The unkown primary tumour: IHC Classification, antibody selection, protocol optimization, controls and EQA (part II)
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1 The unkown primary tumour: IHC Classification, antibody selection, protocol optimization, Mogens Vyberg Professor of Clinical Pathology Director of NordiQC Aalborg University Hospital, Aalborg, Denmark controls and EQA (part II)
2 Secondary panels for carcinoma identification/subclassification Cell adhesion molecules EpCAM Claudin 4 Cytokeratin subtypes Oncofetal proteins Transcription factors Neuroendocrine proteins Hormone receptors Secretory proteins... GI-markers Fem.gen.tract markers Liver cell markers Neuroendocrine cell markers Breast markers Lung markers Urinary tract markers Prostate markers Squamous cell markers Mesothelial markers Adrenal cortical markers Germinal cell markers
3 General epithelial markers: Epithelial cell adhesion molecule (Ep-CAM) Claudin 4
4 Ep-CAM 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 germ cells neural crest cells (except. olfactory neurons) Liver
5 Ep-CAM Often upgraded in malignancies +(-) Adenocarcinomas (of most types) Neuroendocrine neoplasms +/- Germinal cell tumours (seminoma weak) Synovial sarcoma Brenner tumour Desmoplastic small cell tumour Olfactory neuroblastoma SYP EpCAM Olfactory neuroblastoma
6 Ep-CAM -/+ Lobular breast carcinoma Hepatocellular carcinoma Squamous cell carcinoma Renal cell carcinoma Malignant mesothelioma - Adrenal cortical carcinoma Choroid plexus carcinoma Sarcomas (apart from synovial) Lymphomas HCC
7 Ep-CAM Undiff carcinoma
8 Ep-CAM run DAKO TRS Low 3-step polymer Ventana CC1 2-step multimer
9 Diva2 ph6.2 Thermo RCC DAKO TRS Low 3-step polymer Ventana CC1 2-step multimer
10 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. Claudin 4 Ep-CAM Tonsil
11 Claudin 4 vs. Ep-CAM Mesothelioma Claudin 4 Ep-CAM
12 Cytokeratins a multigene family Highly complex family of intermediate filaments > 50 distinct types (excl. trichocytic keratins) Types CK1-20 diagnostically most relevant: Class I (type A - Acidic): CK9 (64 kda) - CK20 (40 kda) Class II (type B - Basic/neutral): CK1 (68 kda) - CK8 (52.5 kda)
13 Pairing of cytokeratins Modified from: Lane & Alexander Semin Cancer Biol 1990, 1:165 One CK class I and one CK class II always paired CK class I in a pair ~ 8 kda smaller than class II
14 Cytokeratin types and cell types Neutral/Basic (B, class II) Acidic (A, class I) Squamous epithelia: - suprabasal, keratinizing (+) suprabasal, non-keratinizing (+) basal cells ( 1 tonsil, 2 mucosa) (+) 1 (++) 12 (+) 1 - (+) 1 Transit. epith.: superficial cells intermed. / 3 basal cells - (+) (+++) 3 - (++) (+) +++ Mesothelium Bronchus, breast, prost., cerv.: - basal/myoepithelial cells luminal cells Biliary/pancr. ducts, lung alv., endometr., renal collect. ducts Stomach (foveola), intestine (+) Hepatocytes, pancr. acini, prox. renal tubules Endocrine cells ( 5 Merkel, 6 thyr.) (+++) 5 (++) 6 (+++) Smooth muscle (vasc., myom.), myofibrobl., 7 sm.ves.endothelia (++)
15 HMW Cytokeratin types - squamous epithelia Neutral/Basic (B, class II) Acidic (A, class I) Squamous epithelia: - suprabasal, keratinizing (+) suprabasal, non-keratinizing (+) basal cells ( 1 tonsil, 2 mucosa) (+) 1 (++) 12 (+) 1 - (+) 1 Transit. epith.: superficial cells intermed. / 3 basal cells - (+) (+++) 3 - (++) (+) +++ Mesothelium Bronchus, breast, prost., cerv.: - basal/myoepithelial cells luminal cells Biliary/pancr. ducts, lung alv., endometr., renal collect. ducts Stomach (foveola), intestine (+) Hepatocytes, pancr. acini, prox. renal tubules Endocrine cells ( 5 Merkel, 6 thyr.) (+++) 5 (++) 6 (+++) Smooth muscle (vasc., myom.), myofibrobl., 7 sm.ves.endothelia (++)
16 HMW Cytokeratin types - squamous epithelia CK5: esophagus CK4: esophagus
17 Cytokeratin types - urothelium Neutral/Basic (B, class II) Acidic (A, class I) Squamous epithelia: - suprabasal, keratinizing (+) suprabasal, non-keratinizing (+) basal cells ( 1 tonsil, 2 mucosa) (+) 1 (++) 12 (+) 1 - (+) 1 Transit. epith.: superficial cells intermed. / 3 basal cells - (+) (+++) 3 - (++) (+) +++ Mesothelium Bronchus, breast, prost., cerv.: - basal/myoepithelial cells luminal cells Biliary/pancr. ducts, lung alv., endometr., renal collect. ducts Stomach (foveola), intestine (+) Hepatocytes, pancr. acini, prox. renal tubules Endocrine cells ( 5 Merkel, 6 thyr.) (+++) 5 (++) 6 (+++) Smooth muscle (vasc., myom.), myofibrobl., 7 sm.ves.endothelia (++)
18 Cytokeratin types - urothelium CK5 (+CK13) CK20
19 Cytokeratin types - mesothelium Neutral/Basic (B, class II) Acidic (A, class I) Squamous epithelia: - suprabasal, keratinizing (+) suprabasal, non-keratiniz.ep (+) basal cells ( 1 tonsil, 2 mucosa) (+) 1 (++) 12 (+) 1 - (+) 1 Transit. epith.: superficial cells intermed. / 3 basal cells - (+) (+++) 3 - (++) (+) +++ Mesothelium Bronchus, breast, prost., cerv.: - basal/myoepithelial cells luminal cells Biliary/pancr. ducts, lung alv., endometr., renal collect. ducts Stomach (foveola), intestine (+) Hepatocytes, pancr. acini, prox. renal tubules Endocrine cells ( 5 Merkel, 6 thyr.) (+++) 5 (++) 6 (+++) Smooth muscle (vasc., myom.), myofibrobl., 7 sm.ves.endothelia (++)
20 Cytokeratin types - mesothelium CK19: mesothelium CK5: mesothelium (adenocarcinoma neg.)
21 Cytokeratin types - complex epithelia Neutral/Basic (B, class II) Acidic (A, class I) Squamous epithelia: - suprabasal, keratinizing (+) suprabasal, non-keratinizing (+) basal cells ( 1 tonsil, 2 mucosa) (+) 1 (++) 12 (+) 1 - (+) 1 Transit. epith.: superficial cells intermed. / 3 basal cells - (+) (+++) 3 - (++) (+) +++ Mesothelium Bronchus, breast, prost., cerv.: - basal/myoepithelial cells luminal cells Biliary/pancr. ducts, lung alv., endometr., renal collect. ducts Stomach (foveola), intestine (+) Hepatocytes, pancr. acini, prox. renal tubules Endocrine cells ( 5 Merkel, 6 thyr.) (+++) 5 (++) 6 (+++) Smooth muscle (vasc., myom.), myofibrobl., 7 sm.ves.endothelia (++)
22 Cytokeratin types - complex epithelia CK5 (14,17): breast CK18 (8,7): breast
23 Cytokeratin types - simple epithelia Neutral/Basic (B, class II) Acidic (A, class I) Squamous epithelia: - suprabasal, keratinizing (+) suprabasal, non-keratinizing (+) basal cells ( 1 tonsil, 2 mucosa) (+) 1 (++) 12 -(+) 1 - -(+) 1 Transit. epith.: superficial cells intermed. / 3 basal cells - (+) (+++) 3 - (++) (+) +++ Mesothelium Bronchus, breast, prost., cerv.: - basal/myoepithelial cells luminal cells Biliary/pancr. ducts, lung alv., endometr., renal collect. ducts Stomach (foveola), intestine (+) Hepatocytes, pancr. acini, prox. renal tubules Endocrine cells ( 5 Merkel, 6 thyr.) (+++) 5 (++) 6 (+++) Smooth muscle (vasc., myom.), myofibrobl., 7 sm.ves.endothelia (++)
24 Cytokeratin types - simple epithelia (liver, kidney) CK7(19): bile ducts CK7(19): renal col.
25 Cytokeratin types - simple epithelia Neutral/Basic (B, class II) Acidic (A, class I) Squamous epithelia: - suprabasal, keratinizing (+) suprabasal, non-keratinizing (+) basal cells ( 1 tonsil, 2 mucosa) (+) 1 (++) 12 -(+) 1 - -(+) 1 Transit. epith.: superficial cells intermed. / 3 basal cells - (+) (+++) 3 - (++) (+) +++ Mesothelium Bronchus, breast, prost., cerv.: - basal/myoepithelial cells luminal cells Biliary/pancr. ducts, lung alv., endometr., renal collect. ducts Stomach (foveola), intestine (+) Hepatocytes, pancr. acini, prox. renal tubules Endocrine cells ( 5 Merkel, 6 thyr.) (+++) 5 (++) 6 (+++) Smooth muscle (vasc., myom.), myofibrobl., 7 sm.ves.endothelia (++)
26 Cytokeratin types - simple epithelia CK20: gastric foveolae CK20: colon
27 Cytokeratin types - simple epithelia Neutral/Basic (B, class II) Acidic (A, class I) Squamous epithelia: - suprabasal, keratinizing (+) suprabasal, non-keratinizing (+) basal cells ( 1 tonsil, 2 mucosa) (+) 1 (++) 12 -(+) 1 - -(+) 1 Transit. epith.: superficial cells intermed. / 3 basal cells - (+) (+++) 3 - (++) (+) +++ Mesothelium Bronchus, breast, prost., cerv.: - basal/myoepithelial cells luminal cells Biliary/pancr. ducts, lung alv., endometr., renal collect. ducts Stomach (foveola), intestine (+) Hepatocytes, pancr. acini, prox. renal tubules Endocrine cells ( 5 Merkel, 6 thyr.) (+++) 5 (++) 6 (+++) Smooth muscle (vasc., myom.), myofibrobl., 7 sm.ves.endothelia (++)
28 Cytokeratin types - simple epithelia CK8: liver CK8: kidney
29 Cytokeratin types - simple epithelia Neutral/Basic (B, class II) Acidic (A, class I) Squamous epithelia: - suprabasal, keratinizing (+) suprabasal, non-keratinizing (+) basal cells ( 1 tonsil, 2 mucosa) (+) 1 (++) 12 -(+) 1 - -(+) 1 Transit. epith.: superficial cells intermed. / 3 basal cells - (+) (+++) 3 - (++) (+) +++ Mesothelium Bronchus, breast, prost., cerv.: - basal/myoepithelial cells luminal cells Biliary/pancr. ducts, lung alv., endometr., renal collect. ducts Stomach (foveola), intestine (+) Hepatocytes, pancr. acini, prox. renal tubules Endocrine cells ( 5 Merkel, 6 thyr.) (+++) 5 (++) 6 (+++) Smooth muscle (vasc., myom.), myofibrobl., 7 sm.ves.endothelia (++)
30 Cytokeratin types - simple epithelia CK20: Merkel cells CK8: Pancreas Islet of Langerhans
31 Cytokeratin types - mesenchymal cells Neutral/Basic (B, class II) Acidic (A, class I) Squamous epithelia: - suprabasal, keratinizing (+) suprabasal, non-keratinizing (+) basal cells ( 1 tonsil, 2 mucosa) (+) 1 (++) 12 -(+) 1 - -(+) 1 Transit. epith.: superficial cells intermed. / 3 basal cells - (+) (+++) 3 - (++) (+) +++ Mesothelium Bronchus, breast, prost., cerv.: - basal/myoepithelial cells luminal cells Biliary/pancr. ducts, lung alv., endometr., renal collect. ducts Stomach (foveola), intestine (+) Hepatocytes, pancr. acini, prox. renal tubules Endocrine cells ( 5 Merkel, 6 thyr.) (+++) 5 (++) 6 (+++) Smooth muscle (vasc., myom.), myofibrobl., 7 sm.ves.endothelia (++)
32 Cytokeratin types - mesenchymal cells CK7: endothelium CK8: lymph node fibroblastic reticulum cells
33 Cytokeratins in epithelial neoplasias Neutral/Basic (B, class II) Acidic (A, class I) Squamous cell carcinoma (+) (+) (+) (+) - (+) Transitional cell tumour Malignant mesothelioma Adenocarcinoma: complex epithelia (lung, breast) (+) (+) (+) Adenocarc.: biliary tract, pancr., endom., ovary (+) (+) (+) (+) ++ Adenocarc.: stomach Adenocarc.: intestine Hepatocellular carcinoma Renal cell carcinoma Endocrine tumours: carcinoids Merkel cell carcinoma Thyroid carcinoma
34 Cytokeratins in squamous cell carcinoma Neutral/Basic (B, class II) Acidic (A, class I) Squamous cell carcinoma Transitional cell tumour Malignant mesothelioma Adenocarcinoma: complex epithelia (lung, breast) (+) (+) (+) Adenocarc.: biliary tract, pancr., endom., ovary (+) (+) (+) (+) ++ Adenocarc.: stomach Adenocarc.: intestine Hepatocellular carcinoma Renal cell carcinoma Endocrine tumours: carcinoids Merkel cell carcinoma Thyroid carcinoma
35 Cytokeratins in squamous cell carcinoma CK5 CK4 esophagus
36 Cytokeratins in urothelial carcinoma Neutral/Basic (B, class II) Acidic (A, class I) Squamous cell carcinoma Transitional cell tumour ! +! Malignant mesothelioma Adenocarcinoma: complex epithelia (lung, breast) (+) (+) (+) Adenocarc.: biliary tract, pancr., endom., ovary (+) (+) (+) (+) ++ Adenocarc.: stomach Adenocarc.: intestine Hepatocellular carcinoma Renal cell carcinoma Endocrine tumours: carcinoids Merkel cell carcinoma Thyroid carcinoma
37 Cytokeratins in urothelial carcinoma CK20 CK5 CK20 CK5
38 Cytokeratins in malignant mesothelioma Neutral/Basic (B, class II) Acidic (A, class I) Squamous cell carcinoma (+) (+) (+) (+) - (+) Transitional cell tumour Malignant mesothelioma Adenocarcinoma: complex epithelia (lung, breast) (+) (+) (+) Adenocarc.: biliary tract, pancr., endom., ovary (+) (+) (+) (+) ++ Adenocarc.: stomach Adenocarc.: intestine Hepatocellular carcinoma Renal cell carcinoma Endocrine tumours: carcinoids Merkel cell carcinoma Thyroid carcinoma
39 Cytokeratins in malignant mesothelioma CK5 CK5
40 Cytokeratins in adenocarcinomas Neutral/Basic (B, class II) Acidic (A, class I) Squamous cell carcinoma (+) (+) (+) (+) - (+) Transitional cell tumour Malignant mesothelioma Adenocarcinoma: complex epith. (lung, breast) (+) (+) (+) Adenocarc.: biliary tract, pancr., endom., ovary (+) (+) (+) (+) ++ Adenocarc.: stomach Adenocarc.: intestine Hepatocellular carcinoma Renal cell carcinoma Endocrine tumours: carcinoids Merkel cell carcinoma Thyroid carcinoma
41 Cytokeratins in adenocarcinomas CK5: Breast lob. carcinoma CK5: Breast duct. carc.
42
43 Cytokeratins in adenocarcinomas CK5: Prostate Breast lob. hyperplasia carcinoma CK5: prostate adenocarc.
44 Cytokeratins in adenocarcinomas Neutral/Basic (B, class II) Acidic (A, class I) Squamous cell carcinoma (+) (+) (+) (+) - (+) Transitional cell tumour Malignant mesothelioma Adenocarcinoma: complex epith. (lung, breast) (+) (+) (+) Adenocarc.: biliary tract, pancr., endom., ovary (+)! (+)! (+)! (+)! ++ Adenocarc.: stomach Adenocarc.: intestine Hepatocellular carcinoma Renal cell carcinoma Endocrine tumours: carcinoids Merkel cell carcinoma Thyroid carcinoma
45 Cytokeratins in adenocarcinomas CK5: Serous ovarian carcinoma CK17 CK20: Gall bladder adenocarcinoma
46 Cytokeratins in adenocarcinomas Neutral/Basic (B, class II) Acidic (A, class I) Squamous cell carcinoma (+) (+) (+) (+) - (+) Transitional cell tumour Malignant mesothelioma Adenocarcinoma: complex epith. (lung, breast) (+) (+) (+) Adenocarc.: biliary tract, pancr., endom., ovary (+) (+) (+) (+) ++ Adenocarc.: stomach Adenocarc.: intestine ! Hepatocellular carcinoma Renal cell carcinoma Endocrine tumours: carcinoids Merkel cell carcinoma Thyroid carcinoma
47 Cytokeratins in adenocarcinomas CK20 CK20 CK7 Colon: Typical Colon: Deviant CK7
48 Cytokeratins in adenocarcinomas Neutral/Basic (B, class II) Acidic (A, class I) Squamous cell carcinoma (+) (+) (+) (+) - (+) Transitional cell tumour Malignant mesothelioma Adenocarcinoma: complex epith. (lung, breast) (+) (+) (+) Adenocarc.: biliary tract, pancr., endom., ovary (+) (+) (+) (+) ++ Adenocarc.: stomach Adenocarc.: intestine Hepatocellular carcinoma Renal cell carcinoma Endocrine tumours: carcinoids Merkel cell carcinoma Thyroid carcinoma
49 Cytokeratins in adenocarcinomas HCC: typical CK7 CK7 HCC: deviant CK7 Mallory body CK20
50 Cytokeratins in endocrine tumours Neutral/Basic (B, class II) Acidic (A, class I) Squamous cell carcinoma (+) (+) (+) (+) - (+) Transitional cell tumour Malignant mesothelioma Adenocarcinoma: complex epith. (lung, breast) (+) (+) (+) Adenocarc.: biliary tract, pancr., endom., ovary (+) (+) (+) (+) ++ Adenocarc.: stomach Adenocarc.: intestine Hepatocellular carcinoma Renal cell carcinoma Endocr. tumours: carcinoids Merkel cell carcinoma Thyroid carcinoma
51 Cytokeratins in endocrine tumours CK20: Merkel cell carc. CK19: Thyr. papill. carc.
52 Cytokeratins: Selection of antibodies optimal too diluted HIER Prot. CK5: XM-26 CK5/6: clone D5/16 B4 34BE12: CK5/14/1/10/19?
53 Cytokeratins: Selection of antibodies CK5: XM-26 34BE12: CK5/6: clone D5/16 B4 CK5/14/1/10/19?
54 NORDIQC RESULTS DEC JULY 2017
55 Breast dc Renal ccc K8: EP17 (Epitomics) K8/18: EP17/EP30 (Agilent) K8/18: B22.1/B23.1 (Roche, CM) K18: DC10 (several) K8(7): CAM5.2 (several) K8: 35βH11 (Roche)
56 Breast dc Renal ccc K8: EP17 (Epitomics) K8/18: EP17/EP30 (Agilent) K8/18: B22.1/B23.1 (Roche, CM) K18: DC10 (several) K8(7): CAM5.2 (several) K8: 35βH11
57 NORDIQC RESULTS DEC JULY 2017 Comparison of clone 5D3 used with optimized protocols on two platforms: Leica Bond (A) and Ventana BenchMark (B) to detect LMW Ks in serial sections of liver (1) and breast ductal carcinoma (2). Clone 5D3 gives strong reactions on Leica Bond but consistently poor reactions when used on BenchMark.
58 Cytokeratins: Selection of antibodies optimal Esophagus CK19: clone A53-B/A2.26 Papillary thyroid carcinoma CK19: clone RCK108 After proteolysis as recommended by the vendor
59 Secondary panels for carcinoma identification/subclassification - Apart from cytokeratins and general epithelial markers (EpCAM, Cl4) GI-markers CDX2, CAD17, SATB2, SMAD4, CEA Fem.gen.tract markers CA125, PAX8, WT1, ER, p53 Liver cell markers Neuroendocr. markers Breast markers Arginase, Glypican3, GlutSynt, cancd66a, cancd10, AFP Synaptophysin, Chromogranin GATA3, ER, GCDFP15, Mam.glob.
60 Secondary panels for carcinoma identification/subclassification Lung markers TTF-1, Napsin, p40 Mesothelioma makers Calretinin, Popoplanin, WT1, CA125 Adrenal cortic.markers Inhibin, Melan A, Synaptophysin Germinal cell markers Prostate markers Urinary tract markers SALL4, OCT3/4, PLAP, AFP, CD117, CD30 NKX3.1, PSA, Prostein, AMACR, ERG Uroplakin II, GATA3, (CK5, CK20)
61 GI markers CDX2 CEA CAD17 SATB2 SMAD4
62 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 heterogenous colon pancreas
63 Colon adenocarcinoma CDX-2 protein in adenocarcinoma Colorectum +( ) Mucinous ovar. +( ) Yolk sac tumour + Esoph./Stom. +/ Pancr./biliary /+ Lung /+ Prostate (+) Urothelial (+) Endometrioid (+)
64 Colon adenosquamous carc. CDX-2 protein in adenocarcinoma Colorectum +( ) Mucinous ovar. +( ) Yolk sac tumour + Esoph./Stom. +/ Pancr./biliary /+ Lung /+ Prostate (+) Urothelial (+) Endometrioid (+) Colon adenoc. medullary adenoc.
65 ER CDX-2 Endometrioid carcinoma: ER & CDX-2
66 (Appl Immunohistochem Mol Morphol 2013;21:64 72)
67 CDX2 Normal colon 1 Optimized protocols EPR = EPR2764Y EPR-CON EPR-RTU DAK-CDX2 AMT28 CDX2-88
68 CDX2 Normal colon 2 Optimized protocols EPR = EPR2764Y EPR-CON EPR-RTU DAK-CDX2 AMT28 CDX2-88
69 CDX2 Normal pancreas Optimized protocols EPR = EPR2764Y EPR-CON EPR-RTU DAK-CDX2 AMT28 CDX2-88
70 CDX2 Colon adenocarc.1 Optimized protocols EPR = EPR2764Y EPR-CON EPR-RTU DAK-CDX2 AMT28 CDX2-88
71 Carcinoembryonic antigen (CD66e) Adhesion molecule espc. associated with intestine
72 metast. colon adenoc Carcinoembryonic antigen (CD66e) in adenocarcinomas Colorectal + Medull. thyroid + Pancreas/biliary tract +/ Stomach +/ Lung +/ Ovary, mucinous +/ Ovary, non-muc. /+ Prostate Kidney Liver (!)
73 Medul. thyroid carc. Breast ductal carc. Carcinoembryonic antigen
74
75 CEA31 LD II-7 RTU Low express. carcinoma
76 Normal liver II-7 TF3H8-1
77 Female genital tract markers PAX8 CA125 WT1 p53 ER
78 PAX8 Crucial to the organogenesis and development of Urogenital tract Adult genital tract Thyroid Neuroendocrine system
79 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 +/- NET - pancreas, duodenum, rectum -(+)? Thyroid carcinoma + Other carcinomas -(+) Malignant mesothelioma -
80 Ovarian serous carcinoma PAX8
81 PAX8 in clear cell renal cell carcinoma
82
83
84 Optimal Too weak Fallopian tube Kidney Ovarian serous carcinoma Clear cell renal cell carcinoma False negative
85 The unkown primary tumour: IHC Classification, antibody selection, protocol optimization, Mogens Vyberg Professor of Clinical Pathology Director of NordiQC Aalborg University Hospital, Aalborg, Denmark controls and EQA (part II)
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