ROSE (Rapid on site evaluation) in EUS-FNA in solid and cystic pancreatic lesions: possibilities and limits
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1 ROSE (Rapid on site evaluation) in EUS-FNA in solid and cystic pancreatic lesions: possibilities and limits Charitini Salla, MD Head of Cytopathology Department, Hygeia Hospital, Athens, Greece
2 There is no conflict of interest
3 Methods of cytological material collection by FNA CT-guided FNA Transabdominal US-guided FNA Transgastric or transduodenal EUS-FNA Intraoperative FNA is a also a possibility
4 Indications for FNA Most of the times, presence of a mass lesion: solid solid and cystic cystic Organ-specific lesions non-organ-specific lesions (lymphomas, metastases, mesenchymal neoplasms) Sometimes, a diffuse lesion can be sampled
5 EUS-FNA
6 ROSE in Cytology is the equivalent of Frozen section in Pathology
7 Why is ROSE necessary? Decreases: number of non-diagnostic samples number of passes in the lesion patient discomfort cost for health care system
8 PANCREATIC LESIONS SOLID CYSTIC
9 ROSE Recommended for solid lesions: Obtain adequate lesional sampling for a definite diagnosis, taking into account the endoscopist s sonographic impression Prepare the sample for ancillary studies: histochemical, immunohistochemical, biochemical, flow cytometry, molecular (as NGS) and cytogenetic (as FISH) The adequacy of the specimen depends on: size of the lesion nature of the lesion Non-cellular smears in: cystic lesions desmoplasia or necrosis number of passes experience of the endoscopist
10 Valuable information before ROSE Demographic data (gender, age) Clinical data Patient history with emphasis on: alcoholism obesity cigarette smoking pancreatic injury medications toxins infections (toxoplasmosis, adenovirus, TB) malnutrition metabolic disorders IgG4-related diseases polyarteritis nodosa family history of cancer cystic fibrosis other cancer diseases of the biliary tract hyperparathyroidism Signs: Courvoisier Virchow Cullen Grey Turner Symptoms: malaise, fatigue anorexia, weight loss abdominal or back pain recurrent acute pancreatitis nausea and vomiting diarrhea fever and chills ascites / pleural effusion malabsorption new-onset diabetes hyperbilirubinemia (jaundice, pruritus, dark urine, pale colored stools) Syndromes VHL MEN I Peutz-Jeghers FAMMM Beckwith-Wiedemann Trousseau syndrome (hypercoagulability) lipase hypersecrection syndrome clinical syndrome due to hormone production Before ROSE, the cytopathologist should be informed about the most striking features concerning the examined patient
11 Valuable information before ROSE Serum biochemical tests: CEA, Ca19-9 IgG4 amylase, lipase, bilirubin Imaging findings other than EUS* CT/MRI: solid/cystic lesion size and borders of the lesion when cystic: presence of capsule, pattern of calcification location in the pancreas invasion of the adjacent organs probable metastasis in the liver or peripancreatic lymph nodes structural abnormalities, such as pancreas divisum ERCP/MRCP: dilation of ductal system detection of stones or mural nodules in the ductal system communication with the pancreatic ductal system Previous cytological examination Transgastric or transduodenal approach Before ROSE, the cytopathologist should be informed about the most striking features concerning the examined patient
12 Diagnostic approach of the pancreatic lesion Normal vs. lesional tissue Lesional tissue vs. gastrointestinal and pancreatic contamination IF POSSIBLE, allocation of the lesion into one of the diagnostic categories proposed by The Papanicolaou Society of Cytopathology System for Reporting Pancreatobiliary Cytology (Nondiagnostic) Negative Atypical Neoplastic: benign / other Suspicious Positive During the ROSE, most of the times we try to classify the lesion as positive, negative or belonging in the grey zone Identification of the lesion The grey zone includes both: premalignant lesions specimens with various degrees of reactive atypia
13 ROSE of solid lesions First step Solid lesions Luminal border formation and columnar configuration Exclude: gastrointestinal contamination metastasis (if compatible with other cancer s morphology) Second step Glandular smear pattern Other lymphoma sarcoma non-aca met Solid cellular smear pattern Cell-rich, stroma-poor Exclude: pancreatic contamination (normal or injured acini) Differential diagnosis: chronic pancreatitis PDA (well, moderately, poorly differentiated) metastatic adenocarcinoma Differential diagnosis: PanNET SPN acinar cell carcinoma PBL
14 EUS findings Chronic pancreatitis Autoimmune pancreatitis Solid cellular lesions PDA Visible side branches Cysts Lobularity MPD: irregular dilated with hyperechoic margins Hypoechoic foci and strands +/- stones Diffuse pancreatic swelling: sausage-like with halo effect Mass lesion Hypoechoic mass Homogeneous Well defined (distinct borders, pushing rather than infiltrating) +/- cystic degeneration Hypoechoic mass Irregular borders Double duct sign : dual dilatation of biliary and pancreatic ducts due to a neoplasm of the pancreatic head: pathognomonic for PDA +/- cystic degeneration EUS features are the most important data for the cytopathologist, as they reflect the endoscopist s impression about the lesion
15 Gastrointestinal contamination: epithelial Gastric epithelium Foveolar cells and mucus neck cells: Sheets Seen on surface: of variable size, with a uniform honeycomb pattern Seen on edge: with apical mucinous cups (occupying the upper 1/3 of the cytoplasm) Nuclei: naked nuclei in a mucoid background, slightly elongated, some with nuclear grooves No goblet cells, no lymphocytes, no brush border Chief cells: Abundant blue granules in the cytoplasm Parietal cells: Abundant dense cytoplasm
16
17 Gastrointestinal contamination: epithelial Duodenal epithelium Enterocytes: large folded sheets of evenly spaced glandular cells studded by goblet cells (fried egg appearance) microvillous border sometimes luminal, sometimes brush border consisting of enterocytes with nonmucinous cytoplasm Intraepithelial lymphocytes: Paneth cells: Coarse, eosinophilic granules in their apical cytoplasm
18
19 Gastrointestinal contamination: mucinous Thin or focally thick, never colloid type Cellular or inflammatory debris are not entrapped within the mucin (clear mucin) In immediate proximity with non-degenerated benign glandular cells Colloid-type mucin: suggestive of mucin-producing cystic neoplasm
20 Pancreatic contamination (in the appropriate clinical setting) Acinar epithelium grape-like acinar structures few naked nuclei Ductal epithelium honeycomb sheets of non-mucinous glandular cells orderly polarized picket-fence cells Islet cells generally not recognized
21 Normal acini
22 Chronic pancreatitis (30%: mass lesion) Mixed population Ductal cells: reactive changes, loss of honeycomb pattern, squamous metaplasia Acinar cells: decrease in late stages Islet cells: single or intact islets Fibrous tissue with spindle cells in interlacing groups Fat but no coagulative necrosis and calcifications Chronic inflammatory cells IgG4-related disease Autoimmune pancreatitis Often not possible diagnosis by cytology alone ROSE: chronic pancreatitis with cellular, fibrous stroma significant atypia of the ductal cells
23 Chronic pancreatitis
24 Chronic pancreatitis
25 Chronic pancreatitis
26 Cellularity Steps in the assessment of PDA Smear pattern (cohesive or loose tissue fragments and dispersed cells) Background (clean or necrotic) Cellular composition (mix of ductal and acinar cells or pure population of ductal cells) Architectural features Nuclear and cytoplasmic features
27 PDA Architectural abnormalities (low power view) Loss of polarity / uneven distribution of cells in the sheet or drunken honeycomb in case of mucinous cytoplasm Crowding / overlapping Cell-in-cell Balls Papillary fragments Nuclear molding Stratification of nuclei when seen on edge More single cells, as dedifferentation occurs +/- tumor diathesis Nuclear abnormalities (high power view) Anisonucleosis (>4:1 nuclear variability in the same cell group) Prominent nucleoli / macronucleoli Chromatin clumping and coarsening Chromatin clearing Nuclear membrane irregularities Nuclear enlargement (>1.5 xrbc) N/C ratio increased +/- Mitoses and karyorrhexis Differential diagnosis between reactive changes in GI epithelium and well-differentiated PDA is based on: nuclear membrane irregularities anisonucleosis 4:1 variation in nuclear size within the same fragment
28 Drunken honeycomb
29 PDA
30 PDA
31 PDA
32 PDA
33 Variants of PDA Colloid carcinoma Undifferentiated carcinoma with osteoclast-like giant cells Undifferentiated/anaplastic carcinoma Adenosquamous carcinoma Medullary carcinoma Hepatoid carcinoma
34 Colloid carcinoma
35 Anaplastic
36 Osteoclastic
37 Adenosquamous carcinoma, squamous component Adenosquamous carcinoma, glandular component
38 ROSE of solid cellular lesions: dd Ar N C PanNET SPN Acinar cell carcinoma PBL (Biphasic and trilineage neoplasm) Small groups Rosettes Clusters Single cells Stripped nuclei Salt & pepper Round nuclei Bland and uniform / endocrine atypia Eccentric Nucleoli inconspicuous (ST: prominent) Branching, papillary-like groups with central fibrovascular core, covered with myxoid stroma, with a third layer of epithelial cells Single cells Naked nuclei Balls of myxoid stroma +/- surrounding thick layer of neoplastic cells (pathognomonic) Round to oval Finely granular chromatin Indentations and grooves Inconspicuous nucleoli Naked nuclei Single cells Cellular fragments Acinar formations Rare syncytia Round to oval Nuclear membrane irregularity (poorly differentiated) Eccentrically placed Single prominent nucleolus Coarsely clumped chromatin Focal anisonucleosis N/C ratio slightly increased Acinar Endocrine Ductal Squamous nests or corpuscles Mesenchymal elements Indistinguishable from acinar cell carcinoma, when squamoid corpuscles and mesenchymal elements are absent, as acinar elements predominate. Cy Scant Dense Variants: clear or oncocytic Basophilic Varying in shape and quantity Perinuclear vacuole Hyaline globules Cytoplasmic tails Mi No No Yes Ne No Yes Rare Granular / vacuolated / dense Often basophilic Bg +/- vascular network Clean or cyst debris Clean or granular
39 Rule of thumb in ROSE ROSE: identification of the solid cellular pattern Definite diagnosis: a matter of immunohistochemistry
40 Acinar cell carcinoma
41 Acinar cell carcinoma
42 Acinar cell carcinoma
43 SPN
44 SPN
45 SPN
46 SPN
47 Neuroendocrine tumor
48 Neuroendocrine tumor
49 Neuroendocrine tumor
50 Metastatic disease Most common sites of origin in clinical series Lung (23%) Kidney (15%) Breast (8%) Melanoma (5%)
51 Metastasis from colon carcinoma
52 Metastasis from small cell lung carcinoma
53 Metastatic squamous cell carcinoma of the lung
54 Metastatic renal cell carcinoma, clear cell
55 Metastatic urothelial carcinoma
56 Metastatic leiomyosarcoma
57 ROSE is not recommended for cystic lesions However, in our Institution ROSE is performed to identify whether we deal with a mucin-producing neoplasm and the aspirated mucin is lesional or not. Characteristics of lesional mucin: Thick, colloid-type mucin Cellular or inflammatory debris within the mucin, either thick or thin
58 Gold standard Intra-laboratory: concordance between ROSE and final cytologic diagnosis Inter-laboratory: concordance between final cytologic diagnosis and pathology report
59 The battle hymn of the endoscopist Dr. Scott Boerner MD, FRCPC Assoc. Prof., University of Toronto I m in!!! Thank you for your attention!
60 Molecular panel for dd between benign and malignant ductal groups K-ras (expressed also in PanIN grade 1) P53 (loss of expression in adenoca) DPC4 (loss of expression in adenoca in only 55% of all PDAs) Chhieng DC, Stelow EB. Pancreatic Cytopathology Molecular mutations: -activation of oncogenes(k-ras, Her2-neu) -inactivation of tumor suppressor genes (p53, p16, DPC4) PCR identified numerous genes implicated in pancreatic carcinogenesis, such as mesothelin, prostate stem cell antigen, S100P, maspin, IMP3 Promising immunohistochemical panel: pvhl(-), maspin(+), S100P(+), IMP3(+) Distinct pattern of mirnas expression in PDAs, applicable in FNA samples
61 Evaluation of cystic lesions EUS features Location Size Wall thickness Unilocular/ oligolocular /multilocular Single / multiple Communication with the larger pancreatic ducts Margins (sharp/nondescript) Calcifications and their location Presence of mural nodules, papillary projections, thick septa, internal debris Gross features of the fluid Viscosity Colour Volume (quantity) Biochemical assessment CEA and amylase: can be assessed on the neat fluid or the supernatant dilution of saline in case of scant samples 1 ml volume is adequate for both CEA and amylase analyses Molecular assessment +/- molecular mutations (KRAS, GNAS) 0.3 ml of the neat fluid after vortexing is adequate
62 Criteria for presence of mucinous cysts Mucin production Thick, colloid-type mucin Cellular or inflammatory debris within the mucin, either thick or thin or Elevated CEA of the cyst fluid 192 ng/ml: accuracy 80% or KRAS/GNAS mutation and/or Neoplastic epithelial cells present Low grade atypia (low and intermediate grade dysplasia) High grade atypia (at least high grade dysplasia, carcinoma in situ) Mucinous cysts are heterogeneous neoplasms and the cytology sample may not reflect the highest grade of the lesion Low CEA / absence of KRAS or GNAS mutations do not preclude a neoplastic mucinous cyst
63 Genetic mutations in neoplastic mucinous cysts IPMNs KRAS (% of cases) Gastric Intestinal Pancreatobiliary Oncocytic Rare Rare GNAS mutation in codon 201 (% of cases) MCNs Only KRAS mutations The KRAS-GNAS combination cannot predict the grade of dysplasia or invasion Next generation sequencing (NGS) can identify genetic mutations that occurs in high grade dysplasia and invasive carcinoma (TP53, SMAD4, CDKN2A)
64 Targeted therapies in pancreatic adenocarcinoma K-Ras, MAP2K and MEK pathway > MAP2K MEK inhibitors Tyrosine kinase receptor pathway > EGFR, VEGFR, IGFR-1, PDFR inhibitors Molecular targets RET pathway > RET, GDNF inhibitors TP53 tumors suppressor pathway PI3K/Akt pathway > PI3K, Akt inhibitors Stromal compartment > Sonic hedgehog (SHH) pathway inhibitors, Hyaluronidase inhibitors, Matrix Metalloproteinases inhibitors mtor signaling pathway > mtor inhibitors JAK/STAT signaling pathway > JAK1/2, STAT3 inhibitors Cancer stem cells (Notch, Wnt and SHH pathways) > Notch 2/3 inhibitors Autophagy Poly (ADP-ribose)polymerase pathway > PARP inhibitors Mosquera C et al. Molecular targeted therapy for pancreatic adenocarcinoma: A review of completed and ongoing late phase clinical trials. Cancer Genet Aug 2. pii: S (16)30223-X. Chadha AS et al. Recent Advances and Prospects for Multimodality Therapy in Pancreatic Cancer. Semin Radiat Oncol Oct;26(4):
65 EGFR pathway inhibition Erlotinib / Tarceva FDA approval in advanced stage disease Phase III trial conducted by National Cancer Institute of Canada showed: Improved overall survival (OS) (HR 0.82; 95% CI ; p = 0.038) Greater one year survival (23% vs 17%), p = in the erlotinib plus gemcitabine group vs gemcitabine only group Phase II trial by ESMO (Pantar Study) also showed survival improvement Currently, this is the only FDA approved targeted therapeutic option for PDA Karandish F and Mallik S. Biomarkers and Targeted Therapy in Pancreatic Cancer. Biomark Cancer Apr 26;8(Suppl 1): Moore MJ, Goldstein D, Hamm J, et al. Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the national cancer institute of canada clinical trials group. J Clin Oncol 2007;25:
66 Future perspectives Radiotherapy + targeted therapies Immunotherapy (checkpoint inhibitors, cancer vaccines, adoptive T-cell transfer) + targeted therapies Biomarkers of response (such as serum biomarkers, surface markers of cancer cells, mirnas) Gene therapy Nanotechnology Personalized cancer treatment Not all people are the same Not all tumors are the same
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