1 Diagnostic Cytopathology of Serous Effusions Session II ( 8.45-9.30 ) To view this session on web copy-paste the following URL into your browser: http://www.slideshare.net/vshidham/02-presentations-ii-vs-14-4-mb-3-30-08 Vinod B. Shidham , MD, FRCPath, FIAC Professor Executive editor & coeditor-in-chief, CytoJournal ( www.cytojournal.com ) Department of Pathology Medical College of Wisconsin 9200 W Wisconsin Av, Milwaukee, WI 53226, USA [email_address] 2008 Wisconsin Society of Cytology SPRING MEETING, 40TH ANNIVERSARY Holiday Inn – Riverwalk, Neenah, WI Saturday, April 19, 2008 (7.30 to 3.30)

2 Outline Session I (40 minutes): Anatomy, histology, cytology, and effusions Collection, transportation, and processing of effusion fluids Factors leading to potential diagnostic pitfalls Approach to diagnostic cytopathology of effusions The panorama of different face of mesothelial cells Session II (45 minutes): Benign conditions with/without specific cellular patterns Mesothelioma Metastatic carcinoma Metastatic sarcoma and melanoma Hematolymphoid disorders (Lymphomas and leukemias) Session III (45 minutes): Evaluation of unknown primary sites of origin- Where do they come from? Immunocytochemistry of effusion fluids: SCIP (Subtractive Coordinate Immunoreactivity Pattern) approach Flow cytometry, molecular techniques, and other special techniques Session IV (45 minutes): Diagnostic cytopathology of peritoneal washings Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

2 Outline Session I (40 minutes): Anatomy, histology, cytology, and effusions Collection, transportation, and processing Factors leading to potential diagnostic pitfalls Approach to diagnostic cytopathology of effusions The panorama of different face of mesothelial cells Session II (45 minutes): Benign conditions with/without specific cellular patterns Mesothelioma Metastatic carcinoma Metastatic sarcoma and melanoma Hematolymphoid disorders (Lymphomas and leukemias) Session III (45 minutes): Evaluation of unknown primary sites of origin- Where do they come from? Immunocytochemistry of effusion fluids: SCIP (Subtractive Coordinate Immunoreactivity Pattern) approach Flow cytometry, molecular techniques, and other special techniques Session IV (45 minutes): Diagnostic cytopathology of peritoneal washings Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

In adults most of the pleural, peritoneal, and pericardial effusions are related to benign conditions like: Congestive heart failure Cirrhosis of the liver Pericarditis. 3

4 Benign conditions with/without specific cellular patterns Session II May mimic carcinoma Extremely reactive mesothelial cells may exhibit atypia overlapping with malignancy Diagnose of malignancy with extreme caution. Immunophenotyping with SCIP approach objectively decide nature of atypical cells. Liver Cirrhosis with activity Uremia Acute Pancreatitis Pulmonary embolism and Infarction With characteristic features Systemic Lupus Erythematosus ( SLE) Rheumatoid Effusions Other less common causes of reactive effusions Fistulous tract associated effusions Endometriosis Asbestos exposure associated effusions Talc associated effusions.

Mesothelial cells vs adenocarcinoma cells Benign conditions with/without specific cellular patterns (continued) 5 Cytologic Features Mesothelial Cells Adenocarcinoma Cell Types Single cell population (spectrum of mesothelial cells) Second population (mesothelial cells and nonmesothelial-noninflammatory cells) Borders of cell groups Hobnail with cytoplasmic Intercellular windows- Present Smooth community borders of nuclei Intercellular windows- May be present Cell in cell pattern Present Rare Vauolated cells (Signet ring appearance) Absence of mucin in cells Nuclear borders not distorted “ Mucin droplet” present Vacuole distorts cell nucleus Cytoplasm Ecto- and endoplasm Usually homogenous-random Cytoplasmic Blebs Present Absent Immunohistochemistry Mesothelial markers- Positive Epithelial markers- Positive Microvilli (EM) Long, slender, bushy Short, stubby, sparse

Benign conditions with/without specific cellular patterns (continued) Reactive mesothelial cells in clusters (ascitic fluid). Mixed with chronic inflammatory cells within the groups and between the mesothelial cells in the background. [Papanicolaou stained ThinPrep preparation (100x Zoomed)]. 6

Benign conditions with/without specific cellular patterns (continued) Reactive mesothelial cells in clusters mixed with chronic inflammatory cells, mostly mature lymphocytes (ascitic fluid). This consult case was initially misinterpreted as positive for malignant cells. Extensive search for primary was negative. [Papanicolaou stained ThinPrep preparation (100x Zoomed)]. 7

Benign conditions with/without specific cellular patterns (continued) a b c RM-b RM-c F2 F1 F2 F1 Degenerative vacuoles in reactive mesothelial cells (ascitic fluid). 8

Benign conditions with/without specific cellular patterns (continued) A thin rim between nuclear border and cell border (1) is seen in mesothelial cells. In comparison, the nuclear border touches the cell border of adenoca cells without a significant cytoplasmic rim (2). [Papanicolaou stained SurePath TM Preparation (b,c,e,f, 100XZoomed)]. Mesothelial cells (a,b,&c) versus adenocarcinoma cells (d,e,&f) with eccentric nuclei . 9 2 2 2 f e d Adenocarcinoma 1 1 1 a c b Mesothelial

10 Peritoneal: Lymphocytic Peritoneal Effusions Cirrhosis Tuberculosis Eosinophilic Peritoneal Effusions Neutrophilic Peritoneal Effusions Spontaneous bacterial peritonitis Extramedullary hematopoiesis / chronic idiopathic myelofibrosis Benign conditions with/without specific cellular patterns (continued) Reactive inflammatory cells in effusion fluids Pleural: Reactive lymphocytic pleural effusions Tuberculosis Eosinophilic Pleural Effusions Neutrophilic Pleural Effusions Pulmonary embolism Parapneumonic effusions Histiocytic Pleural Effusions Rheumatoid arthritis Nodular histiocytic hyperplasia Pericardial Lymphocytic Pericardial effusions Viral pericarditis Eosinophilic Pericardial Effusions Neutrophilic Pericardial Effusions Infectious pericarditis Systemic lupus erythematosus

Benign conditions with/without specific cellular patterns (continued) Session II Most frequently exudative Usually pleural , less frequently pericardial . Cytology: Predominance of inflammatory cells (neutrophils, rarely eosinophils or lymphocytes). Most characteristic features is the presence of LE cells - Inflammatory cells (usually a neutrophil, but could be a macrophage) Containing a homogenous hematoxylin body . The number of LE cells noted in effusion sample can vary. Characteristic but not pathognomonic , other conditions including drug (e.g. procainamide, hydralazine, and isoniazid) associated with an SLE-like syndrome may show LE cells. Not consistent association. Systemic Lupus Erythematosus (SLE) 11

Benign conditions with/without specific cellular patterns (continued) Session II Systemic Lupus Erythematosus (SLE) LE cells 12 Pleural effusion , Diff Quik stain, 40X Pleural effusion , Papanicolaou stain, 40X

Benign conditions with/without specific cellular patterns (continued) Session II Rheumatoid effusions Uncommon but known complication of rheumatoid arthritis. May be concurrent , or occur prior to manifestations of joint disease. Eexudative, more frequently in males than females. Fluids have reduced glucose levels. Cytology Characteristic: Many degenerated cells , necrotic debris , atypical spindled cells resembling spindled squamous cells, histiocytes , and round multinucleated giant cells with many lymphoplasmacytic cells . Mesothelial cells- absent or may be rare. Presence of necrosis in an effusion specimen is almost characteristic for a rheumatoid nodule. Spindled cells with varying degrees of degeneration and frequent pyknosis, may raise the differential diagnosis with squamous cell carcinoma . Non-specific: The characteristic features- not consistent. May only show general reactive pattern - increased neutrophils, lymphocytes and mononuclear cells with reactive mesothelial cells. 13

Benign conditions with/without specific cellular patterns (continued) Session II Rheumatoid effusions Lung nodule and pleural effusion. Multinucleated giant cells with many chronic inflammatory cells. Degenerating as well necrotic cells characteristic of rheumatoid effusion are in the background. (Papanicolaou stain, 40X) 14

Cells in effusion fluid Mesothelial cells Neoplastic- ♦ Quantity- Many cells ♦ Quality- Many large groups Mesothelioma Non-mesothelial cells Reactive- Usually single cells without large 3-D groups Hematopoietic cells (Non-cohesive cells) Neoplastic - (2nd foreign population) ¶ Carcinoma (Cohesive cells) Sarcoma (Spindle cells may be present. Known history of sarcoma is usually crucial for proper interpretation) Melanoma (Non-cohesive cells) Neoplastic- Lymphoma Reactive- Inflammatory cells ¶ Metastatic cancer cells may be the predominant cells without being seen as a ‘second population’. They may be present as scattered solitary cells with cytomorphology overlapping with floridly reactive mesothelial cells. If indicated, i mmunocytochemistry would facilitate confirmation of these cells as non-mesothelial. 1 3 2 4 7 6 5 6b 6a 8 Algorithm for evaluation of a ‘second foreign population’ . 15

Mesothelioma Diagnostic problems in cytological interpretation Cytological features of mesothelioma Rare variants- Clear cell Deciduoid Lymphohistiocytoid Signet-ring cell Small cell Special stains in mesothelioma 16

Mesothelioma (continued) Significant overlap between benign and malignant mesothelial cells, and between mesothelioma and adenocarcinoma cells. Important clues are quantitative and qualitative Quantity- Hypercellular specimns with numerous mesothelial cells Ouality- Large groups of three dimensional mesothelial cells Most of these features are evaluated better on Romanowski stained preparations. Diagnostic problems Large aggregates of reactive mesothelial cells- may be misinterpreted with false positive diagnoses. 17

Mesothelioma (continued) A single malignant mesothelial cell population Multinucleation Articulation between mesothelial cells (intercellular windows) Cell-in-cell arrangements Cytoplasmic vacuoles Peripheral blebs Cluster of atypical cells with knobby outlines (scalloped borders) Variable nuclear enlargement with prominent nucleoli Cytoplasmic metachromasia Cytological features of mesothelioma 18

Mesothelioma (continued) Atypical mitoses may be seen rarely. Background extracellular stromal hyaluronic acid - Metachromatic appearance on Romanowski stains and Fluffy pale green/blue appearance on the Papanicolaou stain . Cytological features of mesothelioma Mesothelial cells (both reactive and neoplastic)- Distinct two-zone appearance with an inner dense ring fading into an outer, delicate, lacy area. Distinguishes mesothelial from adenocarcinoma cells , with diffuse pale cytoplasm with random pattern . General cytological nuclear features of malignancy apply to mesothelioma- Irregular, pleomorphic and enlarged nuclei, prominent nucleoli (including macronucleoli), bi- and multinucleation. However, the features are somewhat subtle and need to be carefully searched. 19

Mesothelioma (continued) Mesothelioma - clusters of cells with irregular, knobby outlines- sometimes resemble papillary architecture . Acinar formation (a feature of adenocarcinoma ) rare in mesotheliomas . Intercellular windows may appear like acini.. Cell engulfment (cell-in-cell) is a common in mesotheliomas . Benign effusions- relatively less cellular, with smaller cell groups, mostly two-dimensional. The individual cells may show variable atypia. Adenocarcinoma cell groups- three-dimensional, complex, with a smooth contour (the so-called “ community border ” ). Cytological features of mesothelioma Spindle cells rarely exfoliate in effusion fluids- they may appear polyhedral owing to the surface tension phenomenon . Scattered atypical spindle cells - suspicious for mesothelioma . 20

Mesothelioma (continued) Cytological features of mesothelioma Mesothelial cell with two zone cytoplasm (white arrow) and fuzzy microvilli (black arrow). Autocyte Prep. Pap stain 40x. 21

Mesothelioma (continued) Cytological features of mesothelioma Loosely cohesive three-dimensional groups of mesothelioma cells with knobby outline. A multinucleate atypical mesothelial cell is seen at the periphery of the group (arrow). Thinprep. Pap stain 40x. 22

Mesothelioma (continued) Cytological features of mesothelioma Mesothelial cell with prominent microvilli (white arrow). Autocyte Prep. Pap stain. 60x. 23

Mesothelioma (continued) Cytological features of mesothelioma Malignant epithelioid mesothelioma (pleural fluid). Mesothelioma cells show numerous large three dimensional groups of cells. The individual mesothelioma cells hardly show any variation from reactive mesothelial cells without remarkable features of malignancy. The mesothelioma cells like reactive mesothelial cells show two zone staining (red arrow 1) with peripheral vacuolation (blue arrow 2). [a-h, Papanicolaou stained ThinPrep preparation (a,b, 20X; c, 100X; d-h, 100XZoomed)]. 24 1 2 a b c e f g h d

Mesothelioma (continued) Cytological features of mesothelioma Cell block from a case of mesothelioma showing hypercellular atypical mesothelial cell groups. Extracellular mucinous material is present in the background (white arrow). H&E. 40X. 25

Mesothelioma (continued) Cytological features of mesothelioma Loosely cohesive small group of spindled mesothelial cells in a case of biphasic mesothelioma. Arrow marks an atypical spindled nucleus. Autocyte Prep. Pap stain. 40x. 26

Mesothelioma (continued) Peritoneal fluid, Deciduoid mesothelioma . Dyscohesive scattered single atypical enlarged cells Pap stain 10X. [Courtesy Dr. Bernard Naylor]. Cytological features of mesothelioma 27

Mesothelioma (continued) Peritoneal fluid, Deciduoid mesothelioma. Enlarged malignant cells with a binucleate form, and glassy cytoplasm. Nuclei are vesicular with prominent nucleoli. Scattered smaller atypical mesothelial cells are present in the background. Pap stain 40X [Courtesy Dr. Bernard Naylor]. Cytological features of mesothelioma 28

Mesothelioma (continued) Special stains in mesothelioma Mesothelial cells produce hyaluronic acid - an acidic mucin. Also seen in the background of effusion smears. Adenocarcinoma cells produce neutral mucin . Mesotheliomas- positive for acidic mucin stains such as Alcian Blue . The staining disappears with hyaluronidase pre-digestion . Adenocarcinomas- epithelial (neutral) mucin- Positive with mucicarmine and PAS (diastase resistant)- PAS positive glycogen in mesothelial cells is hydrolyzed by diastase digestion . A positive stain for an epithelial mucin- is useful for a diagnosis of adenocarcinoma, but a negative result does not exclude it. 29

Cells in effusion fluid Mesothelial cells Neoplastic- ♦ Quantity- Many cells ♦ Quality- Many large groups Mesothelioma Non-mesothelial cells Reactive- Usually single cells without large 3-D groups Hematopoietic cells (Non-cohesive cells) Neoplastic - (2nd foreign population) ¶ Carcinoma (Cohesive cells) Sarcoma (Spindle cells may be present. Known history of sarcoma is usually crucial for proper interpretation) Melanoma (Non-cohesive cells) Neoplastic- Lymphoma Reactive- Inflammatory cells ¶ Metastatic cancer cells may be the predominant cells without being seen as a ‘second population’. They may be present as scattered solitary cells with cytomorphology overlapping with floridly reactive mesothelial cells. If indicated, i mmunocytochemistry would facilitate confirmation of these cells as non-mesothelial. 1 3 2 4 7 6 5 6b 6a 8 Algorithm for evaluation of a ‘second foreign population’ . 30

Metastatic carcinoma Common sources of primary neoplasms causing malignant effusions. Any neoplasm, including rare examples of central nervous system tumor, may involve serous cavity and manifest as malignant effusion. Metastatic adenocarcinoma is, by far, the most common cause of malignant effusions. 31

Metastatic carcinoma (continued) Clinical details may be important for cytologic interpretation of effusions. However, depending on the clinical scenario and cytological picture, the clinical history may be misleading, especially with beginners. Some effusions may develop without any history of cancer and may present a diagnostic challenge. Clinical history 32

Clinical history (continued) The clinical history can play a critical role in ensuring proper triaging and processing of effusion specimens; therefore, to facilitate appropriate processing of effusions, the clinical history should be provided in all the requisitions. This could avoid suboptimal cytopathologic interpretations. Metastatic carcinoma (continued) 33

If the initial findings are equivocal for cancer cells, it is prudent to be extra-cautious by recommending repeat cytologic evaluation on a new specimen. If the initial effusion is caused by cancer, it usually re-accumulates rapidly and may contain unequivocal cancer cells with improved morphology Word of caution!!!! Metastatic carcinoma (continued) 34

Characteristic features of some specific primary sites Metastatic carcinoma (continued) 35

Metastatic adenocarcinoma- NOS , (peritoneal fluid). Cohesive groups of cells in papillary configurations show eccentrically placed nuclei touching the periphery of cells (arrows in b,c,f). The cells in such groups are difficult to study at lower magnification (a,b). However, the cell morphology in such groups can be observed at the periphery, especially under higher magnification (arrow in b). Some groups show gland-like spaces (arrowheads in d,e,f). [a-c: Papanicolaou stained SurePath preparation; d-f: Diff-Quik stained Cytospin preparation. (a, 10X; b, 40X; c, 100XZoomed; d, 10X; e, 40X; f, 100XZoomed)]. 36 Characteristic features of some specific primary sites a b c d e f

a b c d e f w 2 1 Metastatic poorly differentiated adenocarcinoma of lung, (pleural fluid). The Diff-Quik stained preparation demonstrates reactive mesothelial cells (arrowhead 1 in a) mixed with a ‘second population’ of cohesive groups of cells (arrow 2 in a) with eccentric nuclei that touch the periphery of the carcinoma cells (arrowheads in b,c,f). Some cells are less cohesive with nearby small groups or solitary carcinoma cells (arrows in d,e,f). Occasional intercellular spaces, resembling mesothelial window, are present (arrowhead w in c). The patient had poorly differentiated adenocarcinoma of lung. [a-c: Diff-Quik stained Cytospin preparation, d-f: Papanicolaou stained SurePath preparation. (a, 10X; b,c, 100X; d, 10X; e,f, 100X)]. 37 Characteristic features of some specific primary sites (continued)

a c d g h e f i j RM b RM c g j i h d f e Ly NC RM nm nm NC Metastatic small cell carcinoma, (pleural fluid). Cancer cells (a,b) are present as solitary cells (c,d), small groups (g,h,i), and large groups (b & j). The cells are small with high nucleocytoplasmic ratios (c,d,g,h.i). The nuclei are hyperchromatic with salt and pepper chromatin (c,d,i). Solitary cancer cells (NC) (c,d) resemble lymphocytes (blue arrow Ly in f) and may be misinterpreted as lymphoma, especially in PAP stained preparations. However, the presence of cohesive groups (g,h,i,j) with various patterns, including Indian-file pattern (g), typify carcinoma. The nuclear molding (arrows nm in g & i) distinguishes them from other poorly differentiated carcinomas. Mitotic figures (arrowhead in e) and apoptotic cancer cells (arrowheads in d,f,h) are also present. Rare reactive mesothelial cells (blue arrows RM in a,b,e) are present with a few chronic inflammatory cells (blue arrow Ly in f) in the background. Immunostained cell block sections showed immunoreactivity for neuroendocrine immunomarkers (chromogranin, synaptophysin, and CD56). The patient had poorly differentiated small cell carcinoma of lung. (NC, neoplastic cell; RM, reactive mesothelial cell, nm, nuclear molding, Ly, lymphocyte) [a-j: Papanicolaou stained SurePath preparation. (a-b, 100X; c-j, 100XZoomed)]. 38 Characteristic features of some specific primary sites (continued)

a b c d e f g h RM NC NC NC RM TTF-1 Metastatic bronchioloalveolar cell carcinoma of lung , (pleural fluid). Cellular specimen (a) shows three dimensional groups of carcinoma cells (arrowheads in a-f) mixed with reactive mesothelial cells (blue arrow RM in f,g). The two populations are demonstrated more distinctly with the DQ stain (f,g) than with the PAP stain (a-e). However, the morphological details of individual neoplastic cells are superior in the PAP stain (a-e), especially under higher magnification (c-e). The individual cells show features of well differentiated adenocarcinoma (red arrows NC in d,e,g). Although, usually not conspicuous, some of the carcinoma cells have prominent nucleoli (d,e). The neoplastic cells demonstrate nuclear immunoreactivity for TTF-1 (arrowheads in h) consistent with a lung primary. The patient had bronchioloalveolar cell carcinoma of lung. (NC, neoplastic cell; RM, reactive mesothelial cell; TTF-1 Thyroid transcription factor- 1) [a-e: Papanicolaou stained SurePath preparation; f-g: Diff-Quik stained Cytospin preparation; h: Immunostained cell-block section. (a, 10X; b, 40X; c, 100X; d-e, 100XZoomed; f, 40X; g, 100X; h, 40X)]. 39 Characteristic features of some specific primary sites (continued)

Metastatic mammary carcinoma, (pleural fluid). a-g: Proliferation spheres (red arrows NC) with mostly reactive mesothelial cells (blue arrows RM) and inflammatory cells in the background, clearly separated out in the immunostained cell-block sections (a,e). The reactive mesothelial cells (blue arrows RM) stand out distinctly from the neoplastic cells (red arrow NC) in the DQ stain. However, the reactive mesothelial cells in the PAP stain (blue arrows in g) are difficult to distinguish from neoplastic cells (compare with case in h-j). h-j: A different patient with metastatic mammary carcinoma. The effusion predominantly contains solitary adenocarcinoma cells, as highlighted by the BerEP4 immunostained section (red arrows NC in h). The carcinoma cells (red arrows NC in j) can be distinguished easily from reactive mesothelial cells (blue arrow RM in j) in the DQ stain (j), but not in the PAP stain (i). A mitotic figure is present (blue arrow MF in i). Note the resemblance of reactive mesothelial cells in f & g to neoplastic cells in i. Without the help of DQ stain and immunocytochemistry, such fluids, with predominantly one type of cell population, may easily be misinterpreted. [a,e,h: Immunostained cell-block sections; f,g,i: Papanicolaou (PAP) stained SurePath preparation; b,c,d,,j: Diff-Quik (DQ) stained Cytospin preparation. (a, 40X; b, 100X; c,d, 100XZoomed; e, 40X; f, 100X; g, 100XZoomed; h, 40XZoomed; i, 100X; j, 100XZoomed)]. 40 Characteristic features of some specific primary sites (continued) Calretinin RM NC BerEP4 RM NC NC BerEP4 NC NC NC RM g i h f e c a j b d MF RM NC RM RM RM RM RM NC RM

Metastatic adenocarcinoma of colon, (peritoneal fluid). Cohesive groups of cells (a) show high nucleocytoplasmic ratios and eccentric nuclei touching the periphery of the cell (arrow in b). Some adenocarcinoma cells show cytoplasmic vacuoles containing mucin (arrowhead in the inset of b). The cell groups in papillary configurations are difficult to study at lower magnification (c). Peripheral palisading is better observed under higher magnification (d). The patient had colonic adenocarcinoma. [a,b: Diff-Quik (DQ) stained Cytospin preparation, c-d: Papanicolaou (PAP) stained SurePath preparation. (a, 40X; b, 100X; c, 40X; d, 100)]. 41 Characteristic features of some specific primary sites (continued) a b c d

Metastatic gastric adenocarcinoma, (peritoneal fluid). The specimen contained a predominance of solitary neoplastic cells (red arrows a,d) with rare reactive mesothelial cells (blue arrow RM in a). Most of the cancer cells have eccentric nuclei touching the periphery of the cells (a,b,c,d). The reactive mesothelial cells (blue arrow RM) are identifiable more easily in the Diff-Quik stained (a) than in the PAP stained (c) preparation. The solitary carcinoma cells may be misinterpreted as high-grade lymphoma cells, especially in PAP stained preparations (c,d). Apoptotic cancer cells (yellow arrow ‘e’ in c) and mitotic figures (yellow arrow ‘f’ in c) are also present. The predominance of solitary neoplastic cells (a-j) is confirmed in the HE stained (g & h) and BerEP4 (i) immunostained cell-block sections. A few CK 7 immunoreactive mesothelial cells, as intrinsic positive control, are present amongst many neoplastic cells (j). Although, the adenocarcinoma cells in this case were non-immunoreactive for CK 7 (j), a significant proportion of gastric adenocarcinomas are immunoreactive for CK 7. The patient had linitis plastica type diffuse anaplastic gastric adenocarcinoma. (AP, apoptotic cancer cell; CK 7, cytokeratin 7; MF, mitotic figure; NC, neoplastic cell; RM, reactive mesothelial cell) [a-b: Diff-Quik (DQ) stained Cytospin preparation; c-f: Papanicolaou (PAP) stained SurePath preparation; g,h: HE stained cell-block section; i,j: Immunostained cell-block sections. (a, 100X; b, 100XZoomed; c, 100X; d-f, 100XZoomed; g, 40X; h-j 100X)]. 42 Characteristic features of some specific primary sites (continued) a c b d g h e f i j RM d f e b BerEP4 CK 7 NC NC AP MF

Metastatic pancreatic adenocarcinoma, (peritoneal fluid). The neoplastic cells (NC) are as loosely cohesive groups (a) or as solitary cells (b,e) with eccentric nuclei. PAP stained preparations facilitate evaluation of cellular details in cohesive groups (c & d). A few cell groups show gland-like structures (arrow in d). ‘Second population’ (arrows in f,g,&h) of neoplastic cells is highlighted distinctly in immunostained cell-block sections f through h (immunoreactive for BerEP4 in f, non-immunoreactive for vimentin in g and calretinin in h). As inbuilt corresponding positive controls, inflammatory and reactive mesothelial cells (arrowhead in g) are immunoreactive for vimentin and reactive mesothelial cells (arrowhead RM in h) are immunoreactive for calretinin. The patient had pancreatic adenocarcinoma. (NC, neoplastic cell; RM, reactive mesothelial cell) [a-b: Diff-Quik (DQ) stained Cytospin preparation; c-e: Papanicolaou (PAP) stained SurePath preparation; f-h: Immunostained cell-block sections. (a-e, 100X; f-h, 40X)]. 43 Characteristic features of some specific primary sites (continued) BerEP4 f d e c b a bi bii vimentin NC NC NC NC Calretinin RM g h

Metastatic ovarian serous papillary cystadenocarcinoma, (peritoneal fluid). Psammoma bodies (red arrows PSM in a.b,c,e,f,i) are present, isolated and in association with papillary clusters (d,f) of adenocarcinoma cells without stromal cores (g,h). Some carcinoma cells show degenerative vacuoles (blue arrows VAC in h), which should not be misinterpreted as mucinous. Some apoptotic neoplastic cells (blue arrow AP in i) are present. (AP, apoptotic cancer cell; NC, neoplastic cell; PSM, psammoma body; VAC, vacuole) [a-i: Papanicolaou (PAP) stained SurePath preparation. (a, 20X; b, 40X; c-e, 100X, f-i , 100XZoomed)]. 44 Characteristic features of some specific primary sites (continued) a b c d e f g h i PSM VAC PSM PSM PSM PSM AP PSM VAC NC

Primary peritoneal carcinoma, (peritoneal fluid). The specimen shows a predominance of groups of adenocarcinoma cells in papillary configurations (a,d) without stromal cores (g). Solitary neoplastic cells (red arrows NC in a,b,d,e) are easily distinguished from the rare reactive mesothelial cells (blue arrowheads RM in a,c,d,e). However, in PAP stained preparation (d), reactive mesothelial cells (arrowhead RM in e) have significant morphological overlap with neoplastic cells (red arrows NC in d & e). Mitotic figures (yellow arrow MF in a) and apoptotic cells (yellow arrows AP in d,f) are present concurrently. Some cells show degenerative vacuolation (a,b). These vacuoles may resemble secretory vacuoles and lead to misinterpretation of mucinous adenocarcinoma. The cancer cells do not show nuclear immunoreactivity for calretinin (h), but they are immunoreactive for BerEP4 (i). The neoplastic cells show nuclear (and cytoplasmic) immunoreactivity for WT-1(j). The patient had ascites with diffuse peritoneal involvement with omental caking. The ovaries were not enlarged. (AP, apoptotic cancer cell; MF, mitotic figure; NC, neoplastic cell; RM, reactive mesothelial cell) [a-c: Diff-Quik (DQ) stained Cytospin preparation; d-f: Papanicolaou (PAP) stained SurePath preparation; g: HE stained cell-block section; h-j: Immunostained cell-block sections. (a, 100X; b,c, 100XZoomed; d, 100X; e,f, 100XZoomed; h-j, 100X)]. 45 Characteristic features of some specific primary sites (continued) a d b c g h e f i j WT-1 BerEP4 Calret c b RM NC AP e f RM NC AP MF NC RM NC NC RM NC

a b c d Metastatic ovarian mucinous cystadenocarcinoma, (peritoneal fluid). Cohesive groups of neoplastic cells with high nucleocytoplasmic ratios and eccentric nuclei touching the periphery of the cells (arrow in b) are present. Some carcinoma cells have cytoplasmic vacuoles (a,b). The cell groups in papillary configurations are difficult to study at lower magnification (c). However, the cell morphology in such groups can be observed at the periphery of the papillation, especially under higher magnification (arrow in d). The patient had ovarian mucinous cystadenocarcinoma. [a,b: Diff-Quik (DQ) stained Cytospin preparation; c,d: Papanicolaou (PAP) stained SurePath preparation. (a, 40X; b, 100X; c, 40X; d, 100X)]. 46 Characteristic features of some specific primary sites (continued)

Metastatic ovarian adenocarcinoma, (peritoneal fluid). The neoplastic cells show an Indian file pattern. This arrangement is not specific for particular carcinoma (Ref 14); in addition to metastatic mammary carcinoma and small cell carcinoma of lung (see Figure 9.3a,b,g), this pattern may be seen in other non-small cell carcinomas, as in this case with metastatic ovarian adenocarcinoma. [a: Papanicolaou (PAP) stained SurePath preparation; b: Diff-Quik (DQ) stained Cytospin preparation. (a-b, 100X)]. 47 Characteristic features of some specific primary sites (continued) a b

Metastatic carcinoma of endometrium, (pleural fluid). Papillary-like cohesive groups (blue arrow in a) of columnar cells (red arrow in d). The patient had endometrial carcinoma of endometrioid type. [a-d: Papanicolaou (PAP) stained SurePath preparation. (a, 10X; b, 40X; c,d, 100X)]. 48 Characteristic features of some specific primary sites (continued) a b c d

Metastatic prostatic adenocarcinoma, (peritoneal fluid). Cohesive small groups of cancer cells (red arrows in a,e,f,i,j) with mostly eccentric nuclei touching the periphery of the cells are present with some solitary cancer cells (red arrows NC in a,,c,d,f,g,h). Reactive mesothelial cells (blue arrows RM in a,b) are more easily and distinctly identifiable in DQ stained (a) than in PAP stained (f) preparations. Most cancer cells were poorly cohesive, manifested by the presence of small groups (e,i,j) or solitary cells (c,d,g,h). Some cells show vacuolation (f,g,h,i,j) which may be in vitro degenerative or functional with secretion. The patient had prostatic adenocarcinoma with colonic metastasis. The primary tumor also showed a few vacuolated cancer cells in tissue sections. (NC, neoplastic cell; RM, reactive mesothelial cell) [a-e: Diff-Quik (DQ) stained Cytospin preparation; f-j: Papanicolaou (PAP) stained SurePath preparation. (a, 100X; b-e, 100XZoomed; f, 100X; g-j, 100XZoomed)]. 49 Characteristic features of some specific primary sites (continued) RM RM RM a f b c d e g h i j RM RM NC NC NC NC NC NC

Metastatic papillary carcinoma of thyroid, (pleural fluid). ‘ Second population’ of cohesive papillary groups (red arrows NC in b,e,f) of cells associated with Psammoma bodies with concentric lamination (red arrow PSM in g2,h2’) are seen amongst a few reactive mesothelial cells (blue arrows RM in b,i). d1, single tumor cells with eccentric nucleus touching the periphery (blue arrow) DQ stained preparation. d2, single cells may have cytoplasmic vacuole with colloid (arrowhead) in DQ stained preparation. j1, single tumor cells with eccentric nucleus touching the periphery in PAP stained preparation. j2, compare with reactive mesothelial cell (binucleate) with central nuclei in j1 in PAP stained preparation. The patient had papillary carcinoma of thyroid. [NC, neoplastic cell(s); PSM, psammoma body; RM, reactive mesothelial cell(s)] [a-d: Diff-Quik (DQ) stained Cytospin preparation; e-j: Papanicolaou (PAP) stained SurePath preparation. (a, 40X; b,c, 100X; d1,d2, 100XZoomed; e, 10X; f, 40X; g1,g2,h1,h2,i, 100X; j1,j2, 100XZoomed)]. 50 Characteristic features of some specific primary sites (continued) a b c e g1 f d1 d2 g2 Second focus of ‘h1’ ( bright light) h1 h2 i j1 j2 NC RM NC RM NC NC Second focus of ‘g1’ ( bright light) PSM PSM

Metastatic cholangiocarcinoma, (peritoneal fluid). Cancer cells (red arrows in a,h) are present mostly as solitary cells with eccentric nuclei touching the periphery of the cells (b,d,i,k,l) with occasional loosely cohesive groups of cancer cells (a,f) and reactive mesothelial cells (c,e,j and arrowheads in a,h). A few cells show cytoplasmic vacuoles (i) with secretion (yellow arrow in l) which is positive for mucicarmine in cell-block section (n). Some apoptotic cancer cells (yellow arrow AP) are also present (g,m). The morphological features overlap those of other mucinous adenocarcinomas. The patient had cholangiocarcinoma with a mucinous pattern. (AP, apoptotic cancer cell; NC, neoplastic cell; RM, reactive mesothelial cell) [a-g: Diff-Quik stained Cytospin preparation; h-m: Papanicolaou stained SurePath preparation; n: Mucicarmine stained cell block section. (a, 100X; b-g, 100XZoomed; h, 100X; i-m, 100XZoomed); n, 100XZoomed)]. 51 Characteristic features of some specific primary sites (continued) a b h c d e i k b c d e l k i m g l j f f g m n j AP NC RM NC RM NC RM NC AP

A. Sarcomas I. Blue cell tumors Ia. Rhabdomyosarcoma ai. Embryonal rhabdomyosarcoma aii. Alveolar rhabdomyosarcoma Ib. Neuroblastoma Ic. Wilms’ tumor Id. PNET and Ewing’s sarcoma (EWS) Ie. Desmoplastic round cell tumor (DSRCT) II. Fibrosarcoma and other sarcomas B. Malignant Melanoma C. Other malignancies such as germ cell tumors Metastatic non-epithelial neoplasms 52

More frequently encountered in children than in adults. The most common causes in children- ( followed by Lymphoma and leukemia ) Non-epithelial neoplasms including- Wilms’ tumor Neuroblastoma Ewing’s sarcoma , and Embryonal rhab­domyosarcoma Metastatic non-epithelial neoplasms (continued) 53

Metastatic non-epithelial neoplasms (continued) Effusion fluids - Usually sparsely cellular Cells- Single or in loose clusters Tendency to round up due to the surface tension from the surrounding fluid Lack the tissue arrangement and vascular or stromal pattern Cytoplasm - Indistinct cytoplasmic borders May show bipolar cytoplasmic processes Nuclei - Binucleation or multi-nucleation may be present. Round , oval , and sometimes fusiform or spindle shaped Irregular contours , often with nuclear membrane infolding Chromatin clumping with parachromatin clearing Prominent nucleoli 54

Metastatic non-epithelial neoplasms (continued) Metastatic fibrosarcoma (peritoneal fluid). Moderately cellular specimen with predominantly scattered single tumor cells. 55

Metastatic non-epithelial neoplasms (continued) Metastatic melanoma (peritoneal fluid). Dyshesive clusters of malignant cells. ‘Cell-in-cell’ pattern may be present. 56

Metastatic non-epithelial neoplasms (continued) Desmoplastic small round cell tumor (DSRCT) (peritoneal fluid). Dyshesive clusters of small round tumor cells showing rosette-like features (FNAB). DSRCT effusion exhibiting high cellularity. The tumor cells showed nuclear immunoreactivity for WT1. [ With thanks from the article in ‘open access’ cytopathology journal: Granja NM et al CytoJournal 2005, 2:6 Free full text at- http://www.cytojournal.com/content/pdf/1742-6413-2-6.pdf ] 57

Hematolymphoid disorders (Lymphomas and leukemias) (continued) Adults : Lymphoma is the third most common cause of a malignant pleural effusion after lung and breast carcinoma . 58 Pleural fluids, largest group (61%); followed by ascitic fluid (26%) and pericardial fluid (7.5%). Majority from patients with a documented neoplasm (pleural 47.5%, ascitic 23%,, and pericardial 43%) In sporadic Burkitt lymphoma (80-91% of patients) present with abdominal masses which represent involvement of peritoneum, omentum, bowel mesentery, or bowel wall. Children: The most common causes of malignant effusions are hematopoietic neoplasms .

Hematolymphoid disorders (Lymphomas and leukemias) (continued) Air-dried Diff-Quik or Wright-Giemsa stained preparations- Have traditionally been the stains of choice for hematopoietic cells , Especially for examining subtle cytoplasmic details and variations . Papanicolaou-stained preparations- Allow better examination of nuclear details. The hematolymphoid cells-are non-cohesive in cytological specimens. Exceptions can occur- e.g. lymphocytes in tuberculosis effusions may form “ lymphoid aggregates ” Lymphoglandular bodies- Typically are inconspicuous or absent in effusion smears 59

Hematolymphoid disorders (Lymphomas and leukemias) (continued) After determining the lymphoid nature of the cells, the distinction between- low-grade lymphoma and reactive lymphoproliferative process Often difficult to make by morphology alone , Because the cellular atypia is often minimal to absent . For high-grade lymphomas - Differential is usually between undifferentiated sarcoma and carcinoma . For accurate diagnosis and classification- Integration of cytologic examination , clinical history , immunostains , flow cytometry , and molecular studies . 60

After determining the lymphoid nature of the cells, the distinction between-

low-grade lymphoma and reactive lymphoproliferative process

Often difficult to make by morphology alone ,

Because the cellular atypia is often minimal to absent .

For high-grade lymphomas -

Differential is usually between undifferentiated sarcoma and carcinoma .

For accurate diagnosis and classification-

Integration of cytologic examination , clinical history , immunostains , flow cytometry , and molecular studies .

Hematolymphoid disorders (Lymphomas and leukemias) (continued) A. Follicular lymphoma lymphocyte with characteristic cleaved or notched nucleus. B. CLL cells in pleural fluid cytospin preparation. C. Bloody pleural fluid in a patient with CLL. Contamination with peripheral blood must be considered before diagnosing involvement with CLL. Chronic Lymphocytic Leukemia (pleural fluid) 61

Hematolymphoid disorders (Lymphomas and leukemias) (continued) Monomorphous population of medium sized Burkitt lymphoma cells with prominent cytoplasmic vacuolization. Note the mitosis in C. Burkitt lymphoma (peritoneal fluid) A-C. Diff-Quik stain 62

Hematolymphoid disorders (Lymphomas and leukemias) (continued) Diffuse Large B Cell Lymphoma , (ascitic fluid). Large atypical lymphoid cells (A-D). E. Histology section from primary colonic DLBCL of same patient. A-B. Diff-Quik stain, C-D. Pap-stain, E. HE stained section of primary colonic DLBCL. 63

Hematolymphoid disorders (Lymphomas and leukemias) (continued) HHV-8 associated lymphoma cells in ascitic fluid. Primary effusion lymphoma, (ascitic fluid). A,B. Wright-Giemsa stain 64

Thank you Milwaukee Art Museum [email_address] End Diagnostic Cytopathology of Serous Effusions II