Practical Effusion Cytology · Practical Effusion Cytology A Community Pathologist’s Approach to...
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Practical Effusion Cytology
A Community Pathologist’s Approach to Immunocytochemistry
in Body Fluid Cytology
Emily E. Volk, MDWilliam Beaumont Hospital
Troy, MI
© College of American Pathologists 2004. Materials are used with the permission of Emily E. Volk, MD, FCAP.
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Overview
• Prudent use of immunocytochemistry (ICC)• Applications of ICC in body fluid cytology• Pertinent case reviews
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Pre-Analytical
• Form differential diagnosis– Morphology of conventional preparations– Clinical scenario
• Develop question to be answered by ICC• Ensure sample used contains cells in question• Adequate fixation
– Alcohol fixed preparation; Thin Layer; Cell Blocks
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Analytical
• Positive and negative controls • Ideal if cytology sample used for control
– Most labs use tissue control for convenience• Notice the pattern of staining in positive controls
– Membranous, cytoplasmic or nuclear.• Expect heterogeneity of immunostaining within a
sample• Normal cells may have capability to react with
ICC
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ICCCommon Causes of False-Positive Results
• Non-specific antibody binding• Misinterpretation of population as
neoplastic• Necrotic cells• Inappropriate fixation• Antigen diffusion• Antibody concentrations too high
Dabbs DJ. Immunocytology In: Diagnostic Immunohistochemistry. New York; 2002: 630-631.
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ICCCommon Causes of False-Negative Results
• Sample lacks neoplastic population• Antigen expression below sensitivity of antibody• Antibody concentration is too low• Poor fixation• Antigen diffusion
– S100 and GCDFP-15 in alcohol fixatives
• Insufficient antigen retrieval• Papanicolaou decolorization
Dabbs DJ. Immunocytology In: Diagnostic Immunohistochemistry.New York; 2002: 630-631.
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Body Fluid CytologyWBH-Troy 2003
Total Positive for Malignancy
• CSF 88 5• Pericardial 7 2• Pleural 201 41• Peritoneal 89 31• Peritoneal wash 67 4
452 83 (18%)
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Immunocytochemical AnalysisIs it useful?
Portion of workload
Diagnostically useful
WBH-Troy 0.9% 70.4%
Shield et al. Royal Brisbane Hospital
1.6 % 75.8 %
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Immunocytochemical AnalysisWhen is it useful?
• Poorly differentiated malignancy– Subclassification– Primary site determination
• Discrimination of mesothelial cells and metastatic malignancy
• Mesothelioma vs. adenocarcinoma
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Case 1
• 76 year old woman• New onset ascites• Previous history of breast cancer status post
mastectomy and radiation therapy
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Case 1
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Case 1H &E Cell Block
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Case 1BerEp4
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Case 1CK 7
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Case 1WT-1
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Case 1
• Positive ICC: – CK 7 (cytoplasmic)– BerEp4 (membranous)– WT-1 (nuclear)
• Negative ICC:– CK 20– TTF-1 and Surfactant protein A– Calretinin and thrombomodulin
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CK7/CK20 ICC Profile
• CK7+/CK20-– Non-small cell carcinoma of lung– Breast carcinoma (ductal and lobular)– Non-mucinous ovarian carcinoma– Endometrial adenocarcinoma– Mesothelioma
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CK7/CK20 ICC Profile
• CK7+/CK20+– Urothelial carcinoma– Pancreatic carcinoma– Ovarian mucinous carcinoma– Merkel cell carcinoma
• CK7-/CK20+– Colorectal adenocarcinoma
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CK7/CK20 ICC Profile
• CK7-/CK20-– Small cell carcinoma of lung– Squamous cell carcinoma of lung– Prostate adenocarcinoma– Renal cell carcinoma– Hepatoma
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BerEp4
• Monoclonal antibody to two glycopeptides on human epithelial cells
• Usually stains in cell membrane distribution– Adenocarcinomas of various sites (86.7%)*– Epithelial mesothelioma (0.86%)*
*Sheibani et al. AJSP. 1991;15: 779-784.
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WT-1Antibody to Wilms Tumor Suppressor Gene
Products
• Gene resides on 11p13 – Inactivation causes susceptibility to Wilms
tumor– Tissues of mesodermal origin
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WT-1Antibody to Wilms Tumor Suppressor Gene
Products• Nuclear immunostaining
– Mesothelioma (92.9%)– Papillary carcinoma of ovary (100%)– Renal cell carcinoma (100%)
• Negative staining– Adenocarcinoma of lung – Squamous cell carcinoma of lung– Metastatic breast carcinoma– Metastatic colon carcinoma
Kumar-Singh et al. J Pathol. 1997; 181: 67-74.
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Diagnosis:
• Metastatic adenocarcinoma consistent with serous surface or ovarian origin.
• Left ovarian mass later confirmed on pelvic CT scan.
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Case 2
• 73 year old woman with new onset ascites
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Case 2
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Case 2WT-1
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Case 2Cytokeratin AE 1/3
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Case 2Calretinin
Cytoplasmic and nuclear staining
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Case 2CK 5/6
Cytoplasmic staining
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Case 2• Positive ICC
– Calretinin– Cytokeratin AE 1/3– Cytokeratin 5/6 – WT-1 (nuclear)
• Negative ICC– B72.3– LeuM1 (CD15)– CEA
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Case 2Diagnosis
• Malignant mesothelioma
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Epithelial Mesothelioma Positive ICC markers
Cytokeratins• AE 1/3
– Good screening antibody– Broad spectrum
• CAM 5.2 (keratins 8/13)• CK 5/6
– High molecular weight keratin– Epithelial mesotheliomas strongly positive– Pulmonary adenocarcinoma negative to weak positivity– Squamous cell carcinoma +– Urothelial carcinoma (50%) +– Reactive mesothelial cells +
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Epithelial Mesothelioma Positive ICC markers
Calretinin
• Calcium binding protein similar to S-100• Cytoplasmic and nuclear staining • One of the most specific and reproducible
positive markers
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Epithelial Mesothelioma Positive ICC markers
Epithelial Membrane AntigenHuman milk fat globule protein-2 (HMFG-2)
• Cell membrane staining pattern• Adenocarcinomas show cytoplasmic
staining• May see membranous staining in
non-mucinious BAC/ papillary RCC• Benign mesothelial cells usually negative
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Epithelial Mesothelioma Positive ICC markers
Thrombomodulin• Plasma membrane related glycoprotein with
anticoagulant activity• Thick membranous staining in malignant
mesothelioma• Thin membranous staining in reactive
mesothelial cells• Cytoplasmic staining may be seen in
adenocarcinoma
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Epithelial Mesothelioma Positive ICC markers
• N-cadherin– Strong positivity with malignant mesothelioma– Focal weak in pulmonary adenocarcinoma
• WT-1– Nuclear staining of malignant mesothelioma,
papillary ovarian tumors, and RCC
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Epithelial Mesothelioma Negative ICC markers
• Carcinoembryonic antigen (CEA)– 85-100% of pulmonary adenocarcinoma positive
• Leu-M1 (CD 15)– 50-100% of pulmonary adenocarcinoma positive
• B72.3– 84-96% of pulmonary adenocarcinoma positive– 10% epithelial mesothelioma positive
• BerEP4
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Desmin reactivity of mesothelial cells
• Strong cytoplasmic reactivity in 22 of 24 cases (92%) of reactive mesothelial cells
• All cases of malignant mesothelioma and metastatic adenocarcinoma were negative
• Archival paraffin cell block material from body fluids
• ICC to determine malignancy– Exercise caution– More data before widespread use
Affify et al. Applied Immunohistochemistry and Molecular Morphology. 10 (2). 2002: 178-182
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Case 3
• 55yo man with new onset right pleural effusion
• History of smoking-40 pack years
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Case 3
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Case 3EMA
Diffuse cytoplasmic staining
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Case 3CD 15
Cytoplasmic staining
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Case 3Surfactant Protein ACytoplasmic staining
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Case 3ICC
• EMA+ (cytoplasmic)• CD 15 (Leu-M1) +• SPA+• Calretinin-• Thrombomodulin-
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Diagnosis
• Metastatic lung adenocarcinoma
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Surfactant Protein A (SPA)
• Antibodies to surfactant apoproteins• Granular cytoplasmic positivity in primary
lung adenocarcinoma
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Thyroid Transcription Factor 1 (TTF-1)
• Member of the NKx2 family of transcription factors
• Positive nuclear staining– Thyroid tumors– Lung tumors
• Adenocarcinoma• Small cell carcinoma
• Cytoplasmic staining– Hepatocellular carcinoma*
*Pan C-C, et al. AJCP. 2004; 121:343-349.
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SummaryCAVEATS FOR ICC
• Know the antigenic profiles of the tumors in your differential diagnosis.
• Pay attention to controls.• Identify which cells are staining.• Scrutinize the positive cells for their pattern
of reactivity.
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Immunohistochemistry Web site
• www.immunocentral.com• www.immunoquery.com