1Hofmann M, et al. Assessment of a HER2 scoring system for gastric cancer:results from a validation study. Histopathology 2008;52:797-805. 2Lee AH, et al. The effect of delay in fixation on HER2 expression in invasive carcinoma of the breast assessed with immunohistochemistry and in situ hybridization. J Clin Pathol 2014;67:573-5. 3Rakha EA, et al. Updated UK Recommendations for HER2 assessment in breast cancer. J Clin Pathol 2015;68:93-99. 4Babic A, et al. The impact of pre-analytical processing on staining quality for H&E, dual hapten, dual color in situ hybridization and fluorescent in situ hybridization assays. Methods. 2010 Dec;524:287-300. 5HER2. HER2.20144,. 6Wolff AC, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol 2013 Nov 1;3131:3997-4013. 7HER2. HER2.20147,. 8Laboratory General Checklist, CAP Accreditation Program, 09.25.2012, College of American Pathologists HER2 I-VIEWHER24B5 ultraview HER24B5 Dual ISH HER2 22100AMX00667000 22300AMX01167000 22300AMX00002000 0120-868-555 105-0014 2-6-1 http://www.roche-diagnostics.jp VENTANA 1062.TD.TMS.2000.HER01-003B
HER221525 HER2HER2 HER2HER22001 T-DM1 HER2HER2 ToGA 20113 HER2 HER2HER2 2008Hofmann17.619.2HER2 1 HER2 HER2 HER2 HER2 HER2I-VIEW HER24B5 ultraview HER24B5 Dual ISH HER2 IHC I-VIEW / ultraview HER24B5 Dual Color in situ HybridizationDISH Dual ISH HER2 HER2 HER2 I-VIEW / ultraview HER24B5 Dual ISH HER2 3 3 5 5 5 6 7 7 8 9 12 13 13 15 16 16 16 17 18 19 21 23 27 31 31 32 33 33 34 1 2
IHC I-VIEWHER24B5 ultraview HER24B5Roche HER2IHC ultraview HER24B522300AMX01167000 HER24B5518-107918 HER24B5 5mL50 I-VIEWHER24B522100AMX00667000 ultraview DAB 518-109431 25mL250 HER24B5518-107918 HER24B5 5mL50 -HRP DAB H2O2 IgG IgG/IgM 3,3' 25mL250 25mL250 25mL250 COPPER 25mL250 I-VIEW DAB :518-100032 Ig -HRP DAB H2O2 IgG IgG/IgM 3,3' 25mL250 25mL250 25mL250 25mL250 25mL250 HER2HER2 HER2HER24B5 IgG DABH 2O 2COPPER HER2 HRP HRP DAB H2O2 HRP - HRP COPPER 25mL250 GX XT XT ULTRA LSAB HER2HER2 Ig -HRP HER2-HER24B5- IgG- DABH2O2COPPER - HRP HRP HRP DAB H2O2 HRP HRP Ig 518-102982 EZ 10X 2L / p.6 HER2 518-100599 518-108922 518-103033 518-103002 518-108939 HI ULTRA 10X CC1 CC1 ULTRA 2L 2L 2L DS p.6 518-100605 / 250 518-102319 518-100889 250 250 p.7 518-111182 250 518-107895 HER2 4 in 1 10 518-103095 E-bar 1540 ULTRA 3 4
IHC 1. 2. 3.EZ10 4.CC1 New Silane 4m 1mm5mm 406030 3 1 1 23 10%672 456 10 3 5-10mm 3 LOT 601 5SMEZ 1.EZ5 50mLEZ50mL+2.5g 150mLEZ150mL+7.5g 2.10 OK NG LOT 40027 3.5SMEZ IMBR 1.DS41 25mL20mL+5mL 2. 3.Pretreatment24min 4.46 5 6
IHC HER2 4 in 1 518-107895 HER2 4 in 1 518-107895 41 Cell Line MDA-MB-231 T47D MDA-MB-453 BT-474 HER2 IHC 0 1+ 2+ 3+ <10% >10% >10% >10% HER2 HER2 0 1+ 2+ 3+ 10. Running Yes 1EZ 2CC1 3 I-VIEW DAB ultraview DAB 114 2132 3BLOCKER A14 4BLOCKER B14 5Ig18 6HRP18 7DABH2O218 8COPPER14 11 21 11. 518-111182 I-VIEW DAB ultraview DAB Deparaffinization ULTRA Cell Conditioning Antibody AB Block Counterstain / CC1 Standard 37 / 32 min Hematoxylin 8 min / CC1 Standard 37 / 32 min Not Hematoxylin 8 min Post Counterstain Bluing Reagent 4min Bluing Reagent 4min 1.PCE-bar 2.WindowsVSS 3.Instrument View Ready 4. 5. 6.p.6 7.5SMEZp.6 8. 9. 7 8
IHC HER2HER2 HER2 HER2HER2 HER2HER2 57 1. 2. Heterogeneity 03+ 10% 10% 51 51 3+ 2+ equivocal 3.2~4 10~2040 / 10% / 51 1+ 03+ 10% HER2 0 HER2 10% / / 10% 10% / 10% / 10% 3+ 2+ 1+ 0 equivocal HER2 HER2 Score 0 Score 1+ Score 0 Score 1+ Score 2+ Score 3+ Score 2+ Score 3+ 9 10
IHC Heterogeneity HER2 p.7 p.6 p.5 HER24B5 HER2 4m p.6 p.6 p.5 4m p.6 p.5 11 12
Dual Color HybridizationDISH Dual ISH HER2 Silver in situ Hybridization SISHDual Color in situ HybridizationDISH HER2HER217CEN17 Step1 Silver ISH for HER2 Black Signal Silver A Silver B Silver C Step2 RED ISH for Chromosome 17 Red Signal Goat anti-mouse Antibody AP ph Enhancer Naphthol Fast Red Mouse anti-dig Antibody 2 Dual ISH HER2 22300AMX00002000 Rabbit anti-dnp Antibody Goat anti-rabbit Antibody OH O O Dual ISH HER2 518-109509 HER2 DNA 1 HER2 DNA HER2 2 CEN17 DNA CEN17 10mL50 17q11.2-q2 HO DNP NO2 NO2 HER2 DNA Probe DNP-labeled Chromosome17 Centromere Region DIG O H H OH H CEN17 DNA ProbeDIG-labeled HO ultraview SISH DNP 518-109516 DNP HRP A B C Ig 10mL100 10mL100 20mL100 10mL100 10mL100 HER217DNPHER2 DNA DIGChromosome 17 DNA DNPHRPDNPHER2 DNADNP HRPIg HRP ABCHER2 DIGAPDIGCEN17 DNA DIGAPIg APAS-TRNaphtholKLFast RedpH CEN17 FISH ultraview Red ISH DIG 518-109523 DIG AP ph Ig 10mL100 10mL100 20mL100 132DISH FISH98.5% DISH FISH 65 1 66 1 65 66 66 66 132 Naphthol Fast Red AS-TR KL 10mL100 20mL100 146DISH FISH94.5% DISH FISH 123 6 129 2 15 17 125 21 146 13 14
Dual Color HybridizationDISH GX XT XT ULTRA 0120-868-555 XT XT / 518-102982 EZ 10X 2L p.17 1 518-100599 518-108922 518-103033 518-102999 518-108946 518-103026 518-108892 518-109547 HI ULTRA 10X CC2 CC2 ULTRA SSC 10X ultraview Silver Wash 2L 2L 1L 2L 2L 83 DS p.17 p.19 p.20 p.15 20L3 CLSI 518-102289 ISH 3 0.02 units/ml 200 518-102319 518-100889 250 250 p.18 518-109530 HER2 Dual ISH 3-in-1 10 518-103095 E-bar 1540 ULTRA 3 1. 1 2. 3.EZ10SSC5 4.CC2ultraView Silver Wash 1 23 10%672 456 DISHClinical and Laboratory Standards InstituteCLSI Clinical Laboratory Reagent WaterCLRW 8 UV 10 3 5-10mm 3 UV CLRW 10cfu/mL 10M/cm 0.22mTOC 500ppb 15 16
Dual Color HybridizationDISH New Silane HER2 4m HER2 1mm 5mm HER2 Dual ISH 3-in-1 40 518-109530 6030 LOT 601 5SMEZ 1.EZ5 50mLEZ 50mL+2.5g 150mLEZ150mL+7.5g 2.10 3.5SMEZ IMBR 1.DS41 25mL20mL+5mL 2. OK NG xxxxxxxxxxx xxxxxxx xxxxxxx xxxxxxx xxxxxxxxx xxxxxxxxxxxxxxxx xxxx xxxxx xxxxxxxxx LOT 40027 HER2 Dual ISH 3-in-1 518-109530 31 Cell Line MCF 7 ZR-75-1 Calu-3 HER2 IHC 0 1+ 3+ Baking Deparaffinization Cell Conditioning ISH Protease Denaturation 1-2 copies/nuclei 3 copies/nuclei >6 copies/nuclei Hybridization Stringency Wash SISH Multimer SISH Chromogen Red Multimer RED Chromogen Counterstain Post Counterstain FISH 1.0 1.36 5.01 HER2 Dual ISH 3-in-1 DISH 0.85 1.34 5.53 ULTRA 63 / 20 min Depar CC2 / 86 Mild 8 min Standard12 min Extended8 min ISH Protease 3 8 min 20 min 6 hours 76 32 min 4 min 24 min 8 min Hematoxylin - 8 min Bluing Reagent - 4 min - 1-2 / 3 / XT / GX CC2 Mild 8 min Standard12 min Extended8 min ISH Protease 3 8 min 20 min 6 hours 76 16 min 4 min 24 min 8 min Hematoxylin - 8 min Bluing Reagent - 4 min >6 / 3.Pretreatment24min 4.46 17 18
Dual Color HybridizationDISH ULTRA 1.PCE-bar 2.WindowsVSS 3.Instrument View Ready 4.p.21 5. 6.p.17 7.5SMEZp.17 8. 9. 10. Prime2 PrimeInstrument View Function Tests Test-Prime ULTRA Run Prime 11.PrimeRunning Yes 1EZ 2CC2 3ISH 3 4 13 2HER2 DNA 2 3DNP1 4HRP1 5A 2BC1 6DIG1 7AP1 8pH2Naphthol1Fast Red2 LCSEZ LCS Eukitt Entellan New Entellan HSR Malinol Cytoseal XYL Softmount Paramount DPX Cytoseal 60 Permount Histomount Ultramount Thermo EZ Mount SureMount Flo-texx Mountex Shandon Consul mount MM24 Pertex MicroMount Diamount Alcolmount BioMount 2 Acrytol Gel Mount Mount-Quick EMS Merck Merck Sysmex Muto Chemical Richard Allan Scientific WAKO Protaqs Quartett:Dako BDH:Raymond Lamb Richard Allan Scientific Fisher Raymond Lamb Dako Thermo Scientific Triangle Biomedical Sciences Lerner Labs Histolab Thermo Scientific SurgiPath Cell Path SurgiPath Diapath Diapath BBinternational SurgiPath Biomeda Daido Sangyo Co. Lemasol A Alcohol Aqueous Aqueous 11 21 12.EZ 2340-601 11dip30 p.20 19 20
Dual Color HybridizationDISH 3 93% 97% ULTRA Procedure : U Dual ISH CKT Baking Deparaffinization 63 / 20 min Extended Depara 63 / 20 min Depar A. 1ISH Protease 3 2Cell ConditioningCC2 B. 1ISH Protease 3 2Cell ConditioningCC2 Cell Conditioning ISH Protease CC2 / 86 Mild 12 min Standard12 min Extended12 min ISH Protease 3 16 min CC2 / 86 Mild 12 min Standard12 min Extended12 min ISH Protease 3 8 min C. Stringency Wash Denaturation Hybridization Stringency Wash SISH Multimer SISH Chromogen Red Multimer RED Chromogen Counterstain Post Counterstain 20 min 6 hours 72 24 min 8 min 32 min 12 min Hematoxylin - 8 min Bluing Reagent - 4 min 20 min 6 hours 72 16 min 4 min 32 min 12 min Hematoxylin - 8 min Bluing Reagent - 4 min A. B. C. Cell ConditioningCC2 8-16 min 1-3 cycles ISH Protease 3 4-32 min Stringency Wash72-76 SISH Multimer12-60 min Silver Chromogen4-12 min Red Multimer12-60 min Red Chromogen4-12 min 21 22
Dual Color HybridizationDISH HER2CEN17CEN17 HER2HER2 HER2HER2 HER2HER2HER2 57 20~60HER2CEN17 6 12 HER2CEN17 1. 1-2HER2CEN17 2. HEIHC HER2Heterogeneity 3. 4. HER2 HER2Heterogeneity 17Polysomy 17Monosomy HER2 1 CEN171 HER2 2 CEN172 HER21 CEN172 2 1 1 HER2 1 6 28CEN17 2 HER2 1 12 4 16 CEN172 60X HER2 1CEN17 1 HER2 4CEN17 2 HER2 CEN17 60XCEN17 HER2 2CEN17 2 23 24
Dual Color HybridizationDISH 20HER2CEN1720CEN17HER2 HER2/CEN172.02.0HER21 6.0ISHHER2/CEN172.0HER214.0 6.0equivocalHER2/CEN172.0HER214.0 ISHequivocalIHC IHCISH IHC20CEN17HER2 HER2/CEN17HER2/CEN172.02.0HER2/CEN17 1.8-2.22040HER2/CEN17 ISH 20HER2CEN17 HER2/CEN17 2.0 HER2/CEN17 <2.0 HER216.0 CEN17HER2 HER2/CEN17 20HER2/CEN17 1.82.2 HER2/CEN17 <2.0 HER214.0 <6.0 equivocal 20HER2/CEN17 <1.8>2.2 20HER2CEN17 12HER2/CEN17 HER2/CEN17 <2.0 HER21<4.0 HER2 40HER2/CEN17 <2.02.0 1.1 1.2 1.1 1.3 4.9 10.9 3.2 10.3 Heterogeneity HER2 HER2 25 26
Dual Color HybridizationDISH p.29-30 Dual ISH HER2 / p.22 p.16 p.22 p.16 4mp.17 4mp.17 p.17 p.19 p.19 p.20 p.19 p.17 p.22 p.17 p.19 p.20 p.17 p.16 4mp.17 p.22 p.16 4mp.17 DISH p.15 p.17 p.17 p.17 p.19 p.20 p.19 p.16 4mp.17 p.19 p.22 p.16 27 28
29 30 1 Dual Color HybridizationDISH
HER2 HER2HER2 56 HER2 HER2IHC2+ISHIHCISH HER2 HER2HER2 7 HER2IHC IHC IHC HER2 HER2ISHIHC >10 >10 // 10 >10 / 10 / IHC IHC0,1+,2+,3+IHC2+ISH IHC3+IHC2+ISHHER2/CEP172.0 IHC3 IHC2 equivocal IHC1 IHC0 ISHIHCISH IHC ISH 0 1 2 3 HER2/CEP172.0 HER2/CEP17<2.0 ISH HER2 1 4.0 HER2 1 <4.0 HER2 1 6.0 HER2 1 4.0<6.0 HER2 1 <4.0 HER2 ISH ISH ISH ISH equivocal ISH ISH IHCCh17ISH IHCISH Ch17 ISHHER2/CEP172.0HER2/CEP17<2.0 IHC ToGA FISHIHC3+5.1 HER2 HER2 31 32
I-VIEWHER24B5 22100AMX00667000 Dual ISH HER2 22300AMX00002000 790-2991 518-107918 HER24B5 50 2~8 98,000 800-4422 518-109509 INFORM HER2 Dual ISH DNA Probe Cocktail Dual ISH HER2 50 2~8 400,000 760-041 518-100032 760-050 518-100605 Ventana iview DAB Universal Kit I-VIEW DAB g -HRP DAB H2O2 COPPER Endogenous Biotin Blocking Kit 250 250 2~8 2~8 180,000 25,000 800-098 518-109516 800-505 518-109523 ultraview SISH DNP Detection Kit ultraview SISH DNP DNP HRP A B CultraView Red ISH DIG Detection Kit ultraview Red DIG DIG AP ph Naphtol Fast Red 100 2~8 450,000 100 2~8 450,000 ultraview HER24B5 22300AMX01167000 780-4409 518-109547 1:50%25mL HybReady Solution 83 2~8 80,000 1 790-2991 518-107918 HER24B5 50 2~8 98,000 780-4149 518-102289 780-003 518-108892 ISH Protease ISH 30.02 units/ml ultraview Silver Wash ultraview Silver Wash 200 2~8 13,000 2L 15~30 28,000 760-500 518-109431 VENTANA ultraview Universal DAB Detection Kit ultraview DAB -HRP DAB H2O2 COPPER 250 2~8 250,000 950-110 518-103026 950-123 518-102999 950-223 518-108946 SSC SSC 10X CC 2 CC 2 2:5 3:XTGX 4:ULTRA Benchmark ULTRA CC 2 CC 2 ULTRA 2L 15~30 9,000 2 1L 15~30 25,000 3 1L 15~30 40,000 4 781-2991 518-107895 PATHWAY HER-2 4 in 1 Control Slides HER2 4 in 1 10 2~8 88,000 783-4422 518-109530 HER2 Dual ISH 3-in-1 Xenograft Slides HER2 Dual ISH 3-in-1 10 15~30 25,000 790-4795 518-111182 Rabbit Monoclonal Negative Control Ig 250 2~8 13,000 33 34