1. Malignant Mesothelioma in Effusions and Fine Needle Aspirates No relationship exists that represents a possible conflict of interest with respect to the content of this presentation Armando C. Filie, M.D. National Cancer Institute

2. CYTOTELECONFERENCE 2007 - 2008 2

3. 3 OBJECTIVES Objectives Recognize the cytological features of malignant mesothelioma (mesothelioma) in effusion samples Recognize the cytological features of fine needle aspirates of mesothelioma Recognize the cytological features of major lesions in the differential diagnosis of mesothelioma Familiarize with current ancillary studies in the diagnosis of mesothelioma

4. CYTOTELECONFERENCE 2007 - 2008 4 BLANK SLIDE Mesothelioma Malignant neoplasm of pleura, peritoneal cavity and pericardium Incidence of 2,500 cases/year (pleural) Clinical Findings age and presentation: males, 6th-8th decade, unilateral pathogenesis: asbestos exposure (latency of 20- 50 years), ?simian vacuolating virus (SV40) imaging findings: CT scan [pleural masse(s)], invasion by magnetic resonance imaging (MRI) Diagnosis: clinical history + imaging findings + cytology(?)/biopsy

5. CYTOTELECONFERENCE 2007 - 2008 5 Mesothelioma Prognosis and Treatment poor prognosis treatment: surgery (most effective), chemotherapy, radiotherapy (localized recurrences), combine therapy Histologic Types epithelioid (epithelial): up to 17 subtypes (deciduoid, clear cell, small cell, signet ring) sarcomatoid: 8 subtypes (fibrosarcomatous, lymphohistiocytoid, MFH-like) biphasic (mixed) desmoplastic

6. CYTOTELECONFERENCE 2007 - 2008 6 Mesothelioma Cytological Features in Effusions sample preparation: smear, cytocentrifugation, thin layer, cell block (immunostains) stains: Diff-Quik, Papanicolaou

7. CYTOTELECONFERENCE 2007 - 2008 7 Mesothelioma Cytological Features in Effusions patterns: epithelioid (malignant epithelial), sarcomatous (sarcomatoid), anaplastic, biphasic sarcomatoid mesothelioma differential diagnosis: spindle cell sarcomas biphasic mesothelioma differential diagnosis: carcinomas (renal cell carcinoma) anaplastic mesothelioma differential diagnosis: pleomorphic sarcomas epithelioid mesothelioma: most frequent pattern, associated with effusion more frequently than other patterns.

8. CYTOTELECONFERENCE 2007 - 2008 8 Mesothelioma in Effusions Cytological Features of Epithelioid Mesothelioma cellular sample one cell population clusters (scalloped border) cell-in-cell formations intercellular spaces (“windows”) two-tone cytoplasm surface blebs variable N/C ratio multinucleation macronucleoli

9. CYTOTELECONFERENCE 2007 - 2008 9 Mesothelioma in Effusions Cytological Features of Epithelioid Mesothelioma

10. CYTOTELECONFERENCE 2007 - 2008 10 Mesothelioma in Effusions Cytological Features of Epithelioid Mesothelioma

11. CYTOTELECONFERENCE 2007 - 2008 11 Mesothelioma in Effusions Differential Diagnosis Metastatic carcinoma: adenocarcinomas (lung, breast, gynecologic tract, gastrointestinal tract), may be the first manifestation of an occult primary Hematologic neoplasms: B-cell lymphomas (diffuse large B-cell), T-cell lymphomas (anaplastic large cell), plasma cell neoplasms, primary effusion lymphoma (PEL) Melanoma: may be the first manifestation of disease Others: squamous cell carcinoma, mesothelial cell lesions

12. CYTOTELECONFERENCE 2007 - 2008 12 Mesothelioma in Effusions Cytological Features of Metastatic Adenocarcinoma cellular sample two cell population clusters (smooth border) cell-in-cell formations high N/C ratio multinucleation macronucleoli irregular nuclear contours delicate/dense cytoplasm vacuole(s) displacing the nucleus

13. CYTOTELECONFERENCE 2007 - 2008 13 Mesothelioma in Effusions Cytological Features of Metastatic Adenocarcinoma

14. CYTOTELECONFERENCE 2007 - 2008 14 Mesothelioma in Effusions Cytological Features of Metastatic Melanoma cellular sample two cell population (?) aggregates cell-in-cell formations low N/C ratio multinucleation macronucleoli intranuclear cytoplasmic inclusions melanin pigment vacuoles

15. CYTOTELECONFERENCE 2007 - 2008 15 Mesothelioma in Effusions Cytological Features of Metastatic Melanoma

16. CYTOTELECONFERENCE 2007 - 2008 16 Mesothelioma in Effusions Cytological Features of PEL cellular sample two cell population variable N/C ratio multinucleation macronucleoli dense basophilic cytoplasm

17. CYTOTELECONFERENCE 2007 - 2008 17 Mesothelioma in Effusions Cytological Features of PEL

18. CYTOTELECONFERENCE 2007 - 2008 18 Mesothelioma in Fine Needle Aspirates Image-guided fine needle aspiration (FNA) may be used for the initial diagnosis of mesothelioma 4% needle tract seeding for core-needle biopsy with sensitivity of 86% (pleural) FNA of metastatic mesothelioma (rare): scalp, thyroid, cervical lymph node, axillary lymph node, subcutaneous nodules, breast, liver metastasis may be the first indication of mesothelioma inclusions of benign mesothelial cells in lymph nodes Mesothelial cell lesions of pleura: solitary fibrous tumor (most benign, rare malignant), nodular pleural plaque, adenomatoid tumor, simple mesothelial cyst, multicystic mesothelioma, well-differentiated papillary mesothelioma, localized malignant mesothelioma

19. CYTOTELECONFERENCE 2007 - 2008 19 Mesothelioma in Fine Needle Aspirates Cytological Features of Mesothelioma in FNAs cellular aspirate clusters and flat sheets papillary groups (core) acinar/tubular groups single cells intercellular spaces round/polygonal shape spindle cells (sarcomatoid, biphasic) small cytoplasmic vacuoles multinucleation

20. CYTOTELECONFERENCE 2007 - 2008 20 Mesothelioma in Fine Needle Aspirates Cytological Features of Mesothelioma in FNAs

21. CYTOTELECONFERENCE 2007 - 2008 21 Mesothelioma in Fine Needle Aspirates Differential Diagnosis Epithelioid: carcinoma - lung (adenocarcinoma and bronchoalveolar carcinoma [BAC]), ovary and peritoneal serous carcinoma; mesothelial cell lesions; thymoma; epithelioid sarcomas, reactive mesothelial proliferations Sarcomatoid: mesothelial cell lesions, desmoid tumor, schwannoma, spindle cell sarcomas Biphasic: thymoma, synovial sarcoma, desmoplastic small round cell tumor, pleuropulmonary blastoma Anaplastic: pleomorphic sarcomas

22. CYTOTELECONFERENCE 2007 - 2008 22 Mesothelioma in Fine Needle Aspirates Cytological Features of Lung BAC in FNAs monolayer sheets papillae single cells round nuclei nuclear grooves and pseudoinclusions nuclear crowding/overlapping pleomorphic cells mucin (mucinous)

23. CYTOTELECONFERENCE 2007 - 2008 23 Mesothelioma in Fine Needle Aspirates Cytological Features of Lung BAC in FNAs

24. CYTOTELECONFERENCE 2007 - 2008 24 Mesothelioma (Ancillary Studies) Histochemical stains: mucin (Alcian blue, mucicarmin) Electron microscopy: long microvilli (meso), short (adeno) FISH: detection of chromosomal alterations Hyaluronic acid levels in effusion samples Immunocytochemistry: most commonly used may be applied to cytocentrifuged samples, smears, thin layer samples, cell blocks (preferred) panel of mesothelial cell and adenocarcinoma markers: 2 meso and 2 adeno markers or 1/2 meso and 3 adeno markers other markers: hematopoietic markers, melanoma markers, site “specific” markers (TTF-1, PSA, PAP, CDX-2, GCDFP-15, thyroglobulin)

25. CYTOTELECONFERENCE 2007 - 2008 25 Mesothelioma (Ancillary Studies) Mesothelial cell (Mesothelioma) Markers calretinin: neuron-specific calcium binding protein (neural tissues and a few other cell types like mesothelial cells) cytokeratin 5/6: intermediate filament (mainly keratinized and non-keratinized squamous cell carcinoma) Others: HBME-1, WT1,Mesothelin, Podoplanin calretinin

26. CYTOTELECONFERENCE 2007 - 2008 26 Mesothelioma (Ancillary Studies) Mesothelial cell (Mesothelioma) Markers HBME-1CK 5/6

27. CYTOTELECONFERENCE 2007 - 2008 27 Mesothelioma (Ancillary Studies) Adenocarcinoma Markers B72.3: antibody detects a tumor associated protein Ber-EP4: antibody against epithelial adhesion molecule CA19.9: antibody against Lewis a blood group antigen Others: mCEA, CD15, MOC-31 B72.3

28. CYTOTELECONFERENCE 2007 - 2008 28 Mesothelioma (Ancillary Studies) Adenocarcinoma Markers B72.3 Ber-EP4 CA19.9

29. CYTOTELECONFERENCE 2007 - 2008 29 Mesothelioma (Ancillary Studies) Melanoma markers: HMB45, Mart-1, KBA62, S100 Hematopoietic markers: LCA, L26, CD38, HHV8 Others: TTF-1, PSA and PAP, GCDFP-15, thyroglobulin HMB45Mart-1

30. CYTOTELECONFERENCE 2007 - 2008 30 Mesothelioma (Ancillary Studies) Hematopoietic and other markers HHV8TTF-1 CK 7

31. CYTOTELECONFERENCE 2007 - 2008 31 Mesothelioma (Ancillary Studies) Molecular Tests Gene expression (quantitative RT-PCR) Proteomics: protein complement of the genome (serum - early cancer diagnosis), potential in cytopathology Surface enhanced laser desorption/ionization time of flight (SELDI-TOF): protein profile in cytology samples (Fetsch et al, 2002) Initial set: 5 renal cell carcinomas, 9 metastatic melanomas, 6 reactive effusions Unknown set: 4 renal cell carcinomas, 8 metastatic melanomas, 3 reactive effusions

32. CYTOTELECONFERENCE 2007 - 2008 32 Mesothelioma (Ancillary Studies) Molecular Tests SELDI-TOF in FNAs and fluid samples of 8 MM, 4 RCC, 3 reactive effusions

33. CYTOTELECONFERENCE 2007 - 2008 33 Mesothelioma in Effusions and FNAs SUMMARY Cytological features of mesothelioma in effusions and FNAs overlap with those seen in other benign and malignant lesions (adenocarcinoma) Some cytologic features of mesothelioma are not often present in cytology samples of lesions that should be considered in the differential diagnosis Ancillary studies are important in supporting the diagnosis of mesothelioma (immunocytochemistry, electron microscopy) Diagnosis of mesothelioma has prognostic, treatment and legal implications