Bond™ Oracle™ HER2 IHC System for Leica BOND
Contents
1. Presence of strong brown complete cell membrane staining in the 3 Control Cell Line SK BR 3 Presence of weak to moderate brown complete cell membrane staining in the 2 Control Cell Line MDA MB 453 Presence of faint barely perceptible brown incomplete cell membrane staining in the 1 Control Cell Line MDA MB 175 No staining in the 0 Control Cell Line MDA MB 231 Important note A feature of the MDA MB 175 1 control cell line is a distinct growth pattern in which the cells form clusters These clusters give rise to a continuous luminal brush border region across the cell cluster This brush border staining will be stronger than that of the rest of the cell membrane It is the faint barely perceptible incomplete cell membrane staining that is the correct HER2 oncoprotein 1 staining pattern Dot like immunostaining of the Golgi region in the cytoplasm may also be observed in this cell line 2 In house Positive Control Tissue HER2 Primary Antibody The PRESENCE of brown membrane staining should be observed corresponding to the known HER2 oncoprotein status of the chosen positive control 3 In house Negative Control Tissue Component HER2 Negative Control The ABSENCE of membrane staining should be observed A negative control tissue component confirms the lack of detection system cross reactivity to specifically targeted cells cellular components If membrane staining occurs in a negative control tissue component results2. Bond Oracle Negative 269 23 0 292 HER2 IHC 0 or 1 System for 2 10 38 24 72 BOND MAX 3 0 1 66 67 Totals 279 62 90 431 3x3 Concordance 95 Cl 86 54 82 95 to 89 62 96 p 0 0001 Table 7 3x3 concordance of Bond Oracle HER2 IHC System for BOND MAX with HercepTest Page 15 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 In conclusion the data generated in this study demonstrates that the Bond Oracle HER2 IHC System for BOND MAX can be used as an aid in determination of treatment for Herceptin trastuzumab therapy based upon its high concordance with the HercepTest Bond Oracle HER2 IHC System for BOND MAX v PathVysion HER2 DNA Probe Kit Part 2 of the study was designed to examine the concordance between the Bond Oracle HER2 IHC System for BOND MAX and the Abbott Molecular PathVysion HER 2 DNA Probe Kit considered as the gold standard for gene assessment reflex assay used in conjunction with HER2 immunohistochemistry This study was performed at the same investigational sites and used the same study cohort as in Part 1 All cases were stained with the Abbott Molecular PathVysion HER2 DNA Probe Kit according to the manufacturers instructions as specified in the package insert Sequential sections from each case were stained with the Bond Oracle HER2 IHC System for BOND MAX on board a BOND MAX fully automated advanced staining system from Part 1 of t
3. fixation embedding methods or to inherent irregularities within the tissue 15 Excessive or incomplete counterstaining may also compromise correct interpretation of the results Page 12 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 English l English i Nonspecific staining if present usually has a diffuse appearance Sporadic staining of connective tissue may also be observed in sections from excessively formalin fixed tissues Use intact cells for interpretation of staining results Necrotic or degenerated cells often stain nonspecifically 16 False positive results may be seen due to non immunological binding of proteins or substrate reaction products They may also be caused by endogenous enzymes such as pseudoperoxidase erythrocytes or endogenous peroxidase cytochrome C depending on the type of immunohistochemical stain used Tissues from patients infected with Hepatitis B virus and containing Hepatitis B virus surface antigen HBsAg may exhibit nonspecific staining with horseradish peroxidase 17 Unexpected immunohistochemical staining or variations in the staining may be as a result of alterations in the expression levels of the encoding genes or antigens Any change in expected staining patterns should be interpreted in association with all other diagnostic investigations Theinterpretation ofimmunohistochemical staining should be complemented by morphological stud
4. overnight Sections which need further adherence may be incubated at 60 C for a further hour Table 10 Bond Oracle HER2 IHC System for BOND MAX Trouble Shooting Guide If any problems associated with the Bond Oracle HER2 IHC System for BOND MAX fall outside the scope of the troubleshooting guide refer to Table 12 please contact your local Leica Biosystems Technical Services Department or Distributor for assistance Page 21 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 References 1 Corbett IP Henry JA Angus B et al NCL CB11 Anew monoclonal antibody recognizing the internal domain of the c erbB 2 oncogene protein effective for use on formalin fixed paraffin embedded tissue Journal of Pathology 1990 161 15 25 2 Lonardo F Di Marco E King CR Pierce JH Segatto O Aaronson SA et al The normal erbB 2 product is an atypical receptor like tyrosine kinase with constitutive activity in the absence of ligand New Biologist 1990 2 992 1003 3 Carter P Presta L Gorman CM Ridgway JBB Henner D Wong WLT et al Humanization of an anti p185HER2 antibody for human cancer therapy Proceedings of the National Academy of Science USA 1992 89 4285 9 4 Hudziak RM Lewis GD Winget M Fendly BM Shepard HM Ullrich A p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor Molecular amp Cell
5. 14 1477 0 95 occasions represented variations from 0 to 1 or 2 to 3 and as such did not represent a change from clinically positive to clinically negative or vice versa in a 2x2 data assessment Pass value 99 05 95 Cl 98 42 to 99 46 Of the 14 staining events 5 1477 0 34 staining events occurred at Leica Biosystems Newcastle Ltd Site A 8 1477 0 54 occurred at Site B and 1 1477 0 07 occured at Site C The remaining 65 1477 4 40 staining events showed variation from 2 to 1 or 2 to 0 and therefore would represent a change from clinically positive to clinically negative or vice versa in a 2x2 data assessment Pass value 95 6 95 Cl 94 42 to 96 54 Of the 65 clinically significant changes 11 65 16 9 occurred at Leica Biosystems Newcastle Ltd Site A 24 65 36 9 occurred at Site B and 30 65 46 1 occured at Site C Of the clinically significant changes on no occasions did a 3 change to a negative 0 or 1 result or vice versa E Inter Observer Reproducibility 40 randomly selected invasive breast cancer cases providing an equal distribution of each of the HER2 IHC grades resection specimens were consecutively sectioned and provided to Page 19 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev_B 27 10 2014 Leica Biosystems Newcastle Site A Site B and Site C for staining and interpretation The sections were blinded and randomized at each site prior to sc
6. 60 C Tissue specimens should be sectioned between 3 5 um The slides required for HER2 oncoprotein evaluation and tumor verification should be prepared at the same time To preserve antigenicity tissue sections mounted on slides Leica BOND Plus Slides product code S21 2113 should be stained within 4 6 weeks of sectioning when held at room temperature 20 25 C Following sectioning it is recommended that slides are incubated for 12 18 hours overnight at 37 C Sections which require additional adherence may be incubated at 60 C for a further hour In the USA the Clinical Laboratory Improvement Act of 1988 requires in 42 CFR 493 1259 b that The laboratory must retain stained slides for at least ten years from the date of examination and retain specimen blocks at least two years from the date of examination Warnings and Precautions For professional users only One or more components in the product are hazardous As a rule persons under 18 years of age are not allowed to work with this product Users must be carefully instructed in the proper work procedure the hazardous properties of the product and the necessary safety instructions Symptoms of overexposure to ProClin 950 the preservative used in the Oracle reagents may include skin and eye irritation and irritation to mucous membranes and upper respiratory tract The concentration of ProClin 950 in this product is up to a maximum of 0 35 These solutions do not
7. 71 BOND MAX 3 6 61 67 Total 331 97 428 Overall Concordance 95 CI 87 6 84 to 90 Table 8 2x2 concordance of Bond Oracle HER2 IHC System for BOND MAX staining v PathVysion HER 2 DNA Probe kit Page 16 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev_B 27 10 2014 English l English i Immunoreactivity Normal Panel Normal Tissue Type Staining Pattern HER2 Primary Antibody HER2 Negative Control Adrenal egative Negative Brain Cerebellum egative Negative Brain Cerebrum egative Negative Breast egative Negative Bone Marrow egative Negative Colon egative Negative Esophagus egative Negative Eye egative Negative Hypophysis Moderate cytoplasmic staining Negative observed in hypophyseal cells 1 3 Kidney egative Negative Larynx egative Negative Liver egative Negative Lung egative Negative Mesothelium egative Negative Ovary egative Negative Pancreas egative Negative Parathyroid egative Negative Peripheral Nerve egative Negative Prostate egative Negative Salivary Gland egative Negative Skin egative Negative Small Intestine egative Negative Spleen egative Negative Stomach Weak cytoplasmic staining observed Negative in gastric glands 2 3 Striated Muscle egative Negative Testis egative Negative Thymus egative Negative Thyroid egative Negative Tonsil egative
8. Biology 1989 9 1165 72 5 Lewis GD Figari Fendly B Wong WL Carter P Gorman C et al Differential responses of human tumor cell lines to anti p185HER2 monoclonal antibodies Cancer Immunology and Immunotherapy 1993 37 255 63 6 Baselga J Norton L Albanell J Kim Y M Mendelsohn J Recombinant humanized anti HER2 antibody Herceptin enhances the antitumor activity of paclitaxel and doxorubicin against HER2 neu overexpressing human breast cancer xenografts Cancer Research 1998 58 2825 31 7 Nakane PK and Pierce GB Enzyme labeled antibodies Preparations and applications for the localization of antigens Journal of Histochemistry and Cytochemistry 1967 14 929 931 8 Tsutsumi Y Serizawa A and Kawai K Enhanced polymer one step staining EPOS for proliferating cell nuclear antigen and Ki 67 antigen applications to intraoperative frozen diagnosis Pathology International 1995 45 2 108 115 9 Walker RA Bartlett JMS Dowsett M Ellis IO Hanby AN Jasani B Miller K and Pinder SE HER2 Testing in the UK Further Update To Recommendations Journal of Clinical Pathology 2008 1 o Dickson RB and Lippman ME Genes Oncogenes and Hormones Boston Kluwer Academic Publishers 1992 11 Keatings L et al c erbB 2 oncoprotein expression in mammary and extramammary Paget s disease an immunohistochemical study Histopathology 1990 17 234 247 12 The National Committee for Clinical Laboratory Standards NCCLS
9. Oracle HER2 IHC System for BOND MAX contains components required to complete an immunohistochemical staining procedure for formalin fixed paraffin embedded tissues Following incubation with the ready to use HER2 Primary Antibody clone CB11 this System employs ready to use Compact Polymer technology The enzymatic conversion of the subsequently added chromogen results in the formation of a visible reaction product at the antigenic site The tissue sections may then be counterstained dehydrated cleared and mounted Results are interpreted using light microscopy Control slides with four formalin fixed paraffin embedded human breast cancer cell lines are provided to validate staining runs The four cell lines demonstrate HER2 oncoprotein expression at 0 1 2 and 3 intensities The staining intensity of these cell lines correlates to both HER2 oncoprotein receptor load per cell and HER2 gene amplification status The Bond Oracle HER2 IHC System for BOND MAX product code TA9145 is for use on the Leica Biosystems BOND MAX fully automated advanced staining system English Components Provided The materials listed below Table 1 are sufficient to stain 150 slides 60 test slides incubated with HER2 Primary Antibody 60 corresponding test slides incubated with HER2 Negative Control 15 HER2 Control Slides incubated with HER2 Primary Antibody and 15 in house positive tissue controls incubated with HER2 Primary Antibody The number of tests is
10. Primary Antibody ssesee 12 Limitations 555 2 deceive ticescceantstesstsactscoceaneceuoncaanciundsnacciscacdanacsuatouasdgnansnstatanssvnuresucadontcaiassoaissuecsonsa 12 Ac General BImitalioris 3 oem iret ca bik bene osama Anus A ILI 12 B Product Specific Lil mitetiOFis saco coo conteret eroe eva Ce Pedo aede OE Rene VN PAESE EE 13 CEBLnDLcm 14 Clinical Concordance of Bond Oracle HER2 IHC System to Dako HerceprTest 14 2X2 Concordance RESUS 7 5 22 A fae a ned tecto DeL na 15 3x3 Concordance Results 4 15 Immunoreactivity Normal Panel eeseeeeseeeseseeeenne ente nennen nennt n nnn n nnn nnne nnns n nn 17 Reproducibillty Study acc cii0ccccecccecicccccessceccsnccesseccececnctecencatetesstceenstaccesanctecsnccesceectedensneerseceeeries 18 Within and Between Precisiori Testing entere itte tauri enn tna epa sk eae open taba dni aa deas 18 A Within Run Precision Testing zi B Between RUNUPFECISION TeStinig 2 neuen tuetur tnr can Dna tano nuni dn ide Pad n ranae eae rn Rusa ae C bot to Lot Reproducibility 52 2 2 cerro eser eoe aerae erased unadi ssa raisi cip catia D Between Laboratory Reproducibility E Inter Observer Reproducibility terere idest coe tert oe Mice eter tee tant Troubleshooting otio einen ti reir rtt tt onem cte nter e
11. based on the use of a 150 uL automated dispense per slide The kit provides materials sufficient for a maximum of 15 individual BOND MAX staining runs Sections of formalin fixed paraffin embedded human breast cancer HER2 Control Slides cell lines that demonstrate HER2 oncoprotein expression at 0 1 x15 2 and 3 staining intensities when stained in accordance with the protocol provided These sections are fully adhered and do not require further baking HER2 Primary Contains ready to use affinity purified mouse monoclonal IgG Antibody 13 5 mL antibody clone CB11 and 0 35 ProClin 950 HER2 Negative Control 9 mL Contains ready to use mouse IgG at an equivalent concentration to the HER2 Primary Antibody and 0 35 ProClin 950 Peroxide Block 22 5 mL Contains 3 496 hydrogen peroxide Post Primary 22 5 mL Rabbit anti mouse IgG 10 ug mL in Tris buffered saline containing 10 v v animal serum and 0 0996 ProClin 950 Polymer 22 5 mL Poly HRP goat anti rabbit IgG 25 ug mL in Tris buffered saline containing 10 v v animal serum and 0 09 ProClin 950 DAB Part 1 2 25 mL Contains 66 mM 3 3 diaminobenzidine tetrahydrochloride in a stabilizer solution DAB Part B x2 22 5 mL Contains lt 0 1 v v hydrogen peroxide Hematoxylin 22 5 mL Contains 0 196 hematoxylin Table 1 Bond Oracle HER2 IHC System for BOND MAX components Page 4 of 23
12. meet the OSHA criteria for a hazardous substance A Material Safety Data Sheet is available upon request or from www LeicaBiosystems com Specimens before and after fixation and all materials exposed to them should be handled as if capable of transmitting infection and disposed of with proper precautions Never pipette reagents by mouth and avoid contacting the skin and mucous membranes with reagents and specimens If reagents or specimens come into contact with sensitive areas wash with copious amounts of water Seek medical advice Consult federal state or local regulations for disposal of any potentially toxic components Minimize microbial contamination of reagents or an increase in nonspecific staining may occur Page 5 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 Procedure A Reagents required but not supplied BOND Dewax Solution product code AR9222 BOND Epitope Retrieval Solution 1 product code AR9961 BOND Wash Solution x10 Concentrate product code AR9590 Standard solvents used in immunohistochemistry e g ethanol absolute and graded Xylene or xylene substitutes Mounting medium Distilled or de ionized water B Equipment required but not supplied Leica Biosystems BOND MAX fully automated advanced staining system BOND Universal Covertiles product code 821 2001 821 4583 or S21 4611 BOND Mixing Stations product code S21 1971 Drying oven capabl
13. scores were interpreted as negative if the staining was 0 or 1 equivocal for scores of 2 and positive for scores of 3 Data was then analyzed for positive staining agreement and negative staining agreement Page 14 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 English l English i 2x2 Concordance Results In this primary analysis the test results from the two tests Bond Oracle HER2 IHC System for BOND MAX and DAKO HercepTest are categorized as negative 0 1 or positive 2 3 The frequencies of four possible combinations are displayed in a 2x2 table format see Table 6 Then the overall concordance rate based on this 2x2 table was calculated accompanied by a 95 exact confidence interval based on the binomial distribution The null hypothesis H which the success criteria are set against is that concordance is no greater than 75 The observed agreement for 431 samples between the two tests in a 2x2 analysis show a concordance of 92 34 398 431 with a 95 Cl of 89 42 94 67 This data supports rejection of the null hypothesis H that agreement was no greater than 75 with a p value 0 0001 The percentage Positive Agreement sensitivity or the ability of Bond Oracle HER2 IHC System for BOND MAX to correctly identify HercepTest positive cases the percentage of specimens Scored positive by both Bond Oracle HER2 IHC System for BOND MAX and HercepTest out of all th
14. with the patient specimen should be considered invalid 4 Patient Tissue stained using the HER2 Negative Control The ABSENCE of membrane staining verifies the specific labeling of the target antigen by the primary antibody Other brown staining occurring in the cytoplasm of the specimen treated with the HER2 Negative Control such as in connective tissue leukocytes erythrocytes or necrotic tissue should be considered nonspecific background staining and should be noted 5 Patient Tissue stained using the HER2 Primary Antibody HER2 oncoprotein expression levels are determined by the criteria defined in both Table 4 and in the Bond Oracle HER2 IHC System for BOND MAX Interpretation Guide Limitations A General Limitations Immunohistochemistry is a laboratory based multi step technique used to aid in the interpretation and determination of histopathological characteristics It is a technique which requires specialized training in all aspects of procedure including the selection of appropriate reagents tissue fixation processing and IHC slide preparation and interpretation Immunohistochemical staining of tissue is dependent on the handling fixation and processing of the tissue prior to staining Improper fixation freezing thawing washing drying heating sectioning or contamination with other tissues or fluids may produce artifact antibody trapping or false negative results Inconsistent results may be due to variations in
15. 014 English l English i Slide Slide Description Reagent Tissue Slide Icon Position Type 1 Case 1 HER2 Negative Test Te Control smh so te 2 Case2 HER2 Negative Test uq Control WS 3 Case 3 HER2 Negative Test a Control wen ses 4 Case 4 HER2 Negative Test E Control wen co cs 5 Case 1 HER2 Primary Test 2m Antibody IHCH b Higs 6 Case2 HER2 Primary Test ETT Antibody weno no em 7 Case 3 HER2 Primary Test EET Antibody wen p 8 Case4 HER2 Primary Test 2 Antibody IHCH b Hips 9 HER2 Control Slide HER2 Primary Positive 3 Antibody Won co em 10 In house Tissue Control HER2 Primary Positive 4 Antibody SHCH b Wips Table 2 Slide tray layout showing tissue type and reagent E Procedure Steps Follow the steps below to set up a slide tray with the layout described in Table 2 These instructions should be read in conjunction with the BOND System User Manual 1 On the BOND MAX instrument ensure the bulk and hazardous waste containers have enough capacity to perform the required staining runs 2 Ensure there is adequate alcohol distilled or de ionized water BOND Dewax Solution supplied as ready to use BOND Epitope Retrieval Solution 1 supplied as ready to use and BOND Wash Solution supplied as x10 concentrate in the bulk reagent containers to perform the required staining runs 3
16. English i Bond Oracle HER2 IHC System for Leica BOND MAX System Instructions For Use For use on Leica Biosystems BOND MAX fully automated advanced staining system Product Code TA9145 is designed to stain 60 tests 150 slides 60 test slides with HER2 Primary Antibody 60 test slides with HER2 Negative Control 15 HER2 Control Slides with HER2 Primary Antibody 15 positive in house tissue controls with HER2 Primary Antibody C Leica Biosystems Newcastle Ltd Balliol Business Park Benton Lane Newcastle Upon Tyne NE12 8EW United Kingdom 44 191 215 4242 Leica Biosystems Canada 71 Four Valley Drive Concord Ontario L4K 4V8 Canada 3 1 800 248 0123 Leica Biosystems Inc 1700 Leider Lane Buffalo Grove IL 60089 3 1 800 248 0123 Leica Biosystems Melbourne Pty Ltd 495 Blackburn Road Mt Waverly VIC 3149 Australia J 61 2 8870 3500 Page 1 of 23 cl Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev_B 27 10 2014 Contents Intended Use sonne A a recie ei tr deter 3 Summary and Explanation ud Background jJ Expression OFA S 3 Clinical Concordanice Sumtialy ioco he ec Pre ce tr e oer e cad eet cere dire daas 3 Princlple of Procedure Ines 4 Gomponents Provided 2 ttm trece ett atento taie ege te Lope da oH d ee Ea RUE RE acces o Re RU a DOR e dan 4 Di
17. Ensure that a clean BOND Mixing Station is installed Important note The mixing station should never be removed from the BOND MAX instrument while the instrument is turned on Removing the mixing station while the instrument is turned on may have detrimental impacts on staining 4 Turn on the BOND MAX fully automated advanced staining system 5 Turn on the BOND Controller attached to the BOND MAX fully automated advanced staining system 6 Open the BOND software Page 7 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 7 For a new Bond Oracle HER2 IHC System scan the reagent tray barcodes with the handheld scanner to enter the system into the BOND reagent inventory 8 Goto the Slide setup screen and click Add case 9 Enter details for the first case Ensure the dispense volume is set to 150 uL and the preparation protocol is Dewax Click OK 10 With the case highlighted in the Slide setup screen click Add slide 11 First add patient test slides Ensure tissue type is set to Test tissue 12 Confirm the dispense volume is 150 uL and the preparation protocol is Dewax 13 Select staining mode values Single and Oracle do not click Oracle control 14 Select process IHC 15 Select HER2 Negative Control from the marker list The Protocols tab defaults to the correct staining protocol IHC Protocol H and HIER protocol HIER 25 min with ER1 97 16 Click Add slide The ne
18. Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 English i Directions on Use All reagents supplied are formulated specifically for use with this assay and lot numbers are specific for each Bond Oracle HER2 IHC System for BOND MAX For the assay to be valid no substitutions should be made Storage and Stability Store at 2 8 C Do not freeze Return to 2 8 C immediately after use Any deviation from these conditions will invalidate the assay Ensure the Bond Oracle HER2 IHC System for BOND MAX used is within its designated expiry date The signs indicating contamination and or instability of the Bond Oracle HER2 IHC System for BOND MAX are turbidity of the solutions odor development and presence of precipitate Storage conditions other than those specified above must be verified by the user Specimen Preparation All specimens must be prepared to preserve the tissue for immunohistochemical staining Standard methods of tissue processing should be used for all specimens 12 It is recommended that tissues are prepared in formalin based fixatives and are routinely processed and paraffin embedded For example resection specimens should be blocked into a thickness of 3 4 mm and fixed for 18 24 hours in 10 neutral buffered formalin The tissues should then be dehydrated in a series of alcohols and cleared through xylene followed by impregnation with molten paraffin wax held at no more than
19. Negative Uterine Cervix egative Negative Uterus egative Negative Table 9 Normal Panel Staining Page 17 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 Reproducibility Study Within and Between Precision Testing Precision testing was performed at Leica Biosystems Newcastle Ltd The tissue used was a formalin fixed paraffin embedded composite tissue micro array TMA supplied by Isu Abxis Yonsei University Medical Center 134 Shinchon dong Seoul 120 752 Korea comprising of 20 4mm diameter invasive breast carcinoma tissue cores The 20 cases were selected based on previously assigned HER2 scores On this basis x5 cases of HER2 3 x5 cases of HER2 2 x5 cases of HER2 1 and x5 cases of HER2 0 were included A Within Run Precision Testing Within run precision testing of the Bond Oracle HER2 IHC System for BOND MAX was evaluated on a total of 40 consecutive sections from a TMA comprising of 20 invasive breast tumors and 40 HER2 Control Slides All slides were stained with the Bond Oracle HER2 IHC System for BOND MAX on the BOND MAX fully automated advanced staining system Sections were stained during one continuous period using a Bond Oracle HER2 IHC System for BOND MAX from the same manufacturing batch Stained sections were blinded and assessed in a randomized fashion by a single experienced observer to determine within run precision An evaluation of the slides from the wit
20. Quality assurance for immunocytochemistry Approved guideline NCCLS document MM4 A 1 56238 396 5 NCCLS 940 West Valley Road Suite 1400 Wayne Pennsylvania 1999 19087 1898 USA 13 Elias JM Gown AM Nakamura RM Wilbur DC Herman GE Jaffe ES et al Special Report Quality control in immunohistochemistry American Journal of Clinical Pathology 1989 92 836 43 14 Press MF Cordon Cardo C Slamon DJ Expression of the HER 2 neu proto oncogene in normal human adult and fetal tissues Oncogene 1990 5 953 62 15 Nadji M and Morales A R Immunoperoxidase part I the techniques and its pitfalls Laboratory Medicine 1983 14 767 16 Jackson P 2007 Quality Assurance in Immunohistochemistry In Immunohistochemistry 2007 ed Renshaw S PP 205 237 Scion Publishing Ltd 17 Omata M Liew C T Ashcavai M Peters RL Nonimmunologic binding of horseradish peroxidase to hepatitis B surface antigen a possible source of error in immunohistochemistry American Journal of Clinical Pathology 1980 73 626 32 18 Bartlet JMS Ibrahim M et al External Quality Assurance of HER2 FISH Testing Results of a UK NEQAS Pilot Scheme Journal of Clinical Pathology 2006 Amendments to previous issue Equipment required but not supplied Date of issue 27 October 2014 Page 22 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 English l English i Symbol Identification ror Batch C
21. ae trier References cinia esixise aces cesa suda ca EX 22 eas NoEE IR CU a ARI ERR A ARR DP CUR FC FAR NAR FRE Ta Roos 22 Page 2 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 English l English i Intended Use For in vitro diagnostic use Bond Oracle HER2 IHC System for BOND MAX is a semi quantitative immunohistochemical IHC assay to determine HER2 Human Epidermal Growth Factor Receptor 2 oncoprotein status in formalin fixed paraffin embedded breast cancer tissue processed for histological evaluation following automated staining on the BOND MAX slide staining instrument The Bond Oracle HER2 IHC System for BOND MAX is indicated as an aid in the assessment of patients for whom Herceptin trastuzumab treatment is being considered Note All of the patients in the Herceptin clinical trials were selected using an investigational immunohistochemical Clinical Trial Assay CTA None of the patients in those trials were selected using the Bond Oracle HER2 IHC System for BOND MAX The Bond Oracle HER2 IHC System for BOND MAX has been compared to the Dako HercepTest on an independent set of samples and found to provide acceptably concordant results The actual correlation of the Bond Oracle HER2 IHC System for BOND MAX to clinical outcome has not been established Summary and Explanation Background The Bond Oracle HER2 IHC System for BOND MAX contains the mouse monoclonal
22. anti HER2 antibody clone CB11 Clone CB11 originally developed by Corbett et al 1 and manufactured by Novocastra Laboratories Ltd now Leica Biosystems Newcastle Ltd is directed against the internal domain of the HER2 oncoprotein In a proportion of breast cancer patients the HER2 oncoprotein is overexpressed as part of the process of malignant transformation and tumor progression 2 Overexpression of the HER2 oncoprotein found in breast cancer cells suggests HER2 as a target for an antibody based therapy Herceptin is a humanized monoclonal antibody 3 that binds with high affinity to the HER2 oncoprotein and has been shown to inhibit the proliferation of human tumor cells that overexpress HER2 oncoprotein both in vitro and in vivo 4 6 Since the first immunoperoxidase technique reported by Nakane and Pierce 7 many developments have occurred within the field of immunohistochemistry resulting in increased sensitivity A recent development has been the use of polymeric labeling This technology has been applied to both primary antibodies and immunohistochemical detection systems 8 The Compact Polymer detection system utilized by the Bond Oracle HER2 IHC System for BOND MAX is part of a family of novel controlled polymerization technologies that have been specifically developed to prepare polymeric HRP linked antibody conjugates As this polymer technology is utilized in the Oracle product range the problem of nonspecific endogeno
23. d a BOND MAX fully automated advanced staining system Stained slides were masked and assessed in a randomized fashion by a single trained observer to determine Lot to Lot reproducibility An evaluation of the slides tests and controls from the lot to lot investigation indicated that 36 36 data points could be interpreted No variation in staining occurred in the 36 data points between the three different manufacturing lots of the Bond Oracle HER2 IHC System for BOND MAX Staining with the Bond Oracle HER2 IHC System for BOND MAX is consistent across manufacturing batches D Between Laboratory Reproducibility Between laboratory reproducibility testing of the Bond Oracle HER2 IHC System for BOND MAX was evaluated at 3 sites Leica Biosystems Newcastle Site A and two independent laboratories Sites B and C on a total of 192 sections from a TMA comprising of 20 invasive breast tumors and 24 HER2 Control Slides Of the 192 TMA sections stained 96 were stained with the HER2 Primary Antibody and 96 with the HER2 Negative Control reagent All slides were stained with the Bond Oracle HER2 IHC System for BOND MAX on the BOND MAX fully automated advanced staining system The slides were evaluated in 8 independent runs performed within each of the 3 different investigational sites using a Bond Oracle HER2 IHC System for BOND MAX from the same manufacturing batch Stained slides were blinded and assessed in a randomized fashion by a single experienced obser
24. d appropriately to preserve the tissue antigenicity for immunohistochemical staining Standard methods of tissue processing should be employed for all specimens 12 Page 9 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 HER2 Control Slide HER2 Primary Antibody Each of the supplied HER2 Control Slides contains four formalin fixed paraffin embedded human breast cancer cell line cores with staining intensity scores of 0 1 2 and 3 One slide must be included in each test run ie slide tray The correct evaluation of the HER2 Control Slide supplied by Leica Biosystems indicates the validity of the test refer to Bond Oracle HER2 IHC System for BOND MAX Interpretation Guide The HER2 Control Slides supplied with this system validate reagent performance only and do not verify tissue preparation In house Positive Control Tissue HER2 Primary Antibody If in house positive control tissue components are used they should be biopsy or surgical specimens fixed processed and embedded as soon as possible in the same manner as the patient sample s Positive tissue controls are indicative of correctly prepared tissues and valid staining techniques At least one positive control component for each test run should be included The positive control section should demonstrate weak positive staining so as to define subtle changes in primary antibody sensitivity Note Known positive control tissue co
25. e HercepTest positive cases was 84 87 129 152 with a 95 CI of 78 17 90 16 The percentage Negative Agreement specificity or the ability of the test to correctly identify HercepTest negative cases the percentage of specimens scored negative by both Bond Oracle HER2 IHC System for BOND MAX and HercepTest out of all the HercepTest negative cases was 96 42 269 279 with a 95 Cl of 93 51 98 27 HercepTest Negative Positive Totals Bond Oracle HER2 Negative 269 23 292 IHC System for 10 129 139 BOND MAX Positive Totals 279 152 431 2x2 Concordance 95 Cl 92 34 89 42 to 94 67 p 0 0001 Table 6 2x2 concordance of Bond Oracle HER2 IHC System for BOND MAX with HercepTest 3x3 Concordance Results Data was grouped as negative 0 or 1 equivocal 2 or positive 3 for 3x3 analysis and showed a concordance of 86 54 373 431 with a 9596 Cl of 82 95 to 89 62 Therefore the null hypothesis H that agreement was no greater than 75 was rejected with a p value 0 0001 The percentage Positive Agreement for 3 the percentage of specimens scored 3 positive by both Bond Oracle HER2 IHC System for BOND MAX and HercepTest out of all the 3 HercepTest positive cases in this study was 73 33 66 90 with a 95 Cl of 62 97 to 82 11 The percentage Negative Agreement was 96 42 269 279 with a 95 CI of 93 51 to 98 27 See Table 7 HercepTest Negative 2 3 Totals 0 or 1
26. e of maintaining 60 C Light microscope 4 40x objective magnification Slides Leica BOND Plus Slides product code S21 2113 Coverslips BOND Slide Label and Print Ribbon product code 821 4564 BOND Aspirating Probe Cleaning System product code CS9100 C Methodology Prior to undertaking this methodology users must be trained in BOND fully automated immunohistochemical techniques Each test section to be stained with the HER2 Primary Antibody will require an identical section for staining with the HER2 Negative Control The negative control section allows differentiation between specific and nonspecific staining at the antigen site Each BOND staining run should include a HER2 Control Slide At the end of the staining protocol if the cell lines do not demonstrate the correct staining patterns refer to Bond Oracle HER2 IHC Systems Interpretation Guide the run should be regarded as invalid D Slide Layout A new BOND Universal Covertile product code S21 2001 or 821 4583 should be used with each slide The use of BOND Universal Covertiles which have been previously utilized for either immunohistochemical or in situ hybridization staining has not been validated with this test The slide tray layout Table 2 enables optimal performance of the Bond Oracle HER2 IHC System for BOND MAX and the full 60 tests to be obtained Page 6 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2
27. formerly NCCLS Quality Assurance for Immunocytochemistry Approved Guideline 12 and Special Report Quality Control in Immunohistochemistry 13 In addition refer to Table 3 below for the types of immunohistochemical quality controls and their purposes Sample Description HER2 Primary Antibody HER2 Negative Staining Control Staining HER2 Control As supplied in the Bond Controls staining Slide Oracle HER2 IHC System procedure and indicates for BOND MAX the validity of the reagent performance In house Tissue containing target Controls all steps of the Positive Control antigen The ideal control analysis Validates tissue Tissue is weakly positive staining preparation and Bond Detection of tissue so as to define Oracle HER2 IHC System nonspecific subtle changes in primary for BOND MAX staining background antibody sensitivity performance staining In house Tissues or cells expected Detection of nonspecific Negative to be negative could be antibody cross reactivity Control Tissue located in patient tissue or with cells cellular Component positive negative control components tissue components Fixed and processed as per patient sample Table 3 Immunohistochemical quality controls and their purpose Control tissue should be biopsy or surgical specimens formalin fixed processed and paraffin embedded as soon as possible and in the same manner as the patient sample s Specimens must be handle
28. gative control reagent slide is created 17 Still in the Add slide dialog select HER2 Primary Antibody from the marker list Default protocols and all other settings remain unchanged 18 Click Add slide The test slide is created 19 Repeat steps 8 to 18 until all cases and patient test slides have been created 20 Next create the HER2 Control Slide Add it to the last case or create a new case for control slides depending on your standard laboratory practises Important note It is a requirement of the Bond Oracle HER2 IHC System for BOND MAX that a HER2 Control Slide is included in each run ie slide tray in order to validate the assay 21 In the Add slide dialog set tissue type to Positive tissue 22 Click Oracle control 23 Select the lot number of the HER2 Control Slide in the Lot No list The lot number is inscribed on the label area of the slide Important note The HER2 Control Slide must come from the same lot of the Bond Oracle HER2 IHC System for BOND MAX 24 Select HER2 Primary Antibody from the marker list Retain dispense volume staining mode process and protocol settings 25 Click Add slide to add the HER2 Control Slide 26 Finally add a positive in house tissue control slide 27 Deselect Oracle control 28 Select HER2 Primary Antibody from the marker list Retain dispense volume staining mode and process and protocol settings Tissue type remains Positive tissue 29 Click Add slide This completes slide c
29. he clinical study Of the 431 cases stained no result was obtained on three 3 occassions due to insufficient probe hybridization resulting in a total cohort of 428 cases All stained slides were scored by trained observers at two investigational sites For 2x2 concordance analysis the scores were interpreted as negative if the HER2 CEP17 gene amplification ratio was less than 2 0 and positive if greater than or equal to gt 2 0 following a 20 tumor cell count 2x2 Concordance Results The observed agreement for 428 samples between the two tests in a 2x2 analysis show a concordance of 87 6 375 428 with a 95 Cl of 84 to 90 The percentage Positive Agreement sensitivity or the ability of Bond Oracle HER2 IHC System for BOND MAX to correctly identify PathVysion positive cases the percentage of specimens scored positive by both Bond Oracle HER2 IHC System for BOND MAX and PathVysion out of all the PathVysion positive cases was 93 8 61 30 97 with a 95 Cl of 86 8 to 97 4 The percentage Negative Agreement specificity or the ability of the test to correctly identify PathVysion negative cases the percentage of specimens scored negative by both Bond Oracle HER2 IHC System for BOND MAX and PathVysion out of all the PathVysion negative cases was 85 8 284 331 with a 95 Cl of 81 6 to 89 2 See Table 8 PathVysion HER2 DNA Probe Kit Negative Positive Totals Bond Oracle HER2 0 1 284 6 290 IHC System for 24 41 30
30. hin run investigation indicated that 733 800 91 6396 test data points could be interpreted 40 data points were excluded due to presence of DCIS only and a further 27 data points could not be interpreted due to a loss of invasive tumor specific to 3 cores Variation in staining occurred 61 8 3296 out of a possible 733 staining events On 37 occasions variation from 3 to 2 n 20 and from 1 to 0 n 17 was observed and would therefore not represent a change from clinically positive to clinically negative or vice versa in a 2x2 data assessment The remaining 24 3 2796 occasions represented a change from clinically negative 0 or 1 to clinically positive 2 or 3 Pass value 96 7 95 Cl 95 15 to 97 81 B Between Run Precision Testing Between run precision testing of the Bond Oracle HER2 IHC System for BOND MAX was evaluated on a total of 24 consecutive sections taken from a TMA comprising of 20 invasive breast tumors and 24 HER2 Control Slides All slides were stained with the Bond Oracle HER2 IHC System for BOND MAX on the BOND MAX fully automated advanced staining system The slides were evaluated in 8 independent runs performed within the same laboratory on three separate occasions using a Bond Oracle HER2 IHC System for BOND MAX from the same manufacturing batch Stained slides were blinded and assessed in a randomized fashion by a single experienced observer to determine between run precision An evaluation of the slide
31. ideline 12 These quality control procedures should be repeated for each new antibody lot or whenever there is a change in assay parameters Human invasive infiltrating ductal breast carcinoma with known HER2 oncoprotein staining intensities from 0 to 3 and other suitably negative tissues are appropriate for assay verification Interpretation of Staining For the determination of HER2 oncoprotein expression only membrane staining pattern and intensity should be evaluated using the scale presented in Table 4 A pathologist using a bright field microscope should perform slide evaluation For evaluation of the immunohistochemical staining and scoring an objective of 10x magnification is appropriate The use of 20 40x objective magnification should be used in the confirmation of the score Cytoplasmic staining should be considered as nonspecific staining and is not to be included in the assessment of membrane staining intensity 14 To aid in the differentiation of 0 1 2 and 3 staining refer to the Bond Oracle HER2 IHC System for BOND MAX Interpretation Guide for representative images of the staining intensities Only specimens from patients with invasive breast carcinoma should be scored In cases with carcinoma in situ and invasive carcinoma in the same specimen only the invasive component should be scored Immunohistochemical Staining Pattern Score Assessment No staining is observed or membrane staining is observed in 0 Negative les
32. ies and the use of suitable control material and should be evaluated within the context of the patient s clinical history and other any diagnostic tests by a qualified pathologist The performance of the assay ie assessments of adequacy of both positive and negative controls and the interpretation of any immunohistochemical staining or its absence must be carried out in an appropriately accredited licensed laboratory under the supervision of a suitably qualified and experienced pathologist who is responsible for the overall assessment of the immunohistochemical assay and its interpretation B Product Specific Limitations This product is not intended for use in flow cytometry Performance characteristics have not been determined for flow cytometry False negative results may be seen as a result of the degradation of antigens in the tissue section Slides required for HER2 oncoprotein evaluation and tumor verification should be prepared at the same time To preserve antigenicity tissue sections mounted on slides Leica BOND Plus Slides product code S21 2113 should be stained within 4 6 weeks of sectioning when held at room temperature 20 25 C Following sectioning slides are recommended to be incubated for 12 18 hours at 37 C Sections which require further adherence may be incubated at 60 C for a further hour Minimal natural variation of immunohistochemical profile will be seen between growth batches of cell lines utilized within the B
33. mponents should only be utilized for monitoring the correct performance of processed tissues together with test reagents NOT as an aid in formulating a specific interpretation of patient samples If the positive control tissue fails to demonstrate appropriate positive staining results obtained with patient specimens should be considered invalid A multi tissue control block containing tumors representing all 4 HER2 grades may also be effectively utilized as appropriate in house control material In house Negative Control Tissue Component HER2 Primary Antibody If in house negative control components are used they should be fresh biopsy or surgical specimens fixed processed and embedded as soon as possible in the same manner as the patient sample s Use of control tissue known to be HER2 oncoprotein negative with each staining run verifies the specificity of the primary antibody and provides an indication of any nonspecific background staining The variety of different cell types present in most tissue sections offers internal negative control sites this should be verified by the user Normal breast ducts unassociated with tumor may provide a reference to the validity of the assay If specific staining occurs in the internal negative control tissue results with the patient specimens should be considered invalid The use of multi tissue control block representing all four HER2 grades may be utilized for the purposes of negative and postive co
34. ntrol tissues Patient Tissue HER2 Negative Control Use the supplied HER2 Negative Control in place of the HER2 Primary Antibody on a corresponding section for each patient test to evaluate nonspecific staining and allow accurate interpretation of specific HER2 oncoprotein staining at the antigenic site Patient Tissue HER2 Primary Antibody Positive staining intensity should be assessed within the context of any nonspecific background staining with the HER2 Negative Control As with any immunohistochemical test a negative result means that the antigen was not detected not that the antigen was absent in the cells tissue assayed Refer to Slide Screening Order Rationale Limitations Performance Evaluation and Immunoreactivity for specific information regarding Bond Oracle HER2 IHC System for BOND MAX immunoreactivity Assay Verification Prior to the initial use of any antibody or staining system in a diagnostic procedure the user should verify the antibody s specificity by testing it on a series of in house tissues with known immunohistochemical positive and negative profiles Referto Quality Control as previously outlined Page 10 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 English l English i and the quality control requirements of the CAP Certification Program for Immunohistochemistry and or CLSI formerly NCCLS Quality Assurance for Immunocytochemistry Approved Gu
35. ode P4 Storage RE Catalog number wo Invitro ted Manufacturer Fragile diagnostic medical device Ti Consult instructions for use V Contains sufficient for n tests E lt LAN Use by YYYY MM DD SN Serial Number HercepTest is a trademark of and subject to licences held by DakoCytomation Denmark A S Herceptin is a trademark of Genentech Inc and F Hoffmann La Roche Ltd Page 23 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014
36. ond Oracle HER2 IHC System for BOND MAX This natural variation is well within acceptable tolerance levels of a biological entity and does not affect the interpretation or performance of the system Characterization of the cell lines using both flow cytometry and in situ hybridization as presented in Table 5 are also subject to natural biological variation Technical and interpretational variation of control cell lines as assessed by fluorescent in situ hybridization is also reported 18 Assessment of the HER2 Control Slides should take into account all relevant expiry dates Store the Bond Oracle HER2 IHC System for BOND MAX at 2 8 C Do not freeze Return to 2 8 C immediately after use Any deviations from these conditions will invalidate the assay Do not replace Bond Oracle HER2 IHC System for BOND MAX reagents with any other components either supplied by Leica Biosystems or by other manufacturers To do so will invalidate the assay It is essential that all of the steps outlined in sections C to E Procedure are performed in the prescribed order Any deviation from this order will invalidate the assay It is essential that tissues fixed only in formalin based fixatives be used in the assay The use of any other type of fixative will invalidate the assay Tissue sections cut outside of the recommended thickness range have not been validated The use of any other section thickness may invalidate the assay Page 13 of 23 Leica Biosystems Bond O
37. oring Inter observer agreement between the two independent clinical sites Site B and Site C was 87 5 95 Cl 73 3 to 95 8 The agreement between Site B and Site C and Leica Biosystems Newcastle was 92 5 95 Cl 79 6 to 98 4 and 85 95 CI 70 1 to 94 29 respectively The analysis of total concurrence between the three observers A B C is 82 50 English l Troubleshooting Problem Probable Cause Remedial Action No Run aborted prior to Using BOND software confirm the presence immunohistochemical staining completion of any reportable errors during the staining run and address as instructed by the BOND software Incorrect protocol Ensure appropriate default to IHC Protocol selection H in the staining protocol field of the Add slide dialog Inadequate Ensure Dewax mode is selected in the deparaffinization of slides Preparation field of the Add slide dialog Inappropriate bulk reagents dispensed Ensure all BOND reagents have been allocated to appropriate bulk containers and placed into appropriate positions on the instrument Contamination of BOND Wash solution with sodium azide Use fresh BOND Wash solution prepared to appropriate working strength Weak specific immunohistochemical staining Inappropriate epitope retrieval Ensure appropriate BOND epitope retrieval reagents have been allocated into correct bulk containers and BOND software has defaulted to the a
38. pathology department for clinical testing and tested independently of other prognostic and or predictive factors with no bias introduced to the cohort Cohorts of 160 and 292 specimens were tested at Site 1 and Site 2 respectively Each cohort had an equal representation of equivocal positive 2 3 and negative 0 1 cases based on previously assigned HER2 IHC scores resulting in a total study population of 452 samples Twelve 12 samples were considered unsuitable due to lack of sufficient invasive tumor and were removed from the study A further nine 9 samples could not be scored as a result of tissue lifting from the slide surface resulting in a final study population of 431 samples All cases were stained with the HercepTest according to the manufacturer s instructions as specified in the package insert Sequential sections from each case were stained with the Bond Oracle HER2 IHC System for BOND MAX on board an automated Leica Biosystems BOND MAX fully automated advanced staining system All cases were de linked from unique patient identifying information and were accompanied by clinical data relating to tumor size tumor stage tumor grade and estrogen receptor status All stained slides were masked and scored in a randomized fashion by trained observers at two sites For 2x2 concordance analysis scores were interpreted as negative if the staining intensity was 0 or 1 and positive for scores of 2 or 3 For 3x3 concordance analysis
39. ppropriate epitope retrieval protocol HIER 25 min with ER1 97 Inappropriate fixation or processing of test specimen Ensure a formalin based fixative is used and that processing schedules are suitable for the specimen undergoing testing Bond Oracle HER2 IHC System for BOND MAX is being used outside its expiry date Ensure the Bond Oracle HER2 IHC System for BOND MAX used is within its specified expiry date Excessive specific immunohistochemical staining Inappropriate epitope retrieval Ensure appropriate BOND epitope retrieval reagents have been allocated into appropriate bulk containers and the BOND software has defaulted to HIER 25 min with ER1 97 Variation in fixation Ensure a formalin based fixative is used and that processing schedules are suitable for the specimen undergoing testing If possible retest case using another block If this is not possible assess the areas which show best fixation patterns in conjunction with a corresponding H amp E stained section Page 20 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev_B 27 10 2014 English i Problem Probable Cause Remedial Action Nonspecific background staining Inappropriate bulk reagents dispensed Ensure all BOND reagents have been allocated into appropriate bulk containers and placed into appropriate positions on the instrument Inadequate deparaffiniza
40. racle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 Cell Line Data Cell Line Bond Oracle HER2 HER2 Gene Amplification Status HER2 IHC System Receptor Load Profile per Cell HER2 Copy HER2 Chr17 Number Gene Ratio SK BR 3 3 4 3x10 13 35 3 55 MDA MB 453 2 1 4x105 5 73 2 05 MDA MB 175 1 6 3x104 3 33 1 20 MDA MB 231 0 9 3x10 3 15 1 13 HER2 receptor load analysis as assessed by flow cytometry HER2 Gene Amplification Status as assessed by dual probe HER2 Chromosome 17 FISH Table 5 HER2 Control Slide profile Clinical Concordance of Bond Oracle HER2 IHC System for BOND MAX v Dako HercepTest Part one of the study examined the suitability of the Bond Oracle HER2 IHC System for BOND MAX for use as an aid in determination of treatment with Herceptin trastuzumab therapy The study was designed to examine the concordance between the Bond Oracle HER IHC System and the Dako HercepTest considered as the gold standard for this assay The acceptance criterion was defined as greater than 75 overall concordance between the two tests with a 95 confidence interval Cl The study was conducted as a two site US based blinded evaluation Each investigational site was supplied with formalin fixed paraffin embedded breast cancer samples of known HER2 status Cases were selected in reverse consecutive order from the clinical archives representing the consecutive flow of cases into a histo
41. reation 30 Print slide labels All Oracle slide labels have OC printed on them The label for the HER2 Control Slide also includes the Bond Oracle HER2 IHC System for BOND MAX lot number 31 Label slides appropriately Page 8 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 English l English i 32 Open the lids of all Bond Oracle HER2 IHC System for BOND MAX containers and load the reagent tray onto the BOND MAX 33 Place slides onto the slide tray in the order indicated in section D Table 2 Apply new Covertiles 34 Load the slide tray onto the BOND MAX and press the Load Unload button 35 Confirm that the slides have been scanned and click the Run Play button on the System status screen 36 Ensure that the tray indicator field displays Proc OK and batch number and finish time are displayed 37 When the run is completed press the Load Unload button and remove the slide trays from the BOND MAX 38 Remove Covertiles and rinse the slides in de ionized water 39 Dehydrate clear and mount sections Quality Control Differences in tissue fixation processing and embedding in the user s laboratory may produce significant variability in results necessitating regular performance of in house controls in addition to the HER2 Control Slides supplied by Leica Biosystems in the Bond Oracle HER2 IHC System for BOND MAX Consult the quality control guidelines of CLSI
42. rections on Use i Storage andiStabllily terrere tet Lec ge Esa esr Re rers Xue Ere GERI ERES ener 5 Specimen PreparallOri c eerte tta te ett ata oe edax etn CE E EET TE T db EAE RR DICE re RR 5 Warnirigs and PreCautions cic 2 2 tene corte tea te Eee ee as ceca aqaa decedens ea ero Scc pesa f 5 liri 6 A Reagents required but not supplied 6 B Equipment required but not supplied 6 C Methodology ess 6 D Slide Layout 6 E Procedure Steps P Quality Control HER2 Control Slide HER2 Primary Antibody sse In house Positive Control Tissue Component HER2 Primary Antibody In house Negative Control Tissue Component HER2 Primary Antibody Patient Tissue HER2 Negative Control sess rennen Patient Tissue HER2 Primary Antibody Assay VerificallOn 5 ice ho a e ee ia Gl prete thee iei ite d Te dore lir deriv Interpretation of Stalinirig 2 2 imi ette rrr iae teen teer rat Seen Rese Slide Screening Order Rationale 11 1 HER2 Control Slide HER2 Primary Antibody d 2 In house Positive Control Tissue HER2 Primary Antibody 412 3 In house Negative Control Tissue Component HER2 Negative Control 412 4 Patient Tissue stained using the HER2 Negative Control 2212 5 Patient Tissue stained using the HER2
43. s from the between run investigation indicated that 456 480 95 00 test data points could be interpreted 24 data points could not be interpreted due to a loss of invasive tumor specific to 5 cores Variation in staining occurred 42 9 21 out of a possible 456 data points On 30 occasions variation from 3 to 2 n 10 and from 1 to 0 n 20 were observed and would therefore not represent a change from clinically positive to clinically negative or vice versa in a 2x2 data assessment The remaining 12 2 63 represented a change from clinically negative 0 or 1 to clinically positive 2 or 3 Pass value 97 37 95 Cl 95 90 to 98 77 C Lot to Lot Reproducibility To determine Lot to Lot reproducibility 3 lots of Bond Oracle HER2 IHC Systems were manufactured under GMP on 3 separate occasions and evaluated on 24 breast tumor sections 24 test data points taken from four different formalin fixed paraffin embedded tissue blocks representing 0 1 2 and 3 HER2 staining intensities and three HER2 Control Slides 12 control data points Three independent runs were performed within the same laboratory on three separate occasions each using a separate manufacturing lot of Bond Oracle HER2 IHC System for BOND MAX All slides were stained with the Bond Page 18 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev_B 27 10 2014 English l English i Oracle HER2 IHC System for BOND MAX on boar
44. s than 10 of the tumor cells 3 Faint barely perceptible membrane staining is detected in more than 1096 of the tumor cells The cells are only stained in 14 Negative part of their membrane Weak to moderate complete membrane staining is observed 5 Equivocal in more than 1096 of the tumor cells Weakly Positive Strong complete membrane staining is observed in more than 1096 of the tumor cells 3 Strongly Positive Table 4 Interpretation of HER2 staining Bond Oracle HER2 IHC System for BOND MAX staining results are interpreted as negative for HER2 oncoprotein expression with scores of 0 and 1 staining intensity equivocal weakly positive with a score of 2 staining intensity and strongly positive with a score of 3 staining intensity Bond Oracle HER2 IHC System for BOND MAX is not intended to provide prognostic information to the patient and or physician and has not been validated for that purpose For each staining assessment slides should be examined in the order presented below to determine the validity of the staining run and enable semi quantitative assessment of the staining intensity of the sample tissue Page 11 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev_B 27 10 2014 Slide Screening Order Rationale Slides should be screened in the following order 1 HER2 Control Slide HER2 Primary Antibody A valid assay with the Oracle HER2 Control Slide shows the following
45. tion of slides Nonspecific immunohistochemical cross reaction in tissue Ensure Dewax is selected in the Preparation field of the Add slide dialog Refer to Bond Oracle HER2 IHC System description of normal tissue cross reactivity refer to Table 9 Nonspecific immunohistochemical cross reaction with areas of tissue necrosis Ensure a formalin based fixative is used and that processing schedules are suitable for the specimen undergoing testing If possible retest case using another block If this is not possible assess in conjunction with a corresponding H amp E stained section areas which show best fixation patterns Drying artifact following completion of a staining run If slides are to be placed on an overnight run itis recommended that the BOND delayed start functionality is used Ensure that there is an adequate volume of distilled or de ionized water available to dispense on the slides for this period to ensure the slides do not dry out Sections adhered to slides with the aid of starch additives Use unstarched slides e 9 Leica BOND Plus Slides product code S21 2113 Tissue detached from patient control slide s Use of incorrect type of slides or inadequate draining of section Ensure appropriate slides are used for patient control sections e g Leica BOND Plus Slides product code S21 2113 Ensure slides receive adequate draining and are incubated at 12 18 hours at 37 C
46. us biotin staining which may be seen with streptavidin biotin detection systems does not occur Expression of HER2 The HER2 oncoprotein is expressed at levels detectable by immunohistochemistry in up to 2096 of adenocarcinomas from various sites Between 1096 and 2096 of invasive ductal carcinomas of the breast are positive for HER2 oncoprotein 9 9096 of cases of ductal carcinoma in situ DCIS of comedo type are positive 10 together with almost all cases of Paget s disease of the breast 11 Clinical Concordance Summary The Bond Oracle HER2 IHC System for BOND MAX was developed to provide an alternative to the investigational Clinical Trial Assay CTA used in the Herceptin clinical studies The performance of the Bond Oracle HER2 IHC System for BOND MAX for determination of HER2 oncoprotein overexpression was evaluated in an independent study comparing the results of the Bond Oracle HER2 IHC System for BOND MAX to the Dako HercepTest on 431 breast tumor specimens of US origin None of these tumor specimens were obtained from patients in the Herceptin clinical trials The results indicated a 92 3496 concordance in a 2x2 analysis 95 confidence intervals of 89 42 to 94 67 and 86 54 in a 3x3 analysis 95 confidence intervals of 82 9596 to 89 6296 between the results from the two assays Page 3 of 23 Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN US Rev B 27 10 2014 Principle of Procedure The Bond
47. ver at Leica Biosystems Newcastle to determine between laboratory reproducibility An evaluation of the slides from the between laboratory reproducibility investigation indicated that 1477 1920 76 9396 test data points could be interpreted 443 test data points could not be interpreted due to a Inadequate performance of the HER2 Control slide on 2 24 occasions resulting in 2 runs 160 test data points being removed This event occurred once at Site A and once at Site B 80 data test points per investigational site removed b Deviation from the test plan at Site C in which 24 slides in total were manually counterstained with hematoxylin following Bond Oracle HER2 IHC System for BOND MAX staining This resulted in excessive counterstaining of both HER2 control slides and TMA test data points resulting in 240 data points being removed C Loss of invasive tumor resulting in 23 test data points being removed This event occurred on 23 occasions at Site A and was a direct result of loss of tissue in the TMA block on production of the 192 consecutive TMA sections required to complete this investigation d Uninterpretable staining due to inadequate washing by the BOND MAX fully automated advanced staining system resulting in 20 data points being removed An evaluation of the interpretable slides in the between laboratory precision investigation indicated that variation in staining occurred 79 5 2896 out of a possible 1477 staining events Of these
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