English Bond™ Oracle™ HER2 IHC System Instructions For Use For use on Leica Biosystems’ BOND™ fully automated, advanced staining system.
Intended Use..............................................................................................................................3 Summary and Explanation.......................................................................................................3 Background...........................................................................................................................................3 Expression of HER2..........................................................................
English Intended Use For in vitro diagnostic use Bond Oracle HER2 IHC System is a semi-quantitative immunohistochemical (IHC) assay to determine HER2 (Human Epidermal Growth Factor Receptor 2) oncoprotein status in breast cancer tissue processed for histological evaluation. The Bond Oracle HER2 IHC System is indicated as an aid in the assessment of patients for whom Herceptin® (trastuzumab) treatment is being considered (see Herceptin® package insert).
The Bond Oracle HER2 IHC System 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.
English 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 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 used is within its designated expiry date.
English 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.
English Slide Position Slide Description Reagent Tissue Type 1 Case 1 *HER2 Negative Control Test 2 Case 2 *HER2 Negative Control Test 3 Case 3 *HER2 Negative Control Test 4 Case 4 *HER2 Negative Control Test 5 Case 1 *HER2 Primary Antibody Test 6 Case 2 *HER2 Primary Antibody Test 7 Case 3 *HER2 Primary Antibody Test 8 Case 4 *HER2 Primary Antibody Test 9 HER2 Control Slide *HER2 Primary Antibody Positive 10 In-house Tissue Control *HER2 Primary Antibody Positive
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 µL 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.
English 34.Load the slide tray onto the BOND 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. 38. Remove Covertiles and rinse the slides in de-ionized water. 39.Dehydrate, clear and mount sections.
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 Interpretation Guide).
English and/or CLSI (formerly NCCLS) Quality Assurance for Immunocytochemistry, Approved Guideline (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.
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: • 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.
English 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.
Cell Line BOND Oracle HER2 IHC System Profile HER2 Receptor Load per Cell* HER2 Gene Amplification Status+ HER2 Copy Number HER2:Chr17 Gene Ratio SK-BR-3 3+ 4.3x105 13.35 3.55 MDA-MB-453 2+ 1.4x10 5 5.73 2.05 MDA-MB-175 1+ 6.3x104 3.33 1.20 MDA-MB-231 0 9.3x10 3.15 1.13 3 *HER2 receptor load analysis as assessed by flow cytometry. HER2 Gene Amplification Status as assessed by dual probe (HER2:Chromosome 17) FISH. + Table 5.
English 2x2 Concordance Results In this primary analysis the test results from the two tests (Bond Oracle HER2 IHC System 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).
Clinical Concordance of Bond Oracle HER2 IHC System to PathVysion HER-2 DNA Probe Kit Part 2 of the study was designed to examine the concordance between the Bond Oracle HER2 IHC System and the Abbott Molecular PathVysion HER-2 DNA Probe Kit, considered as the ‘gold standard’ for gene assessment reflex assay used in conjunction with HER-2 immunohistochemistry. This study was performed at the same investigational sites and used the same study cohort as in Part 1.
English Immunoreactivity – Normal Panel Normal Tissue Type Staining Pattern HER2 Primary Antibody HER2 Negative Control Adrenal Negative Negative Brain, Cerebellum Negative Negative Brain, Cerebrum Negative Negative Breast Negative Negative Bone Marrow Negative Negative Colon Negative Negative Esophagus Negative Negative Eye Negative Negative Hypophysis Moderate cytoplasmic staining observed in hypophyseal cells (1/3) Negative Kidney Negative Negative Larynx Negative Nega
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.
English masked and assessed in a randomized fashion by a single trained observer to determine Lotto-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. Staining with the Bond Oracle HER2 IHC System is consistent across manufacturing batches. D.
F. Between Instrument Precision (BOND-MAX v BOND-III) Between instrument precision testing using the Bond Oracle HER2 IHC System was performed at a single independent European investigational site. The samples tested were obtained from formalin-fixed, paraffin-embedded whole sections from ninety-nine invasive breast cancer cases (needle core and resection specimens).
English Troubleshooting Problem Probable Cause Remedial Action No immunohistochemical staining Run aborted prior to completion Using BOND software, confirm the presence of any reportable errors during the staining run and address as instructed by the BOND software. Incorrect protocol selection Ensure appropriate default to *IHC Protocol H in the staining protocol field of the Add slide dialog.
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 deparaffinization of slides Ensure *Dewax is selected in the Preparation field of the Add slide dialog.
English 5. Lewis GD, Figari I, 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.