Effusion cytology. Dr Alpha Tsui Royal Melbourne Hospital 2008

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1 Effusion cytology Dr Alpha Tsui Royal Melbourne Hospital 2008 General points: -large unilateral effusion (>1 litre) in the elderly is highly suspicious for malignancy -effusions associated with malignancies may be just a reactive response without presence of any tumour cells -number of neoplastic cells in recurrent effusions often increases. Ask for re-tap if first tap shows no malignant cells or inconclusive -in large suspicious effusions, if initial 4 slides show no malignant cells, re-prep further slides and cell blocks Gross appearance of effusion fluids: -note volume, colour, clarity, malodour, opalescence, high viscosity -fluid containing many cancer cells, if allowed to stand, may form a thick, whitish yellow layer at bottom of container -pleural fluid from rheumatoid pleuritis may contain a heavy, whitish, flocculent sediment and the supernatant may have appearance of fruit juice -fluids containing numerous pigmented melanoma cells may be chocolate brown -fluids containing many haemosiderin-laden macrophages are lighter brown -effusions caused by mesothelioma often contain a high concentration of hyaluronic acid which increases the viscosity (may be a dry-tap clinically) Transudates: -low protein content, usually <3g/dL -low specific gravity, < lower cellular content -lower fibrin content -implies systemic problems e.g. cardiac, liver, renal failure Exudates: -higher protein content, usually >3g/dL -higher specific gravity, > high cellular content -contains many inflammatory cells or neoplastic cells -implies local problems e.g. infection, tumour ADEQUACY: No quantitative rule for number of mesothelial cells on a slide. But at least 50ml of effusion fluid is required for optimal assessment. Normal cells in effusion fluid: -RBCs, leukocytes, mesothelial cells Normal mesothelial cells: -cohesive, monolayered sheets of cells

2 -mesothelial cells forming small cell balls; may have central collagen core (especially when forcibly removed) -collagen cores are more commonly seen (but not diagnostic) in mesothelial cell groups (benign or malignant). They are rare in carcinomatous effusions. -round or oval nucleus centrally or slightly eccentrically located -nucleus may be single or multiple -most often show 1 or multiple small nucleoli -nucleus rarely appears to actually form outer boundary of a cell -abundant dense cytoplasm -lacy collar (due to degeneration of microvilli), forming a vacuolated rim around the entire circumference of the cell -cytoplasmic blebbing (larger vacuole with coalescence of microvilli) -mesothelial window, formed by interdigitating long and slender surface microvilli Reactive mesothelial cells: -the cytoplasm becomes dense with 2-tone endoplasmic-ectoplasmic margin -endoplasm: paler area around the nucleus. Ectoplasm: darker area at the periphery of the cytoplasm -may form cell balls, containing generally fewer than cells per cluster -may also form small papillary structures -mild nuclear enlargement, smooth nuclear membrane -chromatin fine, open, vesicular (not coarse, not hyperchromatic) -nucleoli usually present and centrally located -immunostains generally not helpful: desmin positive; weak or absent membranous staining for EMA; p53 largely negative Vacuolated mesothelial cells: -vacuolation commonly due to degeneration; may be therapy-induced -cytoplasmic vacuoles, initially present at the endo-ectoplasmic border -degenerative mesothelial vacuoles often overlap the nucleus without pushing it aside or distorting it (i.e. not forming a signet-ring cell with compressed nucleus) -vacuoles do not contain mucin -the nuclear morphology of the vacuolated cells is exactly the same as the neighbouring non-vacuolated mesothelial cells -no malignant nuclear features -as effusion becomes chronic, the vacuoles become larger Numerous neutrophils in fluid: -empyema -infarction and rupture of organ -beware broncho or oesophageal pleural fistula by squamous cell carcinoma. Look for malignant cells in necrotic cell debris Numerous lymphocytes in fluid: -chronic pleurisy -TB -viral pneumonia -lymphoma / leukaemia -other malignancies

3 Numerous eosinophils in fluid: Note: eosinophilic cytoplasmic granules not seen on Pap stain -repeated taps, chronic drain tube placement -allergy -autoimmune disorders -pneumonia -pulmonary infarct -fungal infection -parasitic infection -TB -neoplasms -pneumothorax and haemothorax -peritoneal dialysis -idiopathic Differential diagnosis of second benign population of cells in peritoneal fluid: -endosalpingiosis -fallopian tube epithelium -ruptured cysts -adhesions -pelvic inflammatory disease -endometriosis -ectopic pregnancy -ovarian torsion -tubal abscess -hydrosalpinx Specific entities: Rheumatoid arthritis: -spindle cell macrophages: tapering ends, moderately dense cytoplasm, often has a finely granular ground-glass appearance, nuclei frequently multiple -multinucleated giant cells: may contain 20 or more nuclei -necrotic granular background material -ragocytes: neutrophils containing degenerated nuclear debris, appearing as spheric cytoplasmic inclusions -neutrophils may also contain fat droplets -eosinophils, neutrophils, lymphocytes may be present -mesothelial cells are noticeably absent -cholesterol crystals may be present SLE: -LE cell: when a haematoxylin body is phagocytosed by a cell -haematoxylin bodies in the background -neutrophils -tart cell: macrophage which has phagocytosed a bare nucleus of a cell that underwent cytolysis -reactive mesothelial cells

4 Pulmonary infarct: -cellularity ranges from slight to profuse -mesothelial cells, macrophages including haemosiderophages, lymphocytes, neutrophils are present -eosinophils may be seen TB: -high proportion of lymphocytes (80-100%) -very few or no mesothelial cells -numerous mesothelial cells should be regarded as strong evidence that the effusion is not caused by TB -may also contain neutrophils, plasma cells, macrophages, RBCs -multinucleated giant cells and epithelioid cells are very rare Mesothelioma: -2 steps in the diagnosis of mesothelioma: -cells must be recognised as being mesothelial in origin -they must be neoplastic -hypercellular smear with large cell clusters, most clusters >50 cells -cell clusters have scalloped contours (mulberry appearance), may be elongated or papillary. Acinar structures can be seen (more so in FNA samples). -large cell clusters often accompanied by smaller clusters and single cells -single population of cells and all cells are mesothelial-like -individual cells are enlarged with increased N/C ratio -nuclei smoothly round or oval, nucleoli prominent -nuclear chromatin more coarsely granular -dense 2-tone cytoplasm -may see large, crescent-shaped lakes of glycogen in perinuclear position (in Pap stain: yellow). This is not a feature of adenocarcinoma. -lacy collar maintained -cytoplasmic blebbing is seen in degenerate cells with coalescing microvilli -cell-in-cell engulfment seen more often in mesothelioma but also present in reactive mesothelial cells -cytoplasmic vacuolation is non-specific (present in both mesothelioma and adenocarcinoma). However, demonstration of hyaluronic acid in the cytoplasmic vacuoles is characteristic for mesothelioma. Hyaluronic acid presents as magentacoloured intra / extracytoplasmic material with Giemsa stain and grey streaks with Pap stain (needs to do Alcian blue and hyaluronidase to confirm) -cytoplasm is elongated and tapered in sarcomatoid mesotheliomas. Immunostaining is required to confirm the diagnosis. -occasional apoptotic cells with pyknotic nuclei and orangeophilic cytoplasm (squamous-like cells) are present and they are quite specific for mesothelioma (due to abundant keratin filaments in the cytoplasm). They are not generally seen in benign effusions or in adenocarcinomas. -may have psammoma bodies (non-specific: seen in both benign and malignant cases) -always perform immunostains on the cell block (initial panel: Calretinin, Mesothelin, CK5/6, WT1, CEA, TTF-1, BerEP4)

5 Helpful features favouring mesothelioma over adenocarcinoma: One-cell population, windows, lacy collar, dense 2-tone cytoplasm, collagen cores, presence of hyaluronic acid, squamous-like cells. Helpful features favouring mesothelioma over reactive change: Marked cellularity with large cell groups and papillae, high N/C ratio, hyperchromasia, irregular nuclear contour, prominent irregular nucleoli, coarse chromatin, squamous-like cells, necrosis Adenocarcinoma: -tendency to form smoothly contoured cohesive groups -'proliferation spheres' frequently present with breast and to lesser extent with ovarian and lung carcinomas (proliferation spheres not seen in recently developed malignant effusions) -may also be single cells or papillary fragments -large cells with eccentric nuclei, forming edge of cytoplasm -prominent nucleoli -less well-defined, delicate, foamy and vacuolated cytoplasm, containing mucin -signet-ring cells -crowded vacuolated tumour cells may produce irregular intercellular spaces but these are not true windows -metastatic breast, gastric, renal and pancreatic carcinomas can be very difficult to pick out as the tumour cells are often single with only mild atypia. Must do immunostains if there is a strong clinical suspicion or for staging purposes e.g. peritoneal washings. Panel: ER, PR, CDX-2, CK20, CD10. Features suggestive of adenocarcinoma from the breast: Cannonballs with smooth borders; targetoid inclusions; linear files. Serous cystadenoma of the ovary: -usually of low cellularity -clusters, sheets and occasionally, papillary groups -cells have cuboidal or columnar shape -round to oval nuclei containing finely granular chromatin -inconspicuous nucleoli -scant to moderate cytoplasm -cell borders well-defined within sheets -overlapping cells not present -ciliated columnar cells may be found Mucinous cystadenoma: -honeycomb sheets may be seen -round uniform basal nuclei -uniform columnar cells with cytoplasmic vacuolisation -lack cilia -mucin-filled macrophages and mucin in the background

6 Borderline mucinous cystadenoma: -replacement of honeycomb pattern by crowded and palisaded cells which form micropapillae -dissociated cells may be seen Adenocarcinoma of ovary: -usually very cellular -cells in large acinar or papillary clusters mixed with numerous single cells -nuclear pleomorphism with coarse irregularly distributed chromatin, prominent nucleoli -many vacuolated cells -tumour giant cells -necrotic debris -psammoma bodies -mitoses Ovarian borderline tumour vs carcinoma: Features favouring carcinoma: -more cellular fragments with irregular borders -frequent single cells and mitoses -cells larger, more pleomorphic Pseudomyxoma peritonei: -most from tumours of the appendix (adenoma / adenocarcinoma). Some cases from ovary, pancreas -abundant mucin +/- epithelium -epithelium usually at least dysplastic Squamous cell carcinoma: -SCC in serous fluid is uncommon, most from lung, larynx, female genital tract -malignant cells are usually few -more flat, 2-D appearance of fragments -tadpole and fibre cells are rare -well-defined cell boundaries within fragments -nuclei centrally located -coarse chromatin -single malignant cells with 'ink black' nuclei and abundant densely staining cytoplasm characteristic Small cell carcinoma: -cohesive tiny groups, frequently in the form of chains -tumour cells are small but at least 2x the size of lymphocytes -nuclear angulation, nuclear moulding -scant cytoplasm Urothelial carcinoma: -morphology non-specific -cells are epithelioid or spindled -eccentric angulated and hyperchromatic nuclei -some may show squamous differentiation

7 Melanoma: -dyscohesive -large nucleoli -bi or multinucleation -nuclei eccentric and oval -intranuclear pseudoinclusions -abundant cytoplasm -melanin pigment FALSE POSITIVES: -post radiation or chemotherapy - large bizarre multinucleated mesothelial cells -extramedullary haemopoiesis including megakaryocytes -reactive changes from various causes -degenerate benign vacuolated mesothelial cells, mimicking adenocarcinoma FALSE NEGATIVES: -single cell presentation in some carcinomas e.g. breast, stomach, pancreas -bland looking carcinoma e.g. renal cell carcinoma, looking like histiocytes -small cell melanoma with bland morphology -single cell presentation in mesothelioma with only mild nuclear atypia -tumour cells (e.g. small cell carcinoma) obscured by inflammation, reactive mesothelial cells -hypocellular tumours e.g. squamous cell carcinoma, sarcoma -low grade lymphoma -difficult to distinguish borderline ovarian tumours from low grade carcinomas PRACTICAL TIPS: 1. If a foreign population of atypical epithelial cells can be seen, it is malignant until proven otherwise. 2. Should not see many cell balls in normal pleural fluid, some in pericardial fluid, common in ascitic fluid. 3. Large papillary aggregates are significant in pleural effusions. The greater the number and the size of the aggregates, the greater the concern. Large cell balls >50 cells, even with only mild atypia beware neoplastic process. 4. Necrosis and apoptosis are abnormal, often associated with malignancy.

8 5. Presence of thick 3D groups is a warning sign for malignancy, as compared with benign 2-D flat monolayered honeycomb sheets. 6. If there are too many mesothelial cells (markedly cellular on low-power), even if single or in small groups, beware that it may be mesothelioma. Beware single cell presentation in mesothelioma. 7. Even if it is poorly differentiated mesothelioma, the cells are still mesothelial-like. If the tumour cells are bizarre and markedly pleomorphic, think about carcinoma first. 8. Pure population of carcinoma cells with no mesothelial cells can be seen but the tumour cells are not mesothelial-like. 9. Look carefully at the inflammatory cells in the background (reactive lymphocytes are predominantly T-cells). Beware haematopoietic malignancy esp. if the lymphocytes are mainly B-cells. 10. Always do immunostains on a cell block to confirm mesothelioma, even if the cytology is diagnostic. All cases of mesotheliomas (esp. first presentation) should be confirmed by histology.

9 Approach to effusion cytology: 1 cell or 2 cell population one cell population (all cells mesothelial-like) 2 cell population (some cells not mesothelial-like) (see below) yes nuclear atypia no inflammatory background yes Reactive mesothelial cells (small cell ball) Normal no Mesothelioma (large cell ball) 2 cell population Groups or single cell pattern Groups acinar arrangement / linear files Adenocarcinoma small cell Single cell pattern large cell nuclear moulding granular chromatin no nucleoli Small cell ca targetoid inclusions cytoplasmic vacuoles Lobular carcinoma pleomorphic nuclei eccentric nuclei nucleoli moderate cytoplasm Poorly diff. ca no nuclear moulding scant cytoplasm lymphoglandular bodies Lymphoma binucleation prominent nucleoli intranuclear inclusions cytoplasmic pigment Melanoma prominent nucleoli scant cytoplasm lymphoglandular bodies Lymphoma

10 Fig 1. Normal mesothelial cells. Note lacy collar (Arrow) Fig 2. Mesothelial cells showing a window: interdigitating microvilli (Arrow)

11 Fig 3. Reactive mesothelial cells with inflammatory background. Fig 4. Reactive mesothelial cells with low N/C ratio, round nuclei, smooth nuclear membrane and central nucleoli.

12 Fig 5. Reactive mesothelial cells. Fig 6. Reactive mesothelial cells. Mild nuclear enlargement with open chromatin. Small cell groups. Inflammatory background.

13 Fig 7. Benign mesothelial cells with degenerate vacuoles in the cytoplasm (vacuoles not indenting the nuclei; no nuclear features of malignancy). Fig 8. Rheumatoid arthritis. Note ragocytes: Debris within neutrophils (Arrows)

14 Fig 9. Mesothelioma. Markedly cellular with large cell balls. Fig 10. Mesothelioma with papillary groups.

15 Fig 11. Mesothelioma. Nuclear atypia present (red arrow). Compare with normal mesothelial cells (green arrow). Fig 12. Mesothelioma. Note collagen core (Arrow)

16 Fig 13. Mesothelioma. Fig 14. Mesothelioma. Tumour cells with cytoplasmic vacuoles containing magentacoloured hyaluronic acid.

17 Fig 15. Mesothelioma. Note presence of squamous-like cells (Arrow) which is a helpful clue to the diagnosis. Fig 16. Cell block: Mesothelioma.

18 Fig 17. Sarcomatoid mesothelioma. Fig 18. Pericardial effusion. Adenocarcinoma from the breast. Often with large cell groups showing smooth border.

19 Fig 19. Adenocarcinoma from the breast. Nuclei forming edge of cell group. Fig 20. Adenocarcinoma from the breast. Single cell presentation. Small tumour cells with targetoid inclusions (Arrows).

20 Fig 21. Adenocarcinoma from the breast (invasive lobular carcinoma). Note Indian file (Arrow). Fig 22. Adenocarcinoma from the lung.

21 Fig 23. Adenocarcinoma from the stomach with signet-ring cells. Fig 24. Adenocarcinoma from the ovary. Note papillary group with straight edge (Arrow).

22 Fig 25. Adenocarcinoma from the ovary. Note papillary structure with fibrovascular core. Fig 26. Pseudomyxoma peritonei.

23 Fig 27. Metastatic renal cell carcinoma. Single tumour cells with mild nuclear atypia and vacuolated cytoplasm, mimicking foamy macrophages. Fig 28. Metastatic renal cell carcinoma (cell block). Tumour cells CD10 positive.

24 Fig 29. Small cell carcinoma. Note nuclear moulding. Fig 30. Leukaemia. Note myeloid precursors with eosinophilic granules (Arrow).

25 References Pereira TC et al. The diagnosis of malignancy in effusion cytology: A pattern recognition approach. Adv Anat Pathol 2006;13: Bhatti TR et al. Malignant mesothelioma. Fluid cytology and differential diagnostic features. Pathology Case Reviews 2006;11:67-73 Boerner S. Mimicry and pitfalls in effusion cytology. Pathology Case Reviews 2006;11:85-91 Shield P. Peritoneal washing cytology. Cytopathology 2004;15: Shidham VB, Atkinson BF. Cytopathologic diagnosis of serous fluids. Saunders Elsevier. 2007