Immunohistochemical classification of the unknown primary tumour

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1 Immunohistochemical classification of the unknown primary tumour NordiQC Workshop 2014 Prof. Mogens Vyberg Institute of Pathology Aalborg, Denmark PART 2 Secondary panels for carcinoma identification/subclassification Cytokeratin subtypes Cell adhesion molecules Oncofetal proteins Transcription factors Neuroendocrine proteins Hormone receptors Secretory proteins... Breast markers Lung markers GI-markers Fem.gen.tract markers Urinary tract markers Prostate markers Squamous cell markers Mesothelial markers NE cell markers Liver cell markers Adrenal cortical markers Germinal cell markers Cell adhesion molecules Epithelial cell adhesion molecule (Ep-CAM) Claudin 4 (c) Mogens Vyberg

2 Ep-CAM Glycoproteins located on the cell membrane surface (preferentially basolaterally) and in the cytoplasm of virtually all epithelial cells with the exception of squamous epithelia (mucosae focal pos.) hepatocytes (pos. with inflammation and fibrosis) renal proximal tubular cells gastric parietal cells some endocrine cells Also negative mesothelial cells (reactive pos.) mesenchymal cells germ cells neural crest cells (except. olfactory neurons) Liver Ep-CAM +(-) Adenocarcinomas (of most types) Neuroenocrine neoplasms Olfactory neuroblastoma Seminoma/Dysgerminoma Synovial sarcoma Desmoplastic small cell tumour Brenner tumour Large cell lung carcinoma Ep-CAM -/+ Lobular breast carcinoma Hepatocellular carcinoma Squamous cell carcinoma Renal cell carcinoma Malignant mesothelioma Embryonal carcinoma - Adrenal cortical carcinoma Sarcomas (apart from synovial sarcoma) Lymphomas HCC (c) Mogens Vyberg

3 Ep-CAM Undiff carcinoma Ep-CAM Choroid plexus pap. Malignant mesothelioma Yolk sac tumour EpCAM EMA (c) Mogens Vyberg

4 EpCAM protocol Colon adenocarcinoma Lab A Lab B Claudin 4 Integral membrane protein, which belongs to the claudin family. The protein is a component of tight junction strands and may play a role in internal organ development and function. Tonsil Claudin 4 Ep-CAM Claudin 4 vs. Ep-CAM Mesothelioma Claudin 4 Ep-CAM (c) Mogens Vyberg

5 Claudin 4 vs. Ep-CAM Renal clear cell carc Claudin 4 Ep-CAM Breast cancer markers Estrogen receptor GCDFP-15 Mammaglobin GATA3 Estrogen receptor alpha Estrogen sensitive tissues: Breast Ovarium Fallopian tube Endo- and myometrium Uterine cervix Thyroid gland Pituitary gland Pancreas Sweat gland Salivary gland Prostate, Liver (c) Mogens Vyberg

6 Estrogen receptor alpha Adenocarcinomas Breast carcinoma +/ Ovarian (non-muc.) +/ Endometrial carc. +/ Cervix, lung, kidney, endocrine... /+ ERα Sica et al. APLM 132,1889,2008 Estrogen receptor alpha Lung adenocarcinomas ERα Sica et al. APLM 132,1889,2008 Estrogen receptor alpha: The influence of antibody selection 92 lung adenocarcinomas ER+ ( 1% pos. cells) ERα 1D5: 8% 6F11: 14% SP1: 27% Gomez-Fernandez et al. AIMM 2010;18:137 (c) Mogens Vyberg

7 Serial sections stained for Estrogen receptor Lab. A Lab. B 19 ER Run B17/2013: 281 labs 60% optimal 12% insufficient Lab. A Lab. B 20 Serial sections stained for Estrogen receptor Control: uterine cervix Lab. A Lab. B 21 (c) Mogens Vyberg

8 Serial sections stained for Estrogen receptor Control: uterine cervix All Abs but 6F11 6F11 in 15/36 labs Lab. A Lab. B 22 GCDFP-15 (gross cystic disease fluid protein 15) Prolactin induced glycoprotein of apocrine cells Adenocarcinomas Breast +/ Sweat gland +/ Salivary gland +/ Lung (+) Renal cell (+) Ovary (+) GCDFP-15 in sweat glands GCDFP-15 Breast carcinomas (c) Mogens Vyberg

9 Gastric biopsies, 43 y ER GCDFP-15 Mammaglobin Mammary-specific member of the uteroglobin family Adenocarcinomas Breast +/ Sweat gland +/ Salivary gland +/ Lung /+ Stomach, biliary, thyroid, ovary, endom. (+) Mammaglobin in breast Mammaglobin Breast ductal carcinomas (c) Mogens Vyberg

10 61, metastatic adenocarcinoma in the eye lid 61, metastatic adenocarcinoma in the eye lid CK7 61, metastatic adenocarcinoma in the eye lid GCDFP-15 (c) Mogens Vyberg

11 61, metastatic adenocarcinoma in the eye lid: Moll gland ca. MAMMAGLOBIN GATA3 Transcription factor recognizing the G-A-T-A nucleotide sequences in target gene promoters, regulating genes involved in the development/maintenance of T-cells breast epithelium urothelium skin trophoblast endothelia GATA3 Transcription factor recognizing the G-A-T-A nucleotide sequences in target gene promoters, regulating genes involved in the development/maintenance of T-cells breast epithelium urothelium skin trophoblast endothelia (c) Mogens Vyberg

12 GATA3 Transcription factor recognizing the G-A-T-A nucleotide sequences in target gene promoters, regulating genes involved in the development/maintenance of T-cells breast epithelium urothelium skin trophoblast endothelia GATA3 + Breast carcinoma Urothelial carcinoma Skin carcinoma (squam., bas., adnex) Yolk sac tumour Lobular breast carcinoma Urothelial carcinoma Miettinen et al. Am J Surg Pathol. 2014, 38: GATA3 +/- Malignant mesothelioma Pancreas adenocarcinoma Chromophobe renal cell carcinoma Paraganglioma -/+ Non-skin squamous cell carc. Salivary gland carcinomas Adrenal cortical carcinoma Embryonal carcinoma (c) Mogens Vyberg

13 GATA3 -(+) Serous and endometrioid adenocarcinoma Colon and gastric adenocarcinoma Prostate adenocarcinoma Renal cell carcinoma (non-chromophobic) Thyroid carcinoma Sarcomas - Endocrine neoplasms Malignant melanoma Seminoma Lung markers TTF-1 Napsin Thyroid transcription factor-1 Thyroid gland: regulating thyroglobulin, thyroperoxidase and thyrotropin receptor. Lung: regulating surfactant proteins and Clara cell secretory protein. Thyroid follicles and C-cells Lung pneumocytes II and Clara cells Brain Pituitary Parathyroid Normal lung (c) Mogens Vyberg

14 TTF-1 Run 39/2013: 227 labs Optimal: 51% Insufficient: 29% Thyroid transcription factor-1 % pos.: SPT24 8G7G3/1 Lung adenocarcinoma large cell carcinoma small cell carcinoma carcinoid squamous cell carcinoma 15 0 Non-lung small cell carcinoma 20-40? Thyroid carc. (non-anaplastic) Non-lung carcinomas Non-lung carcinoids ~ 0 ~ 0 Matoso et al, AIMM 2010,18: Napsin A Aspartic proteinase Type II pneumocytes Proximal and convoluted renal tubules (Pancreatic acini and ducts) (c) Mogens Vyberg

15 Napsin A Aspartic proteinase Type II pneumocytes Proximal and convoluted renal tubules (Pancreatic acini and ducts) Napsin A: lung adenocarcinoma ~ 80% Napsin A: Lung squamous carcinoma 0 30% (c) Mogens Vyberg

16 Napsin A: Renal cell carcinoma 0 70% 49 Liver adenocarcinoma of unknown primary CK20 49 Liver adenocarcinoma of unknown primary: Lung TTF Napsin A (c) Mogens Vyberg

17 Napsin Run 39/2013: 104 labs 30% optimal 42% insufficient GI markers CDX2 CAD17 CEA SMAD4 CDX-2 protein Drosophila caudal related homeobox gene 2 product: Nuclear transcription factor for intestinal differentiation Intestine all cell types incl. endocrine Intestinal metaplasia chronic gastritis Barrett s esophagus Pancreas/bil.tract colon pancreas (c) Mogens Vyberg

18 CDX-2 protein in adenocarcinoma Colorectum +( ) Mucinous ovar. +( ) Esoph./Stom. +/ Mucinous lung +/ Pancr./biliary /+ Prostate (+) Urothelial (+) Endometrioid (+) Endocrine midgut + Yolk sac tumour + Colon adenocarcinoma CDX-2 protein in adenocarcinoma Colorectum +( ) Mucinous ovar. +( ) Esoph./Stom. +/ Mucinous lung +/ Pancr./biliary /+ Prostate (+) Urothelial (+) Endometrioid (+) Endocrine midgut + Yolk sac tumour + Colon adenoc. medullary adenoc. Colon adenosquamous carc. Endometrioid carcinoma: ER & CDX-2 ER CDX-2 (c) Mogens Vyberg

19 CDX2 Normal colon 1 EPRCON EPRRTU DAK AMT 88 CDX2 Normal pancreas EPRCON EPRRTU DAK AMT 88 CDX2 Colon adenocarc EPRCON EPRRTU DAK AMT 88 (c) Mogens Vyberg

20 Quality assurance of CDX2 detection Mean H-score and proportion of positives N EPR* CONC EPR RTU DAK- CDX2 AMT- 28 CDX2-88 High Ex % 100% 100% 98% 96% Low Ex rmab EPR2764Y (Ventana, CellMarque) 95% 48% 58% 19% 13% Borrisholt M, Nielsen S, Vyberg M. Appl Immunohistochem Mol Morphol Jan;21(1): Cadherin 17 Calcium dependent adhesion molecules CAD17 = Liver-Intestine (LI-) Cadherin Regulated by CDX2 Intestine, pancreas Cadherin 17 Adenocarcinoma, colon + (100%) Adenocarc., esophagus, stomach, pancreas biliary tract +/ (often heterogenous) Adenocarc., lung, endometrium, ovary, breast (+) (focal) Endocrine tumour, small intest + (100%) Endocrine tumour, lung, pancr. /+ Squamous cell carc. (+) (c) Mogens Vyberg

21 CAD17 Appendix Cadherin 17: rmab SP183, CM, 1:50, CC1M/16M/UV CAD17 and CDX2 Colon adenocarcinoma Cadherin 17: rmab SP183, CM, 1:50, CC1M/16M/UV CDX2: rmab EPR2764Y, CM Carcinoembryonic antigen (CD66e) Adhesion molecule espc. associated with intestine (c) Mogens Vyberg

22 Carcinoembryonic antigen (CD66e) in adenocarcinomas Colorectal + Medull. thyroid + Pancreas/biliary tract +/ Stomach +/ Lung +/ Ovary, mucinous +/ Ovary, non-muc. /+ Prostate Kidney Liver metast. colon adenoc Carcinoembryonic antigen Medul. thyroid carc. Breast ductal carc. Carcinoembryonic antigen which antibody? Mal. mesothelioma Lab A Lab B (c) Mogens Vyberg

23 SMAD4 Ep-CAM Similar to Mothers Against Drosophila 4 = Deleted in pancreatic cancer-4 (DPC4) Nuclear transcription activator in all normal cells Pancreatico-biliary carcinomas ~50% Colorectal carcinomas ~10% Other carcinmas: rare 53 liver with ACUP 53 liver with ACUP CDX2 SMAD4 (c) Mogens Vyberg

24 SMAD4 loss in pancreatic and ampullary carcinomas Ep-CAM Female genital tract markers WT1 PAX8 ER HNFB1 Wilms tumour-1 (WT1) protein Transcription factor for development of the genitourinary system Mesothelium Ovary (surface / inclusion cysts) Fallopian tube Female genital tract: stromal cells Bone marrow stem cells Fallopian tube (c) Mogens Vyberg

25 Wilms tumour-1 (WT1) protein in adenocarcinomas Serous carcinoma + Endometrioid carcinoma +/ Other carcinomas (+) Mal. mesothelioma +/ Serous carcinoma PAX8 Transcription factor crucial to the organogenesis and development of Urogenital tract Adult genital tract Thyroid (Neuroendocrine system) PAX8 Ovary - serous carcinoma LG + - serous carcinoma HG / clear cell + - endometrioid carcinoma +/- - mucinous carcinoma -(+) Kidney - clear cell / papillary carcinoma + - chromophobe / collect. duct carc. + - RCC, sarcomatoid +/- Thyroid carcinoma + Other carcinomas -(+) Malignant mesothelioma - (c) Mogens Vyberg

26 PAX8 Run 34/2012: 36 labs Tube RCC Optimal: 26% Insufficient: 36% 48 mediastinal lymph node metastasis Case 2 mediastinal lymph node metastasis Pan-CK CK7 (c) Mogens Vyberg

27 Case 2 Metastatic clear cell renal cell carcinoma VIM PAX8 Markers for endocrine tumours Neuroendocrine Synaptophysin Chromogranin A Thyroid Thyroglobulin Liver Arginase CD66a/CD10 Glypican 3 Adrenal cortex, sex cord Calretinin Inhibin Melan A (A 103) Hormones... Unspecific NE: (PGP 9.5) (neuron specific enolase) (CD56) Synaptophysin An integral-membrane glycoprotein of presynaptic vesicles Neurons Neuroendocrine cells (Adrenal cortical cells) SYP 81 pancreas (c) Mogens Vyberg

28 Synaptophysin in neoplasias Neural tumours NE tumours NE carcinomas Adrenal cortical tumours Lung NE large cell carcinoma 82 Synaptophysin in neoplasias SCLC in skin 83 Adrenal cortical carcinoma Synaptophysin Run 37/2013: 214 labs. NE gr. 2 Optimal: 19% Insufficient: 42% SCLC (c) Mogens Vyberg

29 Mesothelial markers Calretinin Podoplanin Calretinin Calcium-binding protein related to S-100 proteins Neurons Mesothelial cells Steroid producing cells adrenal cortical cells testicular Leydig and Sertoli cells ovarian theca interna cells and surface epithelium Neuroendocrine cells Breast glands... Calretinin Mesothelium Appendix (c) Mogens Vyberg

30 Calretinin in neoplasms Malignant mesothelioma Malignant mesothelioma Calretinin Adrenal gland Granulosa cell tumour Podoplanin Lymphatic endothelium Fibroblasts, osteocytes Smooth and striated muscle cells Myoepithelial cells, Cajal cells Basal squamous epithelial cells Gastric crypt c., prostatic basal c. Immature Sertoli c. and gonocytes Renal glomerular podocytes Mesothelium (reactive) Follicular dendritic cells Some lymphocytes Glial/Schwann cells (c) Mogens Vyberg

31 Podoplanin Mesothelioma Many other tumours but rarely adenocarcinomas Malignant mesothelioma vs. lung carcinoma Claudin Ep- P63 WT1 CR PDP VIM 4 CAM CEA NAPA TTF-1 p40 CK5 Ep. malignant mesothelioma (f) Lung adenocarcinoma 0 10 (f) 10 (f ) 30 5? (5) Lung squamous cell carcinoma 0 50? 10 10? ? 10f Liver cell markers Glypican 3 Arginase cancd66a (biliary glycoprotein) Hepatocyte antigen Glutamine synthetase cancd10 canvillin AFP (c) Mogens Vyberg

32 Glypican 3 Heparan sulphate proteoglycan modifying cell signaling Yolk sac tumour + Choriocarcinoma + Embryonal carc. /+ Hepatoblastoma + Hepatocellular carc. +/ Ovarian clear cell c. /+ Carcinoid a.o. /+ HCC Glypican 3 Heparan sulphate proteoglycan modifying cell signaling Yolk sac tumour + Choriocarcinoma + Embryonal carc. /+ Hepatoblastoma + Hepatocellular carc. +/ Ovarian clear cell c. /+ Carcinoid a.o. /+ Teratoma with Emb. carc. Arginase I The final enzyme of the urea cycle: arginine + H2O ornithine + urea Predominantly located in liver cells Hepatocellular carcinoma +/ (80-90%) Adenocarcinoma, biliary tract, pancreas /+ Adencarcinoma breast, colorectum (+) (c) Mogens Vyberg

33 Arg and hepa Hepatocellular carcinoma I rmab SP156 1:25 CM 16M/CC1S/UV mab OCH1E5 1:400 Dako 32M/CC1S/UV+amp Arg and Hepa Pancreas adenocarcinoma rmab SP156 1:25 CM 16M/CC1S/UV mab OCH1E5 1:400 Dako 32M/CC1S/UV+amp CD66a Biliary glycoprotein CEA-like adhesion molecule Canalicular staining! HCC (c) Mogens Vyberg

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