Immunohistochemistry on cytology specimens from...
Transcript of Immunohistochemistry on cytology specimens from...
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Immunohistochemistry on cytology specimens from pleural and
peritoneal fluid
Dr Naveena Singh Consultant Pathologist Bart health NHS Trust
London United Kingdom
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Disclosures and Acknowledgements
I have no financial disclosures.
I would like to acknowledge the use of images from
Diagnostic Cytopathology
and
Professor Mike Sheaff, my colleague and co-author.
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Learning Objectives
• Problems with pathological assessment of serous effusion fluids
• Role of immunohistochemistry; antibodies/panels useful in routine practice
• Common sense approach for safe reporting
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Introduction
• One of the most challenging areas in cytology reporting
• One of the areas most dependent on good quality immunohistochemistry:
– technical quality
– interpretation
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Anatomy of the serous cavities
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Structure and function of mesothelium
• Mesothelium: Single layer of epithelioid cells on a bed of vascular connective tissue
• Submesothelial cells - progenitors of surface epithelioid cells; possibly the cells of origin of malignant mesothelioma
• Stroma with network of capillaries and lymphatics
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Structure and function of mesothelium Normal serous fluid production: • Blood →parietal stromal matrix →mesothelial layer →serous cavity Normal serous fluid resorption: • into capillaries of visceral serosa • through stomata between mesothelial cells into lymphatics within
the parietal serosa Functions: • Produce hyaluronate-rich fluid • Facilitate transport of fluid and cells across serosal barrier Also: • Phagocytic function • antigen presentation, inflammatory processes, coagulation,
fibrinolysis, repair and tumour cell adhesion
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Structure and function of mesothelium
Mesothelial lining cells
• Flat /cuboidal,
• Small to medium sized nuclei, small nucleoli
• Variable amounts of cytoplasm with luminal microvilli on the luminal
• Pinocytotic vesicles on both apical and basal sides of the cell
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Histology of mesothelial cells - normal
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Calretinin
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D2-40
CD68
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Transudate
Exudate
Protein low <2.9g/dl Protein high (>3g/dl)
Non-fibrinous Fibrin present
Cell content low Inflammatory/tumour cells
LDH low LDH high
Specific gravity low <1.015 Specific gravity higher
Clear Microorganisms or foreign material
Effusions
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Transudate
Exudate
Common Common
Left ventricular failure Liver cirrhosis Hypoalbuminaemia Peritoneal dialysis
Malignancy Parapneumonic effusions
Less common Less common
Hypothyroidism Nephrotic syndrome Mitral stenosis Pulmonary embolism
Pulmonary infarction Rheumatoid arthritis Autoimmune diseases Benign asbestos effusion Pancreatitis Post-myocardial infarction syndrome
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Histology of mesothelial cells - reactive
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Calretinin
D2-40
CD68
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Cytology of mesothelial cells
• round with indistinct cell membranes and fuzzy borders
• single or clumped with so-called ‘windows’ between cells
• large round or elliptical nucleus, often centrally placed, with a single prominent nucleolus
• may contain two or multiple nuclei • cytoplasm is green with Papanicolaou and blue with
MGG • 2-zone cytoplasm • vacuoles due to ‘degeneration’ or pinocytosis or
glycogen
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Other cells in effusion fluid
• Macrophages
• Lymphocytes: T>>B
• Neutrophils
• Eosinophils
• Plasma cells
• Mast cells
• Megakaryocytes
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Challenges in effusion cytology in the presence of malignancy
• Multiple causes of effusions; may be due to a co-morbidity, not the cancer
• May be first manifestation of malignancy
• Even when due to cancer, this may be due to
– lymphatic blockage
– Irritation of mesothelium
– ie NOT tumour cell seeding
• Difficulties in interpretation
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Challenges in effusion cytology
• Interpretation challenges – Reactive mesothelial cells can show a range of atypia: bi-/multinucleation,
nuclear atypia, mitotic activity, large cell aggregates, vacuolation, gland-like arrangement
– Malignant cells are altered in effusions
• Polygonal shape • 3-D aggregates → • Degenerative changes
– Some types deceptively bland
– Number of cells present in the fluid is highly variable
• Do not resemble the primary • Look similar irrespective of
origin
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Role of immunohistochemistry
1. MALIGNANT OR REACTIVE?
– Dual population but only subtle differences or overlapping features
– Single population of only malignant cells without obvious cytological features of malignancy
– Isolated malignant cells in a vast reactive population
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Malignant vs Reactive
Mesothelial Carcinoma
Calretinin B72.3
D2-40 BerEP4
CK 5/6 CD15
Vimentin CEAm
EMA (cytoplasmic)
MOC31
• Use a panel including +ve and –ve markers
• CARE when interpreting which cells are positive for what: SCIP
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Malignant vs Reactive
Mesothelial Carcinoma
Calretinin B72.3
D2-40 BerEP4
CK 5/6 CD15
Vimentin CEAm
EMA (cytoplasmic)
MOC31
• Use a panel including +ve and –ve markers
• CARE when interpreting which cells are positive for what: SCIP
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MOC 31
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Malignant vs Reactive
Reactive mesothelial Mesothelioma
Calretinin Calretinin
D2-40 D2-40
CK 5/6 CK 5/6
Vimentin Vimentin
EMA (negative/weak) EMA (membranous)
Desmin (positive) Desmin (negative)
Others: p53, bcl-2, Glut1
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Calretinin
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EMA: Strong membranous staining is typical in mesothelioma; Normal/reactive mesothelial cells: weak staining; Adenocarcinoma cells: diffuse strong cytoplasmic staining without membranous accentuation
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Desmin: NEGATIVE in mesothelioma; positive in normal/reactive mesothelial cells
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Role of immunohistochemistry
2. Determining primary site
General markers of malignancy and negative markers of mesothelial cells and macrophages
Specific markers for particular malignancies
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Antibodies useful for determining site of origin in effusions
Cancer Antibodies
Lung TTF-1, CD56, CK19
Ovary WT-1 (also positive in mesothelial cells), ER
Gastrointestinal tract CDX2, CK7/20, CA19-9, CEAm
Breast ER, mammoglobin, CRx-A01,
Melanoma S-100, HMB-45, melan A
Lymphoma CD3:CD20, other specific studies
Sarcoma Vimentin, other specific
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Common Examples
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Adenocarcinoma of lung: TTF1 +ve
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TTF 1
CD56
Small cell carcinoma of lung: TTF1 and CD56 +ve
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BerEp4 MOC31
CEA
Gastric signet ring carcinoma: CEA positive
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WT1 +ve Calret -ve
ER +ve
High grade serous tubo-ovarian carcinoma
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CD3 CD20
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Pitfalls in interpreting IHC in effusions
• MIXTURE of cells: take care to report expression in the correct population; particularly in presence of low cellularity
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WT1 expressed in mesothelial cells
TTF1 expressed in malignant cells
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Pitfalls in interpreting IHC in effusions
• PATTERN of expression where appropriate
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EMA expression patterns
Reactive mesothelial cells: weak diffuse cytoplasmic
Mesothelioma: Cytoplasmic with STRONG membrane accentuation
Adenocarcinoma: Diffuse strong cytoplasmic expression, obscures cellular detail
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Question 1
• Which of the following markers would not be useful in distinguishing reactive mesothelial cells from metastatic adenocarcinoma?
1. MOC31
2. BerEp4
3. Calretinin
4. CK7
5. CK 5/6
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Question 2
• Which of the following markers would not be useful in the diagnosis of mesothelioma?
1. MOC31
2. Actin
3. Calretinin
4. Desmin
5. EMA
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Case 1: Pleural fluid in known case of lung cancer: reactive or metastatic?
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Case 2: 19F, Fitz-Hugh-Curtis Syndrome. Rule out malignancy
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General approach to reporting malignancy in effusions
• Only report when CERTAIN: malignancy is easy to overcall and undercall: err on side of caution
• Always rule out malignancy if there is a +ve history • Always rule out malignancy if there is doubt on
morphology • Look at all preparations before issuing final report • Use a PANEL of markers, not just one • Take care when interpreting IHC as there is a mixture of
cells present; coordinate approach on serial sections – Due regard to expression by different cell types, eg WT1 – Due regard to different patterns of expression, eg EMA
• Asking for another sample is not unreasonable