1 Video Microsc copy Tutorial #20 Ashish Chandr ra, MD Disclosur re information The speaker has no relationship that representss a possible conflict of interest with respect to the content of this presentation.
2 SEROUS EFFUSION CYTOLOGY- A PRACTICAL APPROACH Dr Ashish Chandra MD FRCPath DipRCPath (Cytol) Clinical lead, Histopathology and Cytopathology Guy s & St. Thomas Hospitals London SE1 4LG UK
3 Introduction Accumulation of fluid in the pleural, pericardial or peritoneal cavities is referred to as a serous effusion and is always pathological. The pathology may be either neoplastic or non-neoplastic. Cytology is very helpful in confirming the presence of malignant neoplastic cells in serous effusions. It is also performed for effusions secondary to inflammatory and reactive conditions. In these conditions, cytology is useful when taken in conjunction with biochemical and microbiological investigations on the fluid. Malignant effusions may be the initial presentation in 10-15% of patients with malignancy. The diagnostic accuracy of the cytologic examination of malignant pleural effusion ranges from 40 to 87%. Recommended sample volume For diagnostic purposes, variable amounts of fluid are received by the cytology laboratory. The BSCC code of practice document suggested that 20ml of representative sample may be adequate for diagnostic purposes while the British Thoracic Society recommends 50ml of fluid for cytology. However, this does not mean that samples of smaller volumes are unacceptable for processing. All samples including those that are heavily blood-stained or clotted should be processed and examined. Macroscopic examination The volume, colour and presence of clots should be recorded in the lab. Chylous (milky) fluid may indicate lymph in the serous cavity and fistula formation either pathologically or following surgery. A high hyaluronic acid content of the fluid associated with mesothelioma makes it viscous in consistency and should be noted. Processing and staining The usual method involves cytocentrifugation and examination of one Pap and one Giemsa stained preparation. Liquid based cytology (LBC) may be used but some methods allow the use of Pap stain only. Processing of clots and cell block preparations are particularly helpful in blood-stained samples as cytological material may be trapped among the red blood cells. Immunostains and special stains may be performed on any of the above preparations. Pre-screening and reporting by scientists In the UK, Biomedical Scientists (BMS) or cytotechnologists may be trained to screen and even sign out negative serous effusion samples. Adequate training and experience is required for this and there is also a Diploma in Expert Practice in Non-gynae Cytology offered by the Institute of Biomedical Scientists (IBMS). Reporting of serous effusions,
4 urine and respiratory cytology samples by BMS may be practised under the guidance of the lead cytopathologist. Cytological interpretation This should be done in the light of the clinical information and a provisional report may be issued to alert the clinician when malignancy is suspected but awaiting confirmation or further characterisation on immunostains. For reporting purposes, cytological interpretation of serous fluids fall under the following categories- 1. Unsatisfactory. These are usually small volume samples composed entirely of blood, probably reflecting an unsatisfactory procedure (unless hemothorax is suspected clinically). Occasionally, poor cellular preservation due to delay in transport to the lab may compromise the specimen quality, resulting in difficulty in interpretation. Effusions are never acellular and even small numbers of macrophages or inflammatory cells should be reported. The presence of mesothelial cells helps in confirming that the procedure was successful even if paucicellular. 2. Benign. These mostly represent a non-specific reactive picture with a heterogeneous population of macrophages, mesothelial cells and variable numbers and types of inflammatory cells. A more specific diagnosis such as empyema may be suggested if the inflammatory cells are predominantly neutrophils. A chronic infection such as tuberculosis may be associated with a dominant lymphocytic population. 3. Atypical cells present, probably degenerated mesothelial cells or macrophages. The atypical cells in this category usually represent degenerated mesothelial cells or macrophages but immunostains are deemed necessary to exclude the possibility of malignancy, particularly when clinical findings are suspicious. If the representative cells are present on the immunostains, it may be possible to resolve this dilemma, however, this is often not the case when only one or two small groups of these cells are present on the cytospins. These cases may benefit from a multi-headed discussion, combining the collective interpretive skill and experience of pathologists and scientists. In a small number of cases, an inconclusive report may have to be issued, requesting a further sample if possible and seeking any useful clinical clues. 4. Atypical cells present, suspicious for malignancy. This may be provisionally reported to the clinician under a number of circumstances, usually before immunostains have been requested. Many of these cases will be diagnostic once the immunostains become available. i) small numbers of single, dispersed atypical cells that are suspicious but not diagnostic of malignancy ii) atypical cells obscured by inflammatory or other cells in a thick, hypercellular preparation iii) difficulty in distinction between reactive mesothelial cells, malignant mesothelial cells and adenocarcinoma.
5 5. Malignant cells present. A confident diagnosis of malignancy is made, usually metastatic adenocarcinoma and is consistent with the available clinical information. Immunostains may help in characterising the malignant cells further as glandular, squamous, neuroendocrine or lymphoid in type. Other ancillary investigations such as flow cytometry, PCR, FISH, ploidy etc may be useful for detecting signature molecules for specific malignancies. These are useful in the light of clinical and cytomorphological findings, however, their relevance in therapeutic intervention in the absence of identifiable malignant cells remains uncertain at present. For malignant glandular cells (ie. adenocarcinoma), a panel of immunostains to confirm the primary site may be necessary. The choice of stains should be guided by clinical and radiological information. In metastases from known previous primaries eg breast or lung, therapeutically relevant markers such as Her-2, EGFR/K-RAS may be required. Choice of immunostains The choice of immunostains will depend on the clinical question to be answered in a particular scenario. In the category atypical cells present, probably degenerated mesothelial cells or macrophages, the distinction between degenerated mesothelial cells from epithelial cells may be made by using a panel incorporating 2 tried and tested markers for each of the two cell types. However, as pointed out earlier, in this category the atypical cells are few in number and may not be represented on the cell block or immunostains. A discussion with the clinician may help gauge the suspicion of malignancy and the need for a repeat sample. If there is no residual fluid to send for cytology, follow up of these patients with a view to early detection of a recurrent effusion may provide a second chance for cytological diagnosis. In the category atypical cells present suspicious of malignancy, confirmation of metastatic carcinoma would rely on the use of at least 2 generic epithelial markers and if the clinical history points to a particular site or sites, specific markers should be included. Once again, the number of cells may be small (4i above) not allowing certainty of the diagnosis on cytomorphology or immunostains. In densely cellular preparations (4ii), dilution of the sample may unmask the malignant cells. It may also be easier to characterise these cells better in a cell block or clot with confirmation on immunostains. A further sample may be required and is usually available from a clinically suspicious or malignant effusion. Mesothelial markers must always be included for cases in 4iii as degenerate mesothelial cells may mimic malignancy and furthermore, mesothelioma may be missed if these are not performed. In the category malignant cells present, the diagnosis is made with confidence either supported by the knowledge of a known primary or based purely upon the unmistakable
6 cytological features. The purpose of immunostains in this category is to ascertain the primary site of malignancy. The choice of immunostains would be guided by the clinical information but also influenced by the patient s gender and site of the effusion (Table 1). Metastatic adenocarcinoma is the commonest metastatic malignancy in effusions but mesothelioma, poorly differentiated squamous cell carcinoma and other cell lineages must all be considered, particularly if the effusion is the first manifestation of malignancy or when the clinical primary is unknown. There is no value in routinely performing immunostains on cytologically benign effusions or on recurrent effusions where a diagnosis has previously been confirmed by immunostains. Drainage of a recurrent effusion may be for therapeutic purposes only although these samples are reflexly dispatched to the cytology lab. Choice of markers Mesothelial cells Reactive mesothelial cells: EMA (thin membranous staining), Desmin +, diploid Mesothelioma: EMA (thick membranous staining), p53/ki67 ++, aneuploid Calretinin highly sensitive marker for the mesothelium, however, it is also expressed in squamous cell carcinoma and ovarian serous adenocarcinoma. Good for distinguishing mesothelioma from lung adenocarcinoma and RCC. WT-1 distinguishes between mesothelioma and squamous cell carcinoma but is expressed in ovarian serous adenocarcinoma. D2-40 and Podoplanin: Highly sensitive for mesothelial cells, negative in lung adenocarcinoma but squamous cell carcinoma (30%) and ovarian serous adenoca (15%) may be positive Mesothelin: strong cytoplasmic positivity in mesothelioma but focal membranous staining may be seen with lung adenoca and squamous cell ca Tenascin-X: positive in mesothelioma, negative in reactive mesothelial cells and ovarian/peritoneal carcinoma Epithelial markers BerEP4: adenocarcinoma and squamous ca lung, but also10-15% mesotheliomas MOC-31: adenoca and squamous ca lung, <10% mesotheliomas Monoclonal CEA: distinguishes lung adenoca from epithelioid mesothelioma (polyclonal CEA may be positive in a third of cases)
7 PAX-8 (more sensitive) and PAX-2: Mullerian epithelium, thyroid follicular cells and renal tubular cells p63: positive in squamous cell carcinoma, negative in mesothelioma B72.3: expressed in high percentage of adenocarcinomas, but negative for mesothelial cells Melanoma HMB45, Melan A, S-100, MART-1 Neuroendocrine markers CD56, NSE, Chromogranin A, Synaptophysin Germ cell tumours PLAP, CD117 (seminoma), CD30 (embryonal ca), AFP (yolk sac tumour), HCG (chorioca) Renal cell carcinoma RCC antigen, CD10, PAX-2 CK7+ CD15- (Chromophobe RCC) CK7+ AMACR+ (Papillary RCC Type 1) Macrophages CD68 (PGM1) Lymphoid markers LCA, CD3, CD20, kappa and lambda light chains New markers Claudin-4: reported to be highly sensitive and specific single shot marker for metastatic carcinoma, always negative in mesothelioma IMP3: negative in reactive mesothelial cells, positive in carcinomas Male Female
8 Pleural Lung, GIT & pancreas, lymphoma Breast, lung, ovary, GIT, lymphoma Peritoneal GIT, pancreas, lymphoma, lung, genitourinary tract Ovary, breast, endometrium, stomach, lymphoma, GIT, pancreas Table 1. Likely primary sites based on gender and body cavity involved CK7+, CK20+ Urothelial carcinoma, ovarian mucinous carcinoma, upper GI and appendix CK7+,CK20- Breast, non-small cell ca lung, ovarian serous carcinoma, endometrial and cervical adenoca, upper GI, pancreas, thyroid CK7-, CK20+ Colorectal carcinoma, Merkel cell ca CK7-, CK20- Hepatocellular carcinoma, renal cell carcinoma, prostatic carcinoma Table 2. Typical pattern of CK7 and CK20 at various primary sites Lung TTF1 (adenoca), surfactants, p63 (squamous), CK5/6 (squamous) GIT CDX2, mcea, villin Breast GCDFP, mammoglobin, ER Ovary WT-1 (serous), ER, CA125, mesothelin (serous), PAX-8, PAX-2 Pancreas CA19.9, CA125, mesothelin Prostate PSA, PRAP,PSMA, AMACR Thyroid Thyroglobulin, TTF1, calcitonin (medullary ca) Bladder HMW-CK, uroplakin-3, p63 Serous cavities (mesothelium) Calretinin (nuclear), WT-1 (nuclear), D2-40, podoplanin, mesothelin, thrombomodulin, EMA (membranous) Endometrial mucinous ca MUC-1+, ER+, PR+, p16- Endocervical adenoca MUC-1-, ER-, PR-, p16+ Table 3. Typical antibody expression by organ type
9 References Krausz T and Barker F. Chapter 4. Reactive effusions. In Diagnostic Cytopathology, 2 nd ed (2003). Editors Gray and McKee. Schofield J and Krausz T. Chapter 5. Metastatic disease and lymphomas. In Diagnostic Cytopathology, 2 nd ed (2003). Editors Gray and McKee Cibas E. Chapter 4. Pleural, pericardial and peritoneal fluids. In Cytology- diagnostic principles and clinical correlates, 3 rd ed (2008), Editors Cibas and Ducatman Koss Diagnostic Cytology and its histopathologic basis, 5 th ed (2006), Vol II, Chapters 25 and 26. Metzgeroth G, Kuhn C, Schultheis B, Hehlmann R, Hastka J. Diagnostic accuracy of cytology and immunocytology in carcinomatous effusions. Cytopathology Aug;19(4): Chandra A, Cross P, Denton K et al. The BSCC Code of practice- exfoliative cytology. Cytopathology 2009, 20, Investigation of a unilateral pleural effusion in adults: British Thoracic Society pleural disease guideline Thorax 2010;65:ii4-ii17. Prospective Study To Determine the Volume of Pleural Fluid Required To Diagnose Malignancy. Chest January :
10 Pu RT, Pang Y, Michael CW. Utility of WT-1, p63, MOC31, mesothelin, and cytokeratin (K903 and CK5/6) immunostains in differentiating adenocarcinoma, squamous cell carcinoma, and malignant mesothelioma in effusions. Diagn Cytopathol Jan;36(1):20-5. Shabaik A, Lin G, Peterson M, Hasteh F, Tipps A, Datnow B, Weidner N. Reliability of Her2/neu, estrogen receptor, and progesterone receptor testing by immunohistochemistry on cell block of FNA and serous effusions from patients with primary and metastatic breast carcinoma. Diagn Cytopathol May;39(5): doi: /dc Su XY, Li GD, Liu WP, Xie B, Jiang YH. Cytological differential diagnosis among adenocarcinoma, epithelial mesothelioma, and reactive mesothelial cells in serous effusions by immunocytochemistry. Diagn Cytopathol Sep 10. DOI: /dc Mohamed F, Vincent N, Cottier M, Peoc'h M, Merrouche Y, Patouillard B, Paul S, Genin C. Improvement of malignant serous effusions diagnosis by quantitative analysis of molecular claudin 4 expression. Biomarkers Jun;15(4): Lonardi S, Manera C, Marucci R, Santoro A, Lorenzi L, Facchetti F. Usefulness of Claudin 4 in the cytological diagnosis of serosal effusions. Diagn Cytopathol May;39(5): doi: /dc Wiseman W, Michael CW, Roh MH. Diagnostic utility of PAX8 and PAX2 immunohistochemistry in the identification of metastatic Müllerian carcinoma in effusions. Diagn Cytopathol Oct 14. DOI: /dc Laury AR, Perets R, Piao H, Krane JF, Barletta JA, French C, Chirieac LR, Lis R, Loda M, Hornick JL, Drapkin R, Hirsch MS. A Comprehensive Analysis of PAX8 Expression in Human Epithelial Tumors. Am J Surg Pathol Jun;35(6):
11 Tong GX, Devaraj K, Hamele-Bena D, Yu WM, Turk A, Chen X, Wright JD, Greenebaum E. PAX8: A marker for carcinoma of Müllerian origin in serous effusions Aug;39(8): doi: /dc Zhang H, Yang XY, Hong T, Feldman T, Bhattacharyya PK. Kaposi sarcoma-associated herpesvirus (human herpesvirus type 8)-associated extracavitary lymphoma: Report of a case in an HIV-positive patient with simultaneous kaposi sarcoma and a review of the literature. Acta Haematol. 2010;123(4): Epub 2010 May 19. Das DK. Serous effusions in malignant lymphomas: a review. Diagn Cytopathol May;34(5): Chute DJ, Kong CS, Stelow EB. Immunohistochemistry for the detection of renal cell carcinoma in effusion cytology. Diagn Cytopathol Feb;39(2): doi: /dc Gokden N, Gokden M, Phan DC, McKenney JK. The utility of PAX-2 in distinguishing metastatic clear cell renal cell carcinoma from its morphologic mimics: an immunohistochemical study with comparison to renal cell carcinoma marker. Am J Surg Pathol Oct;32(10): Murugan P, Siddaraju N, Sridhar E, Soundararaghavan J, Habeebullah S. Unusual ovarian malignancies in ascitic fluid: a report of 2 cases. Acta Cytol Jul- Aug;54(4): Iqbal J, Liu T, Mapow B, Swami VK, Hou JS. Importance of flow cytometric analysis of serous effusions in the diagnosis of hematopoietic neoplasms in patients with prior hematopoietic malignancies. Anal Quant Cytol Histol Jun;32(3):161-5.
12 Davidson B, Dong HP, Holth A, Berner A, Risberg B. Flow cytometric immunophenotyping of cancer cells in effusion specimens: diagnostic and research applications. Diagn Cytopathol Sep;35(9): Kim JH, Choi YD, Lee JS, Lee JH, Nam JH, Choi C. Utility of thyroid transcription factor-1 and CDX-2 in determining the primary site of metastatic adenocarcinomas in serous effusions. Acta Cytol May-Jun;54(3): Oien KA. Pathologic evaluation of unknown primary cancer. Semin Oncol Feb;36(1):8-37. Yuan Y, Dong HP, Nymoen DA, Nesland JM, Wu C, Davidson B. PINCH-2 expression in cancers involving serosal effusions using quantitative PCR. Cytopathology Feb;22(1):22-9. Davidson B, Stavnes HT, Holth A, Chen X, Yang Y, Shih IeM, Wang TL. Gene expression signatures differentiate ovarian/peritoneal serous carcinoma from breast carcinoma in effusions. J Cell Mol Med Mar;15(3): Yuan Y, Nymoen DA, Stavnes HT, Rosnes AK, Bjørang O, Wu C, Nesland JM, Davidson B. Tenascin-X is a novel diagnostic marker of malignant mesothelioma. Am J Surg Pathol Nov;33(11): Bassarova AV, Nesland JM, Davidson B. D2-40 is not a specific marker for cells of mesothelial origin in serous effusions. Am J Surg Pathol Jul;30(7): Ikeda K, Tate G, Suzuki T, Kitamura T, Mitsuya T. IMP3/L523S, a novel immunocytochemical marker that distinguishes benign and malignant cells: the expression profiles of IMP3/L523S in effusion cytology. Hum Pathol May;41(5):
13 Lin O. Challenges in the interpretation of peritoneal cytologic specimens. Arch Pathol Lab Med May;133(5): Saleh HA, El-Fakharany M, Makki H, Kadhim A, Masood S. Differentiating reactive mesothelial cells from metastatic adenocarcinoma in serous effusions: the utility of immunocytochemical panel in the differential diagnosis. Diagn Cytopathol May;37(5): King JE, Thatcher N, Pickering CA, Hasleton PS. Sensitivity and specificity of immunohistochemical markers used in the diagnosis of epithelioid mesothelioma: a detailed systematic analysis using published data. Histopathology Feb;48(3):