Immunomarkers of the GI Tract
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Immunomarkers of the GI tract Prof. Mogens Vyberg NordiQC Institute of Pathology Aalborg University Hospital Aalborg, Denmark • Nothing to declare GI carcinoma of uncertain origin “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 colon . Intestinal metaplasia . chronic gastritis . Barrett’s esophagus . Pancreas/bil.tract pancreas CDX-2 protein in adenocarcinoma . Colorectum +(–) . Mucinous ovar. +(–) . Esoph./Stom. +/– . Mucinous lung +/– . Pancr./biliary –/+ . Prostate –(+) . Urothelial –(+) . Endometrioid –(+) . Yolk sac tumour + Colon adenocarcinoma CDX-2 protein in adenocarcinoma . Colorectum +(–) . Mucinous ovar. +(–) . Esoph./Stom. +/– . Mucinous lung +/– . Pancr./biliary –/+ ColonColon adenoc.medullary adenoc. Prostate –(+) . Urothelial –(+) . Endometrioid –(+) . Yolk sac tumour + Colon adenosquamous carc. Endometrioid carcinoma: ER & CDX-2 ER CDX-2 CDX2 in endocrine tumours . Foregut –(+) Stomach, lung . Midgut + . Hindgut –/+ Ileum endocrine tumour grade 3 CDX2: Selection of antibodies 2009 CDX2: Selection of antibodies 2013 RTU CDX2 antibody selection Mean H-score and proportion of positives N EPR* EPR* DAK- AMT- CDX2- CONC RTU CDX2 28 88 High Ex 54 265 240 234 171 155 100% 100% 100% 98% 96% Low Ex 52 55 27 27 8 5 95% 48% 58% 19% 13% *rmAb EPR2764Y (Ventana, CellMarque) High Expressor : 150 – 300 with best Ab Borrisholt M, Nielsen S, Vyberg M. Low Expressor : 10 – 150 with best Ab AIMM 2013 Jan;21(1):64-72. CDX2 Normal colon 1 EPRCON EPRRTU DAK AMT 88 CDX2 Normal colon 2 EPRCON EPRRTU DAK AMT 88 CDX2 Normal pancreas EPRCON EPRRTU DAK AMT 88 CDX2 Colon adenocarc.1 EPRCON EPRRTU DAK AMT 88 Cadherin 17 . Calcium dependent adhesion molecules . CAD17 = Liver-Intestine (LI-) Cadherin . Regulated by CDX2 . Intestine (uniform) . Pancreas/biliary tract (heterogenous) Cadherin 17 + Adenocarcinoma, colon (incl. medullary MMR prot. def.) Endocrine neoplasm, small intestine +/- or -/+ Adenocarcinomas of esophagus, stomach, pancreas / biliary tract -(+) Adenocarcinomas of lung, endometrium, ovary, breast Endocrine neoplasms of lung and pancreas Squamous cell carcinoma CAD17 – Appendix Cadherin 17: rmAb SP183, CM, 1:50, CC1M/16M/UV CAD17 – Pancreas Cadherin 17: rmAb SP183, CM, 1:50, CC1M/16M/UV CAD17 and CDX2 – Colon adenocarcinoma Cadherin 17: rmAb SP183, CM, 1:50, CDX2: CC1M/16M/UV rmAb EPR2764Y, CM CAD17 and CDX2 – Colon adenocarcinoma Cadherin 17: rmAb SP183, CM, 1:50, CDX2: CC1M/16M/UV rmAb EPR2764Y, CM CAD17 and CDX2 – Colon adenocarcinoma Cadherin 17: rmAb SP183, CM, 1:50, CDX2: CC1M/16M/UV rmAb EPR2764Y, CM CAD17 and CDX2 – Pancreas adenocarcinoma Cadherin 17: rmAb SP183, CM, 1:50, CDX2: CC1M/16M/UV rmAb EPR2764Y, CM Carcinoma in colon mucosa – primary or secondary? CDX2 – Colon adenocarcinoma CAD17 – Colon adenocarcinoma Carcinoembryonic antigen (CD66e) . Adhesion molecule espc. associated with intestine Carcinoembryonic antigen (CD66e) in adenocarcinomas . Colorectal + . Medull. thyroid + . Pancreas/biliary tract +/– . Stomach +/– . Lung +/– . Ovary, mucinous +/– . Ovary, non-muc. –/+ . Prostate – . Kidney – . Liver (!) – . Mesothelioma (!) – metast. colon adenoc Carcinoembryonic antigen Medul. thyroid carc. Breast ductal carc. Carcinoembryonic antigen – which antibody 2009 Carcinoembryonic antigen – which antibody 2013 RTUs Clone II-7 Clone TF3H8-1 Carcinoembryonic antigen – which antibody? Mal. mesothelioma mAb II-7 pAb or TF3H8-1 Carcinoembryonic antigen Malignant mesothelioma No cross reaction Cross reaction Normal liver SMAD4 EpCAM Similar to Mothers Against Drosophila 4 = Deleted in pancreatic cancer-4 (DPC4) Nuclear transcription activator in all normal cells . Deleted in ~ 50% of pancreatic carcinomas . Deleted much less frequently in other carcinomas ♂ 53 – liver with adenocarcinoma of unknown primary ♂ 53 – liver with adenocarcinoma of unknown primary CDX2 SMAD4 SMAD4 loss in pancreaticobiliary carcinomas EpCAM “Glands” in pancreatic nerve SMAD4 loss in “glands” in panceatic nerve Atrophic pancreas Liver tumour of unknown origin Atrophic pancreas Hepatocellular carcinoma Glypican 3 Atrophic pancreasArginase Hepatocellular carcinoma CD66a (biliary glycoprotein) CDX2 Atrophic pancreas Hepatocellular carcinoma CDX2 in HCC Not previously reported Pancreas control CDX2 Atrophic pancreas Hepatocellular carcinoma CAD17 in HCC reported in 6% Cadherin 17 Atrophic pancreas GI tumours of uncertain origin Carcinoma or sarcoma? General epithelial markers: Epithelial cell adhesion molecule (EpCAM) Claudin 4 [Cytokeratins] EpCAM “Epithelial specific antigen”: 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 and reactive focal pos.) . hepatocytes (pos. in reactive) . renal proximal tubular cells . gastric parietal cells . some endocrine cells Also negative . mesothelial cells (pos. in reactive) . mesenchymal cells . neural crest cells (olfactory neurons are pos.) Liver EpCAM +(-) .Adenocarcinomas (of most types) .Neuroendocrine neoplasms +/- .Synovial sarcoma .Brenner tumour .Desmoplastic small cell tumour .Olfactory neuroblastoma Endocrine carcinoma . EpCAM -/+ .Lobular breast carcinoma .Hepatocellular carcinoma .Squamous cell carcinoma .Renal cell carcinoma .Malignant mesothelioma .Embryonal carcinoma, seminoma - .Adrenal cortical carcinoma .Choroid plexus pap./carcinoma .Sarcomas (synovial & epithelioid sarc. +) .Lymphomas HCC EpCAM in low differentiated carcinoma EpCAM Malignant mesothelioma EpCAM protocol Colon adenocarcinoma (Ber-EP4) Lab A Lab B Retrieval: TRS ph 6.1 DAKO Diva2 pH 6.2 Thermo EpCAM antibodies – renal cell carcinoma SPM491 Ber-EP4 Ab71916 EpCAM antibodies – glioblastoma SPM491 Ab71916 Ab71916 optimized published 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. Not found in mesothelioma Claudin 4 EpCAM Tonsil Claudin 4 vs. EpCAM Mesothelioma Claudin 4 EpCAM GI tumours of uncertain origin - stomach Carcinoma or sarcoma? GIST ASMA CD117 EQA of MMR protein stains MLH1 MLH1 . Improper calibration of Ab titre . Less succesful Ab clone G168-728 (8/9 insuff.) . Less sensitive detection systems: 3-step polymer 2-step polymer MLH1 MLH1 loss False neg. internal control 3-step polymer 2-step polymer MLH1 MLH1 loss False neg. internal control Overstained - FP MSH2 MSH2 . Improper calibration of Ab titre . Insufficient HIER . Poor Ab clone 25D12 (17/17 insuff.): 25D12 – combined false negative and positive MSH6 Optimal Too dilute Ab comp. with Tyramid amplification Clone 44 False pos. and neg.: 38/49 insuff. PMS2 PMS2 . Too low concentration of the primary antibody . Insufficient heat induced epitope retrieval . Use of less sensitive detection systems: 3-step polymer 2-step polymer Short fixation sensitivity Short fixation sensitivity Clone Conc. Protocol RTU Protocol Fix sens. Dako 2- & 3-step MLH1 ES05 1:20-100 3-step (+A) Yes Leica 3-step M1 - - Ventana 3-step+A ? MSH2 FE11 1:10-150 3-step Dako 2- & 3-step ? G219-1129 1:50-800 3-step Ventana 3-step No PMS2 EP51 1:20-50 3-step (+A) Dako 2- & 3-step Yes ERP3947 1:75-300 3-step (+A) Ventana 3-step Yes . Dako: HIER TRS High pH 20M, FLEX / FLEX+ . Leica: HIER ER2 20M, Refine-DAB . Ventana: HIER CC1 48-64M, OptiView (+Amplification) Delayed fixation 3 cm lymph node not grossed CD10 – 56C6 .1 h .16 h .3 cm Immunomarkers of the GI tract Thank you for your attention Prof. Mogens Vyberg NordiQC Institute of Pathology Aalborg University Hospital Aalborg, Denmark .