Immunohistochemical classification of the unknown primary tumour (UPT) Part I. Prof. Mogens Vyberg NordiQC Institute of Pathology Aalborg, Denmark
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1 Immunohistochemical classification of the unknown primary tumour (UPT) Part I Prof. Mogens Vyberg NordiQC Institute of Pathology Aalborg, Denmark
2 Tumours of unknown origin: Histology Brain tumour - biopsy
3 CD45 VIM Tumours of unknown origin: Immunohistochemistry Pan-CK S-100
4 IHC classification of the Unknown Primary Tumour UPT: A tumour appearing in metastatic setting without a histologically proven primary tumour. UPT pose an increasing challenge for the pathologist - due to the progress in surgical and oncological treatment possibilities.
5 IHC classification of the Unknown Primary Tumour New, relatively specific antibodies give the pathologist more and better diagnostic tools. But the diagnostic work also become more complex in terms of planning, optimization of protocols, interpretation of reaction patterns and error trapping.
6 IHC classification of the Unknown Primary Tumour 10-15% of cancers remain UPTs +??% uncertain if primary or metastatic - liver, lung, bone, lymph nodes, brain, peritoneum... Undifferentiated (5-10%) - carcinomas, sarcomas, melanomas, germ cell tumours - malignant lymphomas Adenocarcinomas (80-90%) - lung, breast, prostate, colorectum, ovary, pancreas Squamous cell carcinomas (5-10%) - lung, esophagus, uterine cervix
7 IHC classification of the Unknown Primary Tumour Differences in prognosis Differences in treatment regimes malignant lymphomas carcinomas (breast, prostate, ovary...) sarcomas (GIST, synovial sarcoma...) germ cell tumours Pathology tests cost effective Pathology tests save patient discomfort The patient s right to know The risk of hereditary cancer
8 IHC classification of the Unknown Primary Tumour Most likely diagnoses Relevant differential diagnoses Optimal selection of antibodies for a diagnostic algorithm Primary and secondary antibody panels Turn-around-time Laboratory expenses
9 IHC classification of the Unknown Primary Tumour Pathologist - knowledge, acceptance, skill Tumour material - diagnostic markers Antibodies available - applic. in diagnostic algorithms Methods - protocol: sensitivity, specificity, reliability - interpretation: cut-off level for positivity clinical relevance GIST: S-100B Protease GIST: S-100B MWO
10 IHC classification of the Unknown Primary Tumour Pathologist - knowledge, acceptance, skill Tumour material - diagnostic markers Antibodies available - applic. in diagnostic algorithms Methods - protocol: sensitivity, specificity, reliability - interpretation: cut-off level for positivity clinical relevance positive = any staining CK 20 negative = < 50% stained cells CK 20
11 March 2008
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14 Planning diagnostic immunohistochemistry
15 Planning diagnostic immunohistochemistry
16 Planning diagnostic immunohistochemistry
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29 Human protein Atlas AE1/AE3
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33 Primary panel for the unknown primary tumour Real CD45 CK S-100 VIM Haematolymphoid +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
34 CD45 - Leucocyte common antigen (LCA) Transmembrane protein tyrosin phospatase essential for haematopoietic signal transduction and cell activation Membrane associated component: 5 isotypes Intracellular component: one common type
35 CD45 - Leucocyte common antigen (LCA) Transmembrane protein tyrosin phospatase essential for haematopoietic signal transduction and cell activation Membrane associated component: 5 isotypes Intracellular component: one common type Large majority of haematolymphoid cells Lost in maturing erythocytes, megakaryocytes and plasmacells Never found in non-haematolymphoid cells
36 CD45 - Leucocyte common antigen (LCA) Normal lymph node Malignant lymphoma
37 CD45 - Leucocyte common antigen (LCA) More than 90% of lymphomas are positive Negative on: - some Acute Lymphoblastic Leukaemia/LBL - plasma cell malignancies - HR-S cells in classic Hodgkin Lymphoma - some Anaplastic Large Cell Lymphoma (ALCL) - ALK+ Large B-Cell Lymphoma Exceptionally positive in non haematol. tumours?
38 CD45 - Leucocyte common antigen (LCA) Liver Brain Critical assay performance control
39 CD45 negative HRS cells in classic Hodgkin Lymphoma CD45 positive LH cells in NLPHL CD45 weaker staining in B-CLL CD45 weak reactivity in ALCL 39
40 CD45 NordiQC run assessment-cd45.htm Which is best?
41 CD45 NordiQC run assessment-cd45.htm Optimal Insufficient
42 Optimal Insufficient CD45 NordiQC run assessment-cd45.htm B-CLL 56% of labs 18% of labs
43 CD45 - Leucocyte common antigen (LCA) CD45 RO ~ T-cells Lymph node/tonsil CD45 RA ~ B-cells
44 CD45 - Leucocyte common antigen (LCA)
45 CD45 - Leucocyte common antigen (LCA)
46 CD45 - Leucocyte common antigen (LCA)
47 Pancreas tumour
48 Pancreas tumour CD45
49 Pancreas tumour CD163
50 Pancreas tumour: undifferentiated carcinoma CK
51 Primary panel for the unknown primary tumour Haematolymphoid CD45 CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
52 Cellular filaments # Microfilaments: (6 nm) # Intermediate filaments (7-11 nm) # Microtubuli (23 nm)
53 Intermediate filaments Group of mainly cytoplasmic filaments 7 11 nm in diameter Part of the cytoskeleton in virtually all cells, creating as meshwork and connecting nuclear membrane with cell membrane Often associated with microfilaments (6 nm) and microtubules (23 nm) Important for mechanical strength and cellular functions
54 Intermediate filaments tetrameric units Central core formed by eight tetramers Nagle, AJSP 1988, 12:4
55 Intermediate filaments - 5 classes I acidic cytokeratins II basic-neutral cytokeratins III vimentin, desmin, glial fibrillary acidic protein, peripherin IV neurofilament protein, α-internexin, nestin V lamins
56 Cytokeratins as tonofilaments Cytokeratin intermediate filaments attached to desmosomes Drochmans et al. J Cell Biol. 1978, 79:427
57 Cytokeratins in diagnostic pathology Cytokeratins (CKs) belong to the most fundamental markers of epithelial differentiation CKs comprise a large family of subtypes. Different cell types express different patterns of CK subtypes Cancers generally express CK patterns that at least in part represent the pattern of the putative cell of origin Metastases express CK patterns fairly concordant with those of the primary tumours
58 HE CK Micrometastases identified by cytokeratin
59 HE-frozen CK-frozen Carcinoma in frosen section identified by cytokeratin
60 Sarcomatoid carcinoma CK LMW Cytokeratins in carcinomas with aberrant growth patterns
61 Low molecular weight cytokeratins in carcinomas Carcinomas always LMW-CK-positive, except some cases of Renal cell carcinoma Adrenal cortical carcinoma Small cell carcinoma CK8: Adrenal cortical carcinoma CK8: Renal cell carcinoma CK: renal cell carcinoma
62 Primary panel for the unknown primary tumour Haematolymphoid CD45 CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
63 Cytokeratins in non-epithelial tumours CK8 CD45 42 y, tumour infiltrating retroperitoneum Malignant lymphoma! CD79a
64 Primary panel for the unknown primary tumour Haematolymphoid CD45 CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
65 Cytokeratins in malignant mesothelioma CK8 CK5
66 Primary panel for the unknown primary tumour Haematolymphoid CD45 CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
67 Cytokeratins in sarcomas Synovial sarcoma angiosarcoma
68 Cytokeratins in non-epithelial tumours Leiomyosarcoma
69 Primary panel for the unknown primary tumour Haematolymphoid CD45 CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
70 Cytokeratins in malignant melanoma CK
71 Primary panel for the unknown primary tumour Haematolymphoid CD45 CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
72 Cytokeratins in germ cell tumours CK8: Seminoma CK8: Embr. carcinoma
73 Cytokeratins: retrieval causing false negativity HIER Proteolysis AE1 detects CK8 after HIER only AE1 does not detect CK18 AE3 does not detect CK8/CK18 SCLC Proteolysis CK: renal cell carcinoma
74 Cytokeratins: retrieval causing false negativity TP FN HIER Proteolysis RCC TP CK: renal cell carcinoma FN
75 Primary panel for the unknown primary tumour Haematolymphoid CD45 CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
76 S-100 protein Family of acid calcium binding proteins 9/13 kda Located in nuclei, cytoplasm and cell membranes At least 10 α-chains and one β-chain creating homo- and heterodimers S-100 β-chain mainly found in Melanocytes Glial cells Langerhans cells / interdigitating reticulum cells Fat cells Myoepithelial cells Polyclonal antibodies primarily detects the β-chain
77 S-100 protein brain chondrocytes
78 S-100 protein Tonsil
79 S-100 protein pancreas
80 S-100 in malignant tumours mal. melanoma breast carcinoma
81 S-100 protein To HIER or not.. S100B Frozen IHC Proteolytic HIER
82 Primary panel for the unknown primary tumour Real CD45 CK S-100 VIM Haematolymphoid +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
83 Vimentin Cytoplasmic intermediate filament, 57 kda Present in all mesenchymal cells Present in early stages of all cells, replaced by other intermediate filaments in most non-mesenchymal cells Coexpressed with cytokeratin in some epithelia Endometrium, renal tubules, thyroid gland Coexpressed with cytokeratin in some non-epithelial cells Mesothelium
84 Vimentin in normal tissue HE Normal brain VIM
85 Vimentin in carcinomas renal cell carcinoma endometrioid carcinoma
86 Vimentin in non-epithelial tumours mal. melanoma mal. mesothelioma
87 Secondary panels for carcinoma identification/subclassification Cytokeratin subtypes Oncofetal proteins Transcription factors Neuroendocrine proteins Hormone receptors Secretory proteins Cell adhesion molecules... Breast markers Lung markers GI-markers Fem.gen.tract markers Urinary tract markers Prostate markers Squamous cell markers Mesothelial markers NE cell markers Liver markers Adrenal cortical markers Germinal cell markers
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