular layer) Squamoid layer (basal) Glandular columnar cell layer (luminal) NEOPLASTIC CYSTS OF PANCREAS ABRIDGED CLASSIFICATION OF SOLID AND CYSTIC PANCREATIC NEOPLASMS Gross Configuration Neoplasms % Ductal Adenocarcinoma 85% Solid Neoplasms Pancreatic Neuroendocrine Tumor 3-4% Acinar Cell Carcinoma 1-2% (Solid-Pseudopapillary Neoplasm) 1-2% Pancreatoblastoma <1% Cystic -True cysts Serous Cystadenoma 1-2% Mucinous Cystic Neoplasm 1-2% Cystic - Intraductal Intraductal Papillary Mucinous Neoplasm 3-5% Cystic - Degenerative Solid-Pseudopapillary Neoplasm (Ductal Adenocarcinoma, Acinar Cell Carcinoma, Pancreatic Neuroendocrine Tumor) ModifiedfromKlimstraetal.ArchivesofPathologyandLaboratoryMedicine2009;133(3): Entities in parentheses rarely exhibit this gross configuration 16

17 Case # 4 56 year old female Presented with abdominal pain Imaging studies revealed a complex 6cm solid and cystic mass in the pancreatic body Clusters, sheets, single cells with clear cytoplasm, defined cell borders. PAS+ cytoplasm Serous Cystadenoma Tumor cells were also positive for GLUT1 17

18 Case #4- Serous Cystadenoma Cystic spaces lined by bland cuboidal or low columnar clear epithelial cells LOW-RISK PANCREATIC CYSTS Solid pseudopapillary neoplasm Intraductal papillary mucinous neoplasm Mucinous Cystic Neoplasm Pseudocyst Serous Cystadenoma Reid et al. Serous cystic neoplasms of the pancreas: Clinicopathologic and molecular characteristics. SemDiagPathol. Sep 2, Microcystic variant with characteristic central stellate scar, radiating septa and cyst locules. 18

19 Serous Cystadenoma Cyst fluid is usually thin and clear with low amylase and CEA Aspirates are often hypocellular, non-diagnostic Clear cells have defined borders and bland nuclei Hemosiderin-laden macrophages are seen and are due to tumor vascularity Clear cells are + for PAS, Keratin, GLUT1, α-inhibin Mucicarmine stain is negative Serous Cystadenoma Rare (1-2%) benign pancreatic neoplasm Commonly affects elderly females and body/tail region More frequent in von Hippel Lindau disease (VHL mutation) 50% of sporadic cases also show VHL gene mutations Soap bubble appearance on CT, central scar, calcification Most are managed conservatively (observation) Resected if symptomatic or increased rate of growth Resection is usually curative Limitation: Accuracy of imaging, cytology and chemical analysis is ONLY 20%* Case # 5 35 year old female complained of abdominal fullness for 5 months CT showed a 15cm cystic and solid pancreatic mass Serum CA 19-9 >100,000 U/mL CT-guided FNA performed 19

20 Evenly spaced epithelial cells present in a large flat sheet with cytoplasmic mucin Pap stain cells have cytoplasmic mucin, round nuclei and low N/C ratio 20

21 Focally crowded epithelial cells with apoptotic bodies and debris Cells with mild nuclear pleomorphism, high N/C ratio, background debris Cell block shows clusters of bland mucin-filled epithelial cells 21

22 Cell block also has cells with irregular hyperchromatic nuclei and high N/C ratio Case # 5 Diagnosis: Neoplastic cells present. Neoplastic mucinous cyst with high-grade atypia (at least high-grade dysplasia/carcinoma in situ, suspicious for invasion) HIGH-RISK PANCREATIC CYSTS NEOPLASTIC MUCINOUS CYSTS (NMCs) Intraductal papillary mucinous neoplasm Mucinous Cystic Neoplasm 22

23 1. Mucinous Cystic Neoplasm Note mucin-rich** columnar cells with underlying ovarian-type stroma 2. Intraductal Papillary Mucinous Neoplasm Columnar mucin-filled epithelium with villi Papillary nodules and mucin fill main and branch ducts 23

24 Distinction of IPMN from MCN and sub-classification of IPMNs is not required (and is very difficult) on cytology! Management of Neoplastic Mucinous Cysts (NMCs) Worrisomeradiologic features EUS-FNA Cyst size 3cm, thick wall, mural nodules, main pancreatic duct dilatation 5-9 mm/abrupt change in caliber of duct Some branch duct IPMNs and smaller (<3cm) cysts are managed conservatively (observation) High-risk stigmata of malignancy surgery Obstructive jaundice, cystic pancreatic head lesion, solid component, main duct dilatation 10mm If cytology is suspicious/positive for malignancy/high-grade atypia these should be resected Tanaka et al. International consensus guidelines 2012 for the management of IPMN and MCN of the pancreas. Pancreatology.2012;12: IPMN of the main duct with duct dilatation (>1.0 cm) and luminal debris 24

25 Key Differences Between the Two Cysts MCN Not connected to main pancreatic duct or its branches Amylase levels usually low but may be high >90% arise in the tail then body and head (10%) F >> M, F:M 20:1 Usually perimenopausal40-50 yrs Has sub-epithelial ovarian-type fibrous stroma in cyst wall Ovarian stroma is almost never seen on cytologic samples IPMN Always arise (either) from ductal system (main pancreatic duct and/or branches) High amylase levels >80% arise in the head of the pancreas M = F or are slightly > F Most patients are over 60 yrs No sub-epithelial ovariantype stroma seen in cyst wall Cytologic Similarities Both IPMN and MCN containabundant thick mucin Difficult to express from the needle Difficult to spread on the slide Smear cellularity is variable Note abundant thick colloid-like mucin Diff-Quik stain Pap stain Similarities between MCN and IPMN Mucin-filled epithelial cells are typical. Mucin fills the cytoplasm and displaces the nuclei peripherally. 25

26 Similarities between MCN and IPMN Mucin-filled epithelial cells are seen with greater nuclear membrane irregularity. Pap stain. NEOPLASTIC MUCINOUS CYSTS (NMCs) Papillary structure in an IPMN Papillary clusters may be seen in both but are more frequent in IPMN Low-grade and high-grade atypia occur in both MCN and IPMN Note nuclear atypia with nuclear crowding and hyperchromasia Low-Grade Atypia Mild Dysplasia High-Grade Atypia Severe Dysplasia/Invasion 26

27 Low-Grade Atypia in NMCs Mucinous cells in sheets Bland epithelium Cytoplasmic mucin Round nuclei Even chromatin Inconspicuous nucleoli Sheets of cells with low N/C ratio, even chromatin, round nuclei High-Grade Atypia in NMCs < 30% of malignant NMCs have malignant cells on FNA Term that incorporates spectrum of cytologic changes that fall short of a definitive malignant diagnosis is: High-grade atypia Incorporates high-grade dysplasia AND invasive carcinoma Accuracy 80% Sensitivity 72% Specificity 85% NMCs with HGA have a high risk of having invasive carcinoma Pitman et al. Cytological criteria of high-grade epithelial atypia in the cyst fluid of pancreatic intraductal papillary mucinous neoplasms. Cancer Cytopathol. 2014;122:40-7. High-Grade Atypia in NMCs 3-Dimensional clusters Atypical epithelial cells Small single dysplastic cells Increased nuclear/cytoplasmic (N/C) ratio Irregular nuclear membranes Abnormal chromatin pattern (hyper/hypochromasia) Necrosis Pitman et al. Cytologic criteria of high-grade epithelial atypia in the cyst fluid of pancreatic intraductal papillary mucinous neoplasms. Cancer Cytopathol. 2014;122(1):

28 Features of High-Grade Atypia Single cells (small cells with high N/C and irregular nuclei) 3-D clusters of small cells with high N/C, hypochromasia Necrosis Follow-Up of Case # 5 5months after FNA distal pancreatectomy, splenectomyand partial hepatectomy were performed Cyst had grown from 15 cm to 25 cm and had solid and cystic areas Ovarian-type stroma 28

29 Low-grade dysplasia High-grade dysplasia Cyst lining with high-grade dysplasia mucin 29

30 Mixture of benign and malignant multinucleated giant cells Adjacent hemorrhage and malignant glands Diagnosis on Resection Invasive carcinoma, mixed ductal and undifferentiated subtypes with osteoclast-like giant cells (2 cm) Invasive carcinoma arose in a mucinous cystic neoplasm (MCN) with extensive highgrade dysplasia Adhesion to, but no invasion of, liver or spleen Cyto-histologic correlation: Benefits: NMC diagnosis correct High-grade atypia was correct Limitation: FNA missed the undifferentiated component which was very small Likely due to sampling 30

31 Other Limitations in the Cytologic Diagnosis of NMCs Liquid-Based Preparations Mucin is more difficult to see in background Compared to smears Background often clean Necrotic debris is also more difficult to see Some IPMN subtypes are less mucinous than others Oncocytic type Pancreatobiliary type Both subtypes (by definition) are considered high-grade dysplasia Oncocytic IPMNs (IOPN) may appear less cystic/more solid on imaging misdiagnosis as adenocarcinoma Case # 6 56 year old female with cystic mass in head of pancreas Dilated main pancreatic duct EUS-FNA performed 31

32 Note the absence of cytoplasmic mucin, sheets of oncocytic cells with prominent peripheral nucleoli Cell block Cell block: Complex papillae are lined by oncocytic cells with abundant cytoplasm. Case # 6 Cytologic Diagnosis: Neoplastic cells present. IPMN, oncocytic type, with high-grade atypia. It was resected. 32

33 Diagnosis: IPMN, oncocytic type (IOPN), involving main and branch ducts, with focal invasion (0.3cm) Pancreatic Cysts Cytologic Analysis of Pancreatic Cysts Is the cyst mucinous or non-mucinous? Stretchy gelatinous material on aspiration? Yes Colloid-like mucin on smear? Yes Is CEA elevated? Yes Is there high-grade atypia? SOLID PANCREATIC LESIONS CAN UNDERGO CYSTIC DEGENERATION 33

34 Case # 7 37 year old male 7 cm cystic and solid pancreatic head mass EUS-FNA was performed Diff Quik Hypercellular smear with long finger-like papillae and prominent fibrovascular cores Fibrovascular cores Magenta colored myxoid stroma on Diff Quik Bluish-grey myxoid stroma on Pap 34

35 Cells have high N/C ratio with open, vesicular chromatin on Papanicolaou stain Monomorphic cells with vesicular nuclei and rare nuclear grooves Cell Block with branching papillae β-catenin is + (nuclear) PR is + 35

36 Case # 7 Cytologic Diagnosis: - Solid-pseudopapillary neoplasm (SPN) Case # 7 Follow-up Tumor resected 1 month later Diagnosis of SPN was confirmed Solid-Pseudopapillary Neoplasm Rare low-grade malignancy Typically large, solid and cystic pancreatic tail tumors Female predominance (F:M 9:1) Third decade (mean age 28 years) or adolescence Indolent clinical behavior Most are (85%) confined to pancreas Rarely metastasizes to liver and LNs Resection is usually curative Cytologic features are distinctive Accurate FNA diagnosis often made before resection 36

37 Solid-Pseudopapillary Neoplasm Immunohistochemistry is characteristic Positive for (nuclear) β-catenin in 95% Also vimentin positive Frequently negative or focally positive for cytokeratin Loss of e-cadherin (100%) CD56 (variable expression) CD10 + Progesterone receptor + CD99 (paranuclear dot-like +) Case # 8 39 yomale with family history of MEN1 syndrome Multiple 2-3 cm pancreatic masses EUS-FNA was performed Loosely cohesive groups of bland epithelioid cells with plasmacytoid features 37

38 Pap stain with single plasmacytoid cells with salt and pepper chromatin Diagnosis: Well Differentiated Pancreatic Neuroendocrine Tumor Keratin Synaptophysin Well Differentiated Pancreatic NET Cell Block Ki-67 index was 3% (grade 2) 38

39 Pancreatic Neuroendocrine Neoplasms Typically solid, stroma-poor tumors 5% may show cystic degeneration Range from well - poorly differentiated Well differentiated = Pancreatic Neuroendocrine Tumor (PanNET) which is more common Poorly differentiated = Pancreatic Neuroendocrine Carcinoma (PanNEC) is extremely rare 2 subtypes, small cell and large cell neuroendocrine carcinoma Prognosis for PanNETs is variable Prognosis for PanNECs is poor Pancreatic NET with Cystic Degeneration Well differentiated PanNET Well Differentiated Pancreatic NET Uniform, discohesivecells Eccentric nuclei plasmacytoid appearance Salt-n-pepper chromatin on Pap/H&E stain Nucleoli usually indistinct May have prominent nucleoli Variable nuclear atypia Plasmacytoid cells PanNET with prominent nucleoli 39

40 Well Differentiated PanNET- Pleomorphic Variant Single plasmacytoid cells and cells with focal degenerative atypia. May be misdiagnosed as carcinoma on cytology. Pancreatic Neuroendocrine Neoplasms Immunohistochemistry: Positive for neuroendocrine markers Synaptophysin, chromogranin, CD56 Diffusely positive for pancytokeratin, CAM5.2 Negative for DAXX and ATRX protein (applies to PanNETs) Ki67 stain is helpful in grading Keratin Synaptophysin Jiao Y, Shi C, EdilBH, et al. DAXX/ATRX, MEN1, and mtor pathway genes are frequently altered in pancreatic neuroendocrine tumors. Science. 2011;331: World Health Organization Grading System for Pancreatic Neuroendocrine Neoplasms Tumor Grade Mitoses/10 HPFs Ki67 Index (%) Well differentiated Grade 1 PanNET <2 3% Grade 2 PanNET % Poorly differentiated Grade 3 PanNEC >20 OR >20% The WHO recommends that when counting mitoses cells should be counted 40

41 Well Differentiated Pancreatic NET Cell Block Ki-67 index is low Small Poorly Cell Differentiated Carcinoma of Lung Neuroendocrine Metastatic to the Carcinoma Pancreas Small cells with nuclear molding, salt and pepper chromatin Synaptophysin + Keratin + Ki67 index is very high= 80% How Should Ki67 Index be Calculated? 1. Eye-ball estimation (usually inaccurate) 2. Manual Count using the microscope in real time 3. Automated counting Technician-dependent, costly Count can be confounded by: Background lymphocytes which also stain positive Pigmented macrophages 4. Manual count, check off cells on a camera-captured photomicrograph image (most accurate & reproducible method) Number of cells counted is very important ( tumor cells are recommended) Add statement stating that grade may increase in final resection specimen Ki67 is positive in lymphocytes and negative tumor cells Reid et al.calculation of the Ki67 Index in pancreatic neuroendocrine tumors: A comparative analysis of four counting methodologies. Mod Pathol. Nov

42 Manual Count on Photograph of Tumor High Ki67 index=nec Low Ki67 index=net Highlight the Ki67 positive cells Case # 9 81 year old male Abdominal CT revealed 14cm heterogeneous pancreatic tail mass EUS-FNA performed Reid. Cytopathology of the pancreas. Surg Pathol Clinics

43 Prominent nucleoli Case # 9 Immunohistochemistry: Positive for: Pancytokeratin Trypsin, chymotrypsin Negative for: Synaptophysin, chromogranin, β catenin Cytologic diagnosis: Malignant cells present. Acinar cell carcinoma Case # 9 Follow-up Whipple resection performed Diagnosis: Acinar cell carcinoma, 15cm 2 years later patient is alive with liver metastases 43

44 Acinar Cell Carcinoma Rare malignant solid tumor (1-2% of solid tumors) Up to 50% can be extensively cystic or necrotic Raman et al. Acinar cell carcinoma of the pancreas: computed tomography features. Astudy of 15 patients. Abdominal Imaging. February 2013; 38; % are metastatic (often to liver) at diagnosis Age range yrs(mean 62) M > F Tumors are typically large (average 10cm), solid, well circumscribed and stromapoor Survival rates of ~ 18 months when metastatic Acinar Cell Carcinoma Hypercellularsmears Sheets, clusters and single cells Cells are larger than normal acini Nuclei have smooth contours, fine to coarse chromatin, single prominent (sometimes cherry red) nucleolus PAS+ cytoplasmic granules Naked nuclei and background granular debris on smear Acinar Cell Carcinoma Tumor cells stain positively for: Pancytokeratin Pancreatic enzymes: Trypsin (almost 100%), lipase (77%), chymotrypsin (40%), amylase (30%) Don t confuse trypsin with α-1- antitrypsin α-1-antitrypsin is a non-specific stain in pancreatic tumors Because it also stains solidpseudopapillary neoplasm and pancreatic neuroendocrine tumors BCL10 is usually positive even in trypsin-negative cases Trypsin Hosodaet al. BCL10as a useful marker for pancreatic acinar cell carcinoma, especially using endoscopic ultrasound cytology specimens. PatholInt Mar;63(3):

45 Summary Cytologic evaluation of pancreatic cysts is complex Ancillary studies (CEA, viscosity and molecular) have limited specificity and should be used judiciously Always grade atypia when evaluating NMCs Solid tumors (NETs, acinar cell carcinoma) may also undergo cystic degeneration Grading of neuroendocrine neoplasms is recommended Accuracy of grading is limited by specimen cellularity and may increase on final resection specimen 45