The unknown primary tumour: IHC classification part I, the primary panel - Antibody selection, protocol optimization, controls and EQA
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1 The unknown primary tumour: IHC classification part I, Mogens Vyberg Professor of Clinical Pathology Director of NordiQC Aalborg University Hospital, Aalborg, Denmark the primary panel - Antibody selection, protocol optimization, controls and EQA
2 Tumours of unknown origin: Histology Brain tumour - biopsy
3 CD45 VIM Tumours of unknown origin: Immunohistochemistry Pan-CK S-100
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11 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 ~50% GIST: S-100B Protease ~50% GIST: S-100B MWO
12 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
13 Primary panel for the unknown primary tumour Haematolymphoid CD45 Pan-CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
14 CD45 - Leucocyte common antigen (LCA) Transmembrane protein tyrosin phosphatase essential for haematopoietic signal transduction and cell activation Membrane associated component: 5 isotypes Intracellular component: one common type
15 CD45 - Leucocyte common antigen (LCA) Large majority of haematolymphoid cells and Lost in maturing erythocytes, megakaryocytes and plasmacells Never found in non-haematolymphoid cells and
16 CD45 - Leucocyte common antigen (LCA) Normal lymph node Malignant lymphoma
17 CD45 - Leucocyte common antigen (LCA) Liver Brain Kupffer cells: Critical assay performance control
18 CD45 NordiQC run assessment-cd45.htm Which is best?
19 CD45 NordiQC run assessment-cd45.htm Optimal Insufficient
20 Optimal Insufficient CD45 NordiQC run 37 B-CLL assessment-cd45.htm 56% of labs 18% of labs
21 CD45 NordiQC run % 82%
22 CD45 NordiQC run % 82% They followed the vendor s protocol recommendation: no HIER
23 CD45 - Leucocyte common antigen (LCA) CD45 RO ~ T-cells Lymph node/tonsil CD45 RA ~ B-cells
24 CD45 - Leucocyte common antigen (LCA)
25 CD45 - Leucocyte common antigen (LCA)
26 CD45 - Leucocyte common antigen (LCA)
27 Primary panel for the unknown primary tumour Haematolymphoid CD45 Pan-CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
28 Cellular filaments # Microfilaments: (6 nm) # Intermediate filaments (7-11 nm) # Microtubuli (23 nm)
29 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
30 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
31 Cytokeratins as tonofilaments Cytokeratin intermediate filaments attached to desmosomes Drochmans et al. J Cell Biol. 1978, 79:427
32 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
33 HE CK Micrometastases identified by cytokeratin
34 HE-frys CK-frys Carcinoma in frosen section identified by cytokeratin
35 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
36 Primary panel for the unknown primary tumour Haematolymphoid CD45 Pan-CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
37 Cytokeratins in non-epithelial tumours CK8 CD20 & CD3 42 y, tumour infiltrating retroperitoneum Malignant lymphoma! CD45 & CD79a
38 Primary panel for the unknown primary tumour Haematolymphoid CD45 Pan-CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
39 Cytokeratins in malignant mesothelioma CK8 CK5
40 Primary panel for the unknown primary tumour Haematolymphoid CD45 Pan-CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
41 Cytokeratins in sarcomas Synovial sarcoma angiosarcoma
42 Primary panel for the unknown primary tumour Haematolymphoid CD45 Pan-CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
43 Cytokeratins in malignant melanoma CK
44 Primary panel for the unknown primary tumour Haematolymphoid CD45 Pan-CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
45 Cytokeratins in germ cell tumours CK8: Seminoma CK8: Embr. carcinoma
46 Cytokeratins: proteolysis causes false negativity TP FN HIER Proteolysis PAN-CK - AE1/AE3 clone cocktail: AE1 detects CK8 after HIER only AE1 does not detect CK18 AE3 neither detects CK8 or CK18 SCLC Proteolysis CK: renal cell carcinoma
47 Cytokeratins: retrieval causing false negativity TP FN HIER Proteolysis RCC TP CK: renal cell carcinoma FN
48 Misleading datasheets Giving false negative results when only LMW-CKs are present 48
49 Misleading datasheets
50 IHC - NordiQC 2016 AE1/AE3 : Optimal results only obtained by HIER in NordiQC runs AE1/AE3/PCK26: Optimal results mainly obtained by HIER+protelysis Dako: RTU HIER Leica: RTU Proteolysis Thermo: Conc: Proteolysis or HIER Conc: HIER Conc: HIER Quanto Proteolysis UltraVision Misleading data sheets + Wrong control material used!
51 Improved datasheets By 17 th October 2014
52 NordiQC run Fra: Galloway, Mary Sendt: 13. november :14 Til: Søren Nielsen / Region Nordjylland Emne: RE: Changes Made to Package Inserts Sören, Thanks for identifying and alerting us to the issues with...anti-pan Keratin. The package inserts are now changed (see links below). I hope we can continue to learn of any future staining problems you may uncover. Much appreciated! False positive: EP700Y Mary RCC TP FN
53 IHC - NordiQC 2016
54 IHC Controls and CSQI for the primary panel Liver Leica: AE1 (CK19) + AE3 (CK8) 1:10? Dako: AE1 (CK19) + AE3 (CK8) 1:3? AE1AE3, Leica, Enzyme 1, 5 min - Bond AE1AE3, Leica ER 2, 20 min. - Bond AE1AE3, Dako, ER 2, 20 min - Bond 54 Ref.: AE1AE3, Dako BenchMark Ultra
55 Primary panel for the unknown primary tumour Haematolymphoid CD45 CK S-100 VIM +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
56 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
57 S-100 protein Tonsil
58 S-100 protein pancreas
59 S-100 in malignant tumours >90% +++ mal. melanoma breast carcinoma
60 S100 run : 299 labs Conc.+RTU 117 labs
61 Primary panel for the unknown primary tumour Real CD45 CK S-100 VIM Haematolymphoid +/(-) -/(+) -/(+) +/(-) Epithelial - +/(-) -/+ -/+ Mesothelial Mesenchymal and neuronal Non-neuronal neuroepithelial - -/(+) -/ /(+) + + Germ cell - -/+ -/+ +
62 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
63 Vimentin in normal tissue HE Normal brain VIM
64 Vimentin in carcinomas renal cell carcinoma endometrioid carcinoma
65 Vimentin in non-epithelial tumours mal. melanoma mal. mesothelioma
66 Vimentin run : 308 labs RTUs 159 labs
67 The unknown primary tumour: IHC classification part I, Mogens Vyberg Professor of Clinical Pathology Director of NordiQC Aalborg University Hospital, Aalborg, Denmark the primary panel - Antibody selection, protocol optimization, controls and EQA
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