State of the Art Symposium

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1 State of the Art Symposium Syed Z. Ali, MD There is no disclosure necessary Martha B. Pitman, MD There is no disclosure necessary Lester Layfield, MD There is no disclosure necessary Joseph Herman, MD There is no disclosure necessary

2 State Of The Art Symposium 2012 Pancreatic Cytopathology: Past, Present And Future Syed Z. Ali, Martha B. Pitman, Lester Layfield and Joseph Herman American Society Of Cytopathology State Of The Art Symposium 2012 Pancreatic Cytopathology: Past, Present And Future Conflict of interest Drs. Ali, Pitman, Layfield and Herman have no relevant financial relationships with commercial interest to disclose. 1

3 Pancreatic Cytopathology: Past, Present And Future Fine Needle Aspiration of the Pancreas 60 80% of FNA diagnoses are MALIGNANT 85 90% are ACA Pancreatic Cytopathology: Past, Present And Future Pancreatic Cancer New Cases: 43,920 *NCI 2012 SEER Cancer Statistics Review Pancreatic Cytopathology: Past, Present And Future Pancreatic Cancer New Cases: 43,920 Deaths: 37,390 (85%) *NCI 2012 SEER Cancer Statistics Review 2

4 Pancreatic Cytopathology: Past, Present And Future Pancreatic Cancer New Cases: 43,920 Deaths: 37,390 (85%) 4% live 5 years after the diagnosis *NCI 2012 SEER Cancer Statistics Review Pancreatic Cytopathology: Past, Present And Future Pancreatic Cancer New Cases: 43,920 Deaths: 37,390 (85%) 4% live 5 years after the diagnosis 80 85% present with advanced unresectable disease *NCI 2012 SEER Cancer Statistics Review Pancreatic Cancer A Snapshot NCI 2012* Pancreatic Cancer 4 th leading cause of cancer related death in both sexes An estimated $ 1.9 billion is spent annually on treatment NCI invests close to $ 100 million annually on research *NCI SEER Cancer Statistics Review 3

5 Pancreatic Cancer Some Hard Facts Pancreatic Cancer Current Trends The annual percentage change (APC) +1.4* ( 0.5%) Overall mortality +0.5* ( 1.6%) *Joinpoint Trend since 2009 Pancreatic Cancer Beginning to identify noninvasive precursors Reasonably good understanding of genetic and epigenetic changes (KRAS CDKN2A, p53, SMAD4 and BRCA2) In < 20%, a number of other oncogenes involved (BRAF, EGFR) Molecular evolution estimates 10 yrs to evolve from precursor neoplastic clone > several more years to develop metastatic subclones Window of opportunity for a potential screening test American Society Of Cytopathology State Of The Art Symposium 2012 Pancreatic Cytopathology: Past, Present And Future Ali Solid Neoplasms Pitman Cystic Lesions Layfield Pancreatobiliary Brushings Poster and Exhibitor Break ( pm) Pitman Proposed Nomenclature Herman Multidisciplinary Approach, New markers and Therapies 4

6 Cytopathology of Solid Pancreatic Neoplasms Pancreatic Cytopathology The Fear Factor 3 rd most common site where normal cells are misinterpreted as neoplastic/malignant ~ 10% of Whipple surgeries performed for presumed malignancy (clinical and/or FNA findings) reveal benign disease on histopathology ~ 25% of these have autoimmune pancreatitis (AIP) or lymphoplasmacytic sclerosing pancreatitis (LSP) 5

7 Cytopathology Of The Pancreas - A Multi Disciplinary Approach Surgeon Radiologist Clinical Findings Imaging Chemistry Cytomorphology Gastroenterologist Cytopathologist Solid Neoplasms Of The Pancreas [Exocrine Pancreas] Ductal Carcinoma Variants Acinar Cell Carcinoma Pancreatoblastoma Solid Pseudopapillary Neoplasm 6

8 Ductal Adenocarcinoma Adenocarcinoma Cytologic Grading Well Moderate Poor Flat Sheets More crowded 3 D Tissue fragments More single cells Pleomorphism (Focal) Hypochromatic Micronucleoli Pleomorphism (Extensive) Hyperchromatic Macronucleoli 7

9 Why do we grade? Fine tune our diagnosis and differential diagnoses Optimize cancer therapy (if PD, neoadjuvant therapy is often considered) Prognostication (indicator of poor outcome in resectable disease) Grading should preferably be two tiered (not five) 8

10 Immunoperoxidase Profile CK 7 +, CK 20 CEA +, CA MUC1 +, MUC4 + MUC2 (Cf IPMN And Mucinous Cystic Neo) Chromogranin+ (Focal, Scattered Cells) DPC4 (SMAD4) (Loss/Inactivation 55%) Other Markers Mesothelin Fascin 9

11 Ductal Carcinoma Variants Mucinous Non cystic Adenocarcinoma (Colloid) Signet ring Cell Carcinoma Medullary Carcinoma Adenosquamous Carcinoma Undifferentiated Carcinoma With Osteoclast like Giant Cells Anaplastic (Undifferentiated) Carcinoma Mucinous Non cystic Adenocarcinoma (Colloid) Majority arise in association with intestinal type IPMN Overall prognosis better than conventional tubular ductal ACA (not stage adjusted) 10

12 Signet ring Cell Carcinoma Highly aggressive and rare cancer Rule out metastasis to the pancreas (gastric, breast lobular) Medullary Carcinoma Associated with microsatellite instability, colonic adenocarcinoma Usually no KRAS mutations, 20% have inactivation of DNA mismatch repair system Adenosquamous Cell Carcinoma 11

13 Poorer Prognosis median overall survival 10 mo The proportion of squamous differentiation is usually not associated with median overall survival (< 30% versus > or = 30%) Undifferentiated Carcinoma with Osteoclast like Giant Cells Often a component of an in situ or invasive ACA Poor prognosis (< one year) 12

14 Anaplastic (undifferentiated) Carcinoma Extremely bad prognosis Rule out metastasis to the pancreas (melanoma) Acinar Cell Carcinoma 13

15 Trypsin (100%), lipase (80%), chymotrypsin (40%), and amylase (30%) Focal neuroendocrine marker immunoexpression (upto 43%) Overall median survival 19 months (age, size and stage) 14

16 Pancreatoblastoma Solid Pseudopapillary Neoplasm 15

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18 Beta Catenin CD99 Endocrine Pancreas Pancreatic Neuroendocrine Tumor (PanNet) High-grade NE Carcinoma Small cell type Large cell type Pancreatic Neuroendocrine Tumor (PanNet) 17

19 NE markers +, CK7, PAX 8 + Size (>0.5cm are malignant) Grading with Ki 67 % (G1 0 2%, G2 3 20%, G3 >20% or NE Carcinoma) 18

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21 High grade NE Carcinoma (Small Cell Carcinoma) Differential Diagnosis Metastatic Small Cell Carcinoma ( TTF 1?) NHL PNET (O 13?) 20

22 Metastatic Tumors Adenocarcinomas Lung, Stomach, Colon, Breast Renal Cell Carcinoma Hepatocellular Carcinoma Malignant Melanoma Sarcomas Leiomyosarcoma MFH MPNST 21

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24 Cystic Lesions: Cytomorphology and Role of Cyst Fluid Analysis Martha Bishop Pitman, M.D. Director, Cytopathology Massachusetts General Hospital Associate Professor of Pathology Harvard Medical School Boston, MA Differential Diagnosis Pancreatic Cysts Pseudocyst Lymphoepithelial cyst Serous cyst Mucinous cyst (MCN and IPMN) Cystic degeneration of typically solid tumors PanNET SPN other Other more rare cysts radiologyassistant.nl Preoperative Diagnosis Requires a Multimodal Approach Radiology EUS features Size, location, wall thickness, septations, mural nodule/mass Cyst Fluid Analysis Gross fluid quality and quantity amount Thick, mucoid, thin, clear, white, brown, yellow CEA, amylase Cytology Extracellular mucin-thick, thin, diffuse, focal, special stains Epithelial cells-low grade, high grade Background necrosis and inflammation Molecular KRAS, GNAS 1

25 Two basic questions for Cyst analysis 1) Is the cyst mucinous or non mucinous? 2) Is the cyst benign or malignant? Pancreatic Pseudocyst Clinical Common cystic lesion in the pancreas Associated with pancreatitis, trauma, surgery (almost always) Radiology Unilocular, non-septated Thick >> thin walled No mural nodule Histology Cyst lining of histiocytes and inflammatory cells Pancreatic Pseudocyst cyst debris with blood, proteinaceous material and yellow hematoidin-like pigment variable inflammation NO cyst lining epithelium (beware of contamination, mucin and epithelium) CEA low; amylase usually in the 1000 s; no KRAS 2

26 Serous Cystadenoma Clinical Benign, slow growing neoplasm women>>men, mean age 7 th decade Assocaited with VHL with deletion of 3p25 in most cases Often asymptomatic, but can hemorrhage and cause pain Radiology circumscribed, multi-lobulated Microcystic with fibrous septae, central scar, calcifications in ~30-40% Histology glycogen-rich dpas+ cuboidal epithelium Serous Cystadenoma: Variants Unilocular and Oligocystic Serous Cystadenoma Cuboidal non mucinous epithelial cells Hemosiderin laden macrophages in a clean or bloody, nonpseudocyst like background CEA and amylase low NO KRAS 3

27 Neoplastic Mucinous Cysts MCN IPMN Mucinous Cystic Neoplasm Clinical F:M=20:1 Most are benign Prognosis excellent for noninvasive completely resected tumors Resection recommended despite grade Radiology body and tail (90%) do not communicate with the pancreatic ductal system thick walled (Ca++ in 20%) thin or thick septa Mucinous Cystic Neoplasm Not associated with the pancreatic ducts Lined by mucinous, generally non-papillary epithelium Subepithelial ovarian-like stroma required Atypia may be very heterogeneous; invasion may be very focal, so the entire cyst should be submitted for histology 4

28 Mucinous Cystic Neoplasm is not an Aggressive Entity (Crippa, et.al. Annals of Surgery 2008; 247: Collaborative study between MGH and University of Verona 163 patients with MCN, strictly defined Non invasive Invasive MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION HARVARD MEDICAL SCHOOL Intraductal Papillary Mucinous Neoplasm Intra-ductal Branch duct IPMN Combined disease Intraductal Papillary Mucinous Neoplasm Main duct type Diagnosed clinically Dilated main pancreatic duct (definition varies, but >5mm) Pancreatic head mostly, but occur all through the pancreas Intestinal type lining most common 60% have HGD 45% have invasive carcinoma Symptoms common but 25% asymptomatic Treatment-resection AFIP 4 th Series Fascicle 5

29 Intraductal Papillary Mucinous Neoplasm Branch Duct Type Most often in head/uncinate 1/3 with multiple cysts Supports clinical dx Most patients asymptomatic Imaging: bunch of grapes ; single cyst may not be diagnostic for BD-IPMN unless visualized connection to the MPD Most lined by gastric type epithelium Most low grade Treatment-depends. AFIP 4 th Series Fascicle Intraductal Papillary Mucinous Neoplasm Various papillarity of mucinous epithelium of variable cell type and heterogenous atypia No association with ovarian-like stroma under the epithelium AFIP 4 th Series Fascicle Gastric Foveolar Type Cells: Most BD IPMN Usually LGD MUC 5AC+, MUC 6+, MUC1, MUC2, CDX2 AFIP 4 th Series Fascicle 6

30 Intestinal Type Cells: Most Main Duct IPMN Moderate/Intermediate dysplasia MUC 5AC+, MUC 6 weak, MUC1, MUC2+, CDX2+ AFIP 4 th Series Fascicle Pancreatobiliary Type Cells: Less common type High grade dysplasia MUC 5AC+, MUC 6 focal, MUC1+, MUC2, CDX2 Shi and Hruban. Human Pathol 2011.epub 20 July 2011 Oncocytic Type Cells: Uncommon type MUC 5AC goblet cells+, MUC1, MUC2 High grade dysplasia goblet cells+, MUC 6 +, CDX2 AFIP 4 th Series Fascicle 7

31 Mucinous Cysts of the Pancreas WHO Classification 2010 Premalignant Lesions PanIN-3 Intraductal papillary mucinous neoplasm with low to intermediate grade dysplasia Intraductal papillary mucinous neoplasm with high grade dysplasia Mucinous cystic neoplasm with low to intermediate grade dysplasia Mucinous cystic neoplasm with high grade dysplasia Malignant Intraductal papillary mucinous neoplasm with invasive carcinoma Mucinous cystic neoplasm with invasive carcinoma Changes in Nomenclature Adenoma (WHO 2000) Low grade dysplasia (AFIP and WHO 2010) Borderline carcinoma (WHO 2000) Moderate dysplasia (AFIP) Intermediate grade dysplasia (WHO 2010) Carcinoma in-situ (WHO 2000) High grade dysplasia/cis (AFIP) High Grade dysplasia/premalignant (WHO 2010) Mucinous Cystadenocarcinoma (WHO 2000) MCN with invasive carcinoma (AFIP and WHO 2010) IPMN carcinoma (WHO 2000) IPMN with invasive carcinoma (AFIP and WHO 2010) International Consensus Guidelines for Management of IPMN and MCN (Tanaka, et.al. Pancreatology. 2006; 6:17) 8

32 The International Consensus Guidelines 2012 for the Management of IPMN and MCN of the Pancreas Tanaka M, Fernandez del Castillo C, Adsay V, Chari S, Falconi M, Jang J Y, Levy P, Pitman MB, Schmidt MC, Shimizu M, Wolfgang CL, Yamaguchi K, Yamao K. Pancreatology, 2012 May Jun;12(3): High Risk Stigmata Surgery if clinically feasible Obstructive jaundice in a patient with a cyst in the pancreatic head Enhancing solid component of the cyst Main pancreatic duct dilatation 10mm Worrisome Features EUS FNA Cyst 3cm Thickened/enhancing cyst walls Main duct 5 9mm Non enhancing mural nodule Abrupt change in MPD size with distal pancreatic atrophy EUS FNA Susp/Pos cytology Surgery Cytological Preparations No-ROSE Cysts Direct smears If fluid thick enough Fresh undiluted cyst fluid CEA; Amylase Molecular Cytology Cytospin Cellblock Pancreas Cyst Fluid Triage Protocol EUS Requisition form with detailed information ~volumes required All, fresh to molecular lab Warren 5 by endoscopist ~0.3cc KRAS (fresh) <0.5cc or ~0.3cc CEA (fresh) ~0.3cc All, fresh to cytology lab Warren 113 vortex 0.5 cc 0.3cc &/or Centrifuge residual supernatant ~0.3cc ~0.3cc cells KRAS (clinical) 1. CEA 2. Amylase 3. Tissue Bank Cytospin 9

33 Cytological Analysis of Cyst Fluid Challenges Scant fluid Low to no cellularity GI contamination Lack of experience with these types of specimens duodenum Two basic questions for Cyst analysis 1) Is the cyst mucinous or non mucinous? 2) Is the cyst benign or malignant? Is the Cyst Mucinous or Non mucinous? Gross exam by endoscopist thick, viscous fluid Light microscope Thick colloid-like mucin is diagnostic Special stains Alcian Blue (ph2.5): acid mucin Mucicarmine: neutral mucin 10

34 CEA by biochemical analysis ng/ml 10 1 Serous Inflammatory Mucinous Borderline Malignant athology Brugge, et al. Gastroenterology 2004 CFA cut-off levels lab and study dependent (van der Waaij, et. al. Cystfluid analysis in the differential diagnosis of pancreatic cystic lesions: a pooled analysis. Gastrointes Endosc. 2005; 62:383) CEA >800ng/ml CEA <5ng/ml Amylase <250 U/L Neoplastic mucinous cysts Serous cystadenoma Pseudocyst Not a pseudocyst Cyst Fluid CEA is an Accurate Marker of Pancreatic Mucinous Cysts Pancreas 2011 Oct;40(7): Sevdenur Cizginer, M.D., Brian Turner, M.D., A. Reyyan Bilge, PhD, Cetin Karaca, M.D., Martha B. Pitman, M.D. and William R Brugge, M.D. Massachusetts General Hospital, Harvard Medical School Sensitivity= 89/110 (81%) Specificity= 43/44 (98%) Accuracy= 132/154 (86%) PATHOLOGY CEA (using cut-off value of 109.9) Mucinous Total Nonmucinous Nonmucinous 43 (97.7%) 1 (2.3%) 44 Mucinous 21 (19.1%) 89 (80.9%) 110 Total

35 Two basic questions for Cyst analysis 1) Is the cyst mucinous or non mucinous? 2) Is the cyst benign or malignant? Cyst Fluid CEA is an Accurate Marker of Pancreatic Mucinous Cysts Pancreas 2011 Jul 14. [Epub ahead of print]. Sevdenur Cizginer, M.D., Brian Turner, M.D., A. Reyyan Bilge, PhD, Cetin Karaca, M.D., Martha B. Pitman, M.D. and William R Brugge, M.D. Massachusetts General Hospital, Harvard Medical School TABLE 4. Relative Accuracy of EUS Morphology, Cytology, and CEA in the Diagnosis of a Malignant Cyst EUS Morphology Cytology CEA* Sensitivity 33/72 (45.8%) 27/72 (37.5%) 43/55 (78.2%) Specificity 98/126 (77.8%) 121/126 (96%) 54/102 (52.9%) Accuracy 131/198 (66.2%) 148/198 (74.7%) 97/157 (61.8%) * cut off 109 ng/ml MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION HARVARD MEDICAL SCHOOL Intraductal Papillary Mucinous Neoplasm of the Pancreas: Cytologic Analysis and Correlation with Histologic Grade PJ Michaels, EF Brachtel, BC Bounds, WR Brugge, and MB Pitman (Cancer Cytopathol 2006; 108: ) Low grade dysplasia Moderate dysplasia HGD/Carcinoma 12

36 Atypical Epithelial Cells High-Grade Atypical Epithelial Cells in Pancreatic Mucinous Cysts are a More Accurate Predictor of Malignancy than Positive Cytology Martha Bishop Pitman M.D, Muriel Genevay, M.D, Kurt Yaeger, Ivan Chebib, Brian G. Turner, Mari Mino-Kenudson, William R Brugge (Cancer Cytopathol 118: ; 2010) Table 3. Performance Characteristics of Cytology with Various Thresholds of Predicting Malignancy Sensitivity Specificity PPV NPV Accuracy Malignant cells only (all NMC) AEC only* HGA (all NMC) HGA (small BD-IPMN) HGA (MD as TP, all NMC) HGA (MD as TP, small BD-IPMN) MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION HARVARD MEDICAL SCHOOL Cytology Adds Value to Imaging Studies for Risk Assessment of Malignancy in Pancreatic Mucinous Cysts Muriel Genevay, M.D, Mari Mino Kenudson, M.D, Kurt Yaeger, Ioannis T. Konstantinidis, M.D., Cristina R. Ferrone, M.D., Sarah Thayer, M.D.,Ph.D, Carlos Fernandez del Castillo M.D., Dushyant Sahani M.D., Brenna Bounds, M.D., David Forcione, M.D., William R Brugge M.D., and Martha Bishop Pitman M.D. (Annals of Surgery, in press) Table 5: Performance Characteristics of Significant High Risk Stigmata in Predicting Malignancy (CIS or invasion) in Small Branch-duct Intraductal Papillary Mucinous Neoplasms Sensitivity Specificity PPV NPV HGA MN Dilated MPD MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION HARVARD MEDICAL SCHOOL 13

37 Potentially Useful Molecular Tests KRAS Mutations support mucinous etiology GNAS Mutations present in ~60% IPMN but not MCN Sci Transl Med 20 July 2011 ** KRAS and GNAS mutations do not predict malignancy Benign/Low-Grade Glandular Epithelium High Grade Atypical Epithelial Cells in Pancreatic Mucinous Cysts are a More Accurate Predictor of Malignancy than Positive Cytology Martha Bishop Pitman M.D, et.al. (Cancer Cytopath 2010) High-Grade Atypical Glandular Epithelium High Grade Atypical Epithelial Cells in Pancreatic Mucinous Cysts are a More Accurate Predictor of Malignancy than Positive Cytology Martha Bishop Pitman M.D, et.al. ( Cancer Cytopath 2010) 14

38 Grading Epithelial Atypia in EUS-FNA of Pancreatic Mucinous Cysts: An International Interobserver Concordance Study MASSACHUSETTS HARVARD GENERAL HOSPITAL MEDICAL SCHOOL Martha B Pitman MD 1, Barbara A Centeno MD 2, Muriel Genevay MD 3, Ricardo Fonseca, MD 4 and Mari Mino-Kenudson MD 1. PATHOLOGY Departments of Pathology 1 Massachusetts General Hospital, Harvard Medical School, Boston, MA; 2 H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL; 3 Hopital de Pathologie Clinique (CMU), Geneva, Switzerland; 4 Instituto Português de Oncologia de Lisboa Francisco Gentil, Lisbon, Portugal Background: Pancreatic cyst fluid (PCF) analysis provides cytological and biochemical information regarding cyst type and grade. We have demonstrated that the identification of atypical epithelial cells (AEC) in PCF is a sensitive marker for malignancy and is specific for a mucinous cyst with at least moderate dysplasia. Here we assess the interobserver concordance among an international panel of cytopathologists with varying experience and expertise in grading epithelial atypia in PCF. Design: A test set of 60 cell groups was selected from EUS-FNAs of histologically confirmed mucinous cysts of various grades. The cell groups were circled on the glass slide, numbered and paired with digital images to ensure that each reviewer was evaluating the same cells under the microscope. Blinded to the histological grade, observers graded the cell groups as either: 0: gastrointestinal (GI) contamination, benign pancreatic tissue or histiocytes; 1: low grade dysplasia (LGD); 2: moderate dysplasia (MD); 3: high grade dysplasia (HGD); or 4: malignant. Proportion of agreement and kappa coefficient was calculated among all reviewers for low- grade (0-1 or 0-2) versus high-grade (2-4 or 3-4). Agreement was also determined among the two most experienced reviewers. Results: There was perfect agreement among the 4 reviewers in only 8/60 (13%) interpretations, with 6 of these 8 in the 0 classification, and the remaining 2 in grades 3 and 4. Three of 4 reviewers agreed in 16/60 (27%), and 2 reviewers agreed in 33/60 (55%) classifications. There were 3 cell groups that garnered a different classification from all reviewers. Grouping moderate dysplasia in the low and high grade categories did not affect agreement with 75% agreement grouping 0-2 and 3-4, and 71% agreement grouping 0-1 and 2-4. Similarly, the kappa coefficient was moderate and similar between the two classification groupings, 0.45 and 0.42, respectively. The two most experienced reviewers agreed 87% of the time for the 0-2 vs. 3-4 grouping, and 85% for the 0-1 vs. 2-4 grouping; kappa coefficient was substantial and similar between the two groupings, 0.74 and 0.68 respectively. However, only 25% agreement was noted with the grade of moderate dysplasia between these two reviewers. Conclusion: Agreement in classification of dysplasia in mucinous cysts into low-grade and high-grade groups is fairly good among all reviewers and excellent among the two most experienced reviewers. Moderate dysplasia is the most difficult to classify. We recommend grouping moderate dysplasia in the low-grade category and using a 3 tiered classification system of interpreting lesional epithelium: low-grade, high-grade and malignant when possible. Gastrointestinal Contamination Low-grade dysplasia Low-grade Atypia: Nonspecific for origin High-grade Dysplasia High-grade Atypia: Nonspecific for grade Malignant Two experienced reviewers agree as High-grade Complete discordance Concordant Discordant #179 MASSACHUSETTS Endoscopic Ultrasound-Guided Fine Needle Aspiration HARVARD GENERAL HOSPITAL MEDICAL SCHOOL (EUS-FNA) Contributes to a Triple Negative Test in PATHOLOGY Preoperative Screening of Pancreatic Cysts Roseann I. Wu, MD, MPH 1, Won Jae Yoon, MD 2, William R. Brugge, MD 2, Mari Mino-Kenudson, MD 1, Martha B. Pitman, MD 1 1 Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 2 Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts #187 Background EUS-FNA of pancreatic cysts obtains cyst fluid for cytological, biochemical and molecular analysis that can determine if the cyst is mucinous and if cells are present that either suggest or confirm the presence of malignancy. Despite this added value, the utility of EUS-FNA remains controversial in preoperative assessment of pancreatic cysts. International consensus guidelines for the management of intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN) of the pancreas note that small ( 3 cm) pancreatic cysts without high-risk stigmata of malignancy or worrisome features may be observed with repeat imaging only. We assessed the utility of EUS-FNA in a cohort of small pancreatic cysts that were benign by imaging. Results EUS-FNA provided a diagnosis of a mucinous cyst in 37 of 92 patients (40%): 27 (73%) by cytology and 10 (27%) by elevated CEA. The mean follow-up period was 3.5 years (range, years), during which six cases were surgically resected and 15 cases had at least one subsequent EUS-FNA. The resected cysts were five IPMNs (three with intermediate-grade dysplasia {IGD}, one with HGD, one with associated invasive carcinoma) and one MCN with low-grade dysplasia (LGD). Cytology confirmed the absence of HGA in 89 of 92 cases (97%). The 3 cyst fluids with HGA included: CASE 1 CASE 2 CASE 3 Figure 1. Algorithm for the management of suspected BD-IPMN Reference: Pancreatology May-Jun;12(3): Figure 2a. 9 x 7 mm cystic lesion, pancreatic head Figure 2c. 9 x 6 mm cystic lesion, pancreatic head Figure 2e. 20 x 20 mm multi cystic lesion, pancreatic head Figure 2b. Malignant IPMN by EUS, obstructive jaundice and lung masses at 11 months Figure 2d. BD-IPMN with IGD by EUS and resection Figure 2f. Stable cyst size 1 year after FNA then lost to F/U Design We analyzed all pancreatic cysts with EUS-FNA performed between 2006 and 2007 in order to have sufficient clinical follow-up. Of 226 cases, we excluded cases with initial EUS-FNA prior to 2006 or with any high-risk (obstructive jaundice and cyst in the pancreatic head, enhancing solid component, main pancreatic duct {MPD} 10 mm) or worrisome (pancreatitis, cyst 3 cm, thickened/enhancing cyst walls, MPD 5-9 mm, non-enhancing nodule, abrupt change in caliber of pancreatic duct with distal pancreatic atrophy) features by imaging. 92 patients with pancreatic cysts met the inclusion criteria. CEA >192 ng/ml supported a mucinous cyst. Cytology detected extracellular mucin and either did or did not identify cells with high-grade atypia (HGA) consistent with high-grade dysplasia (HGD) or carcinoma. Outcome was determined by either surgical resection or clinical follow-up. 84 of the 92 (91%) patients had uneventful follow-up (i.e. without development of high-risk or worrisome imaging features). The patient with an invasive IPMN progressed from acellular cyst FNA to HGA cytology to adenocarcinoma on successive EUS- FNAs over a two year period. The overall negative predictive value of cytologic examination for HGA was 97.8%. Conclusions The risk of malignancy in small pancreatic cysts with benign imaging is low. EUS-FNA is a screening test that contributes to a triple negative test for pancreatic cysts-- no high-risk stigmata, no worrisome features, and no HGA on cytology-- providing a negative predictive value of 97.8% for conservative management. Cytology may also be helpful in monitoring malignant transformation of mucinous pancreatic cysts over time. 15

39 Algorithmic Guide to Diagnosis Cyst No Epithelial Cells Epithelial Cells Non-Mucoid Mucoid Cuboidal Mucinous Amylase, CEA CEA CEA SCA Not Atypical Atypical Amylase Amylase CEA CEA ND (SCA, PanNET) PCT IPMN MCN Thick ECM Thin ECM IPMN MCN; GI contamination IPMN MCN IPMN MCN, At least Moderate dysplasia IPMN MCN CEA CEA ND IPMN MCN Thank You! 16

40 Lester J. Layfield, MD Professor and Chair Department of Pathology and Anatomical Sciences University of Missouri at Columbia While a binary characterization of benign or malignant is of greatest value to clinicians: Most cytopathologists use a three or four category scheme. Intermediate categories have been designated: Dysplasia Atypical cells Low grade or high grade dysplasia Atypia most likely reactive or Atypia suspicious for malignancy 1

41 Proposed Categorization: Non diagnostic Benign Atypical Ductal Cells of Uncertain Significance, most likely reactive Atypical Ductal Cells of Uncertain Significance, suspicious for malignancy Malignant Characteristics of Benign Biliary Epithelium Regular honeycomb sheets (en face) Orderly palisades (from side) of cuboidal or columnar cells Single cells round up Nuclei are round or oval and lie near base of cell (polarity) Smooth nuclear membranes with even chromatin Small or absent nucleoli Cytoplasm is pale and moderately abundant Mitotic figures and necrotic debris absent Goblet cells may be present Golden brown bile pigment common in background 2

42 The pattern of benign epithelium can be modified by: Biliary tract stones Stents Acute inflammation 3

43 Benign reactive cells (ADCUS, R) are characterized by: Variation in nuclear size but usually less than three fold in any cell group Hyperchromasia Prominent nucleoli Normal mitotic figures Relatively normal N/C ratio Squamous metaplasia Degenerative changes Degenerative changes associated with reactive change: Nuclear pyknosis and karyorrhexis Development of dense eosinophilic cytoplasm (Pap) Degenerating cells usually single Pencil or match stick cells Nuclear crenation (irregular membranes may occur but grooves and popcorn like irregularities are uncommon Nuclear irregularities usually associated with loss of nuclear detail and cytoplasmic eosinophilia (Pap) 4

44 5

45 Differential Diagnostic Tips Reactive cells retain well ordered cohesive clusters and sheets Reactive cells show little nuclear crowding or overlap Reactive cells retain relatively abundant cytoplasm with normal N/C ratio While reactive cells can show considerable variation in nuclear size, variation in size in any single group is rarely over 3 fold Unlike malignant cells, the atypical features of reactive cells can be seen in some adjacent clearly benign cells ADCUS, Suspicious for Malignancy Cells and cell groups have some but not all the features of malignancy 6

46 Cytologic Features of Atypical Ductal Cells of Uncertain Significance, Suspicious for Malignancy Cells form groups and clusters with prominent nuclear crowding Coarse chromatin Variably enlarged nucleoli Increased N/C ratio Nuclear membrane irregularity Up to 3 or 4 fold variation in nuclear size in single cell groups 7

47 Cytologic Features of Adenocarcinoma Increased N/C ratio + Three dimensional cell clusters with marked nuclear crowding Loss of polarity + Nuclear molding + Papillary groups, syncytial groups Cell in cell groups + Large pleomorphic nuclei Atypical mitotic figures Necrotic debris Four fold or greater variation in nuclear size + 8

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50 Major Diagnostic Pitfalls Cloaking of malignant epithelium by overlying benign epithelium Insufficient sampling Degeneration due to bile or duodenal contents Primary sclerosing cholangitis Metaplasia (stones, stents) How do we do? Follow up Cytologic diagnoses Benign histology and/or clinical follow up Probable cancer death or severe deterioration, radiographic picture of metastases Histologically proven cancer Histologically proven dysplasia No diagnostic change Reactive 8 1 Squamous metaplasia 4 Low grade dysplasia High grade dysplasia Carcinoma

51 Reported Accuracy Rates for Pancreatic Biliary Duct Brushing Cytology Author Sensitivity (%) Specificity (%) Desa Foutch Foutch Layfield Lee Rabinovitz 40 Rupp Scudera Venu Ryan 44 Sawada 85 Final diagnosis of a bile duct stricture requires correlation of findings in ERCP and cytology. How can we improve results of brushing cytology? 12

52 Ancillary Tests Intraductal ultrasound CEA K ras point mutation Modified UroVysion FISH DNA analysis Panel of tumor suppressors linked microsatellite marker allelic loss Loss of heterozygosity Intraductal Ultrasound (IDUS) Safe and readily performed Produces detailed images of duct system Generally good diagnostic sensitivity and specificity Results poorer in PSC K ras Point Mutations Common in biliary tract carcinoma (70%) (91% sensitivity, 93% specificity) K ras point mutations are associated with malignancy (p<.oo1) Can occur in benign lesions (17%) Literature remains divided on utility of K ras mutational analysis 13

53 From: Hruban RH, Goggins M, Parsons J, Kern SE. Progression Model for Pancreatic Cancer. Clin Cancer Res Aug;6(8): ken Modified UroVysion Looks for numerical changes in chromosomes 3, 7, 17 and band changes 9p21 Polysomy often seen in adenocarcinoma 80% of carcinomas demonstrate aneuploidy with gains in multiple chromosomes Trisomy 7 may occur but can be seen in reactive lesions and PSC FISH Analysis Significantly improves diagnostic sensitivity without loss of specificity Probes are commercially available Most useful in cases where cytology is negative or inconclusive 14

54 Recommendation: FISH testing should be performed on clinically suspicious but cytologically negative brushing specimens and all equivocal brushing specimens to optimize diagnostic accuracy. 15