HER2 FISH pharmdx. Assay Kit

Transcription

1 PATHOLOGY HER2 FISH pharmdx Assay Kit

2 HER2 FISH pharmdx Clarity You Can Count On Reliable Results at a Glance The robust HER2 FISH pharmdx assay offers bright and distinct signals for easy and fast reading of slides, yielding consistent counting accuracy. HER2 FISH pharmdx kit is a direct fluorescence in situ hybridization (FISH) assay designed to quantitatively determine HER2 gene amplification in formalin-fixed, paraffin-embedded breast cancer tissue specimens. The HER2 FISH pharmdx kit is FDA approved as an aid in the assessment of patients when Herceptin (trastuzumab) treatment is being considered. IHC and FISH Predictor of Clinical Benefit from Herceptin Accurate assessment of a patient s HER2 status is vital for correctly identifying women likely to benefit from Herceptin. Patients most likely to benefit are those whose tumors have high HER2 receptor overexpression and/or amplification of the HER2 gene.1 Herceptin increases the benefits of chemotherapy in HER2 positive breast cancers.2,3,4 Initial trials treating women with tumors scoring IHC 2+ and IHC 3+ demonstrated clinical benefit from Herceptin. Among those patients, those who tested IHC 3+ or FISH-positive showed the greatest benefit.4 60 FISH + IHC3+ IHC2+/ CT Alone Herceptin + Chemotherapy (CT) by HER2+ Status Overall response rate (%) CT + Herceptin 2,3

3 Most clinical guidelines recommend HER2 testing with IHC and/or FISH at the time of initial breast cancer diagnosis and confirmation of IHC 2+ results with FISH. 5,6,7 Dako s HercepTest is a semi-quantitative immunohistochemical (IHC) assay to determine HER2 protein overexpression. It is FDA approved as an aid in assessing patients considered for Herceptin therapy. HercepTest, in combination with HER2 FISH pharmdx, delivers a complimentary IVD HER2 assay package to aid in id entifying these patients. Use of HER2 FISH pharmdx in a HER2 Testing Algorithm 5,6,7 Patient Tumor Sample HercepTest HER2 FISH pharmdx SCORE 0 1+ Negative Negative 2+ Weakly Positive (Equivocal) 3+ Positive NEGATIVE Non-amplified (Score <2) POSITIVE Amplified (Score 2) HER2 FISH pharmdx NEGATIVE Non-amplified (Score <2) POSITIVE Amplified (Score 2) Report to Oncologist for Herceptin Consideration HercepTest Highly Concordant to FISH Breast Cancer Specimen IHC Status Percentage of All Cases 6 Concordance IHC/FISH 8 (HercepTest/PathVysion) (n = 426 from 37 Centers) Reliability of HER2 Status 9 0/ % Reliably Negative % Weakly Positive (Equivocal), Require Confirmatory Testing %* Reliably Positive * Of the 102 IHC 3+ tumors, 6 were found to be FISH negative, 5 of these had FISH scores between 1.75 and 2.00 and 1 had a higher number of copies of chromosome 17. PharmacoDiagnostic and pharmdx are registered trademarks of Dako A/S. HercepTest and Herceptin are registered trademarks of Genentech, Inc., subject to licenses held by Dako and F. Hoffmann-La Roche Ltd.

4 Clearly Effective from a Scientific View HER2 Testing: Biology The HER2 gene is located on chromosome 17 and encodes the HER2 protein (p185 HER2). It is present in two copies in all normal diploid cells. In some breast cancer tumors the HER2 gene amplifies as part of the malignant transformation. HER2 Testing: IHC and FISH Immunohistochemistry (IHC) measures the level of HER2 receptor overexpression, while fluorescence in situ hybridization (FISH) quantifies the level of HER2 gene amplification. Together they are the most commonly used methods of determining HER2 status in routine diagnostic settings.6,10 IHC and FISH Targets for HER2 Testing FISH HER2 Receptors DNA IHC mrna Transcript Ligand Amplified result, Score >2. Breast cancer specimen stained with HER2 FISH pharmdx. Breast Cancer Cell Positive result, Score 3+. Breast cancer specimen stained with HercepTest. HER2 gene amplification is the underlying biological change that results in HER2 overexpression. HER2 FISH pharmdx Assay HER2 FISH pharmdx quantitatively determines interphase HER2 gene amplification in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue. HER2 FISH pharmdx uses a probe mix to quantitatively determine HER2 gene amplification. Dako s HER2 FISH pharmdx includes a chromosome 17 reference probe to correct for HER2 signal number in 11 the event of chromosome 17 aneusomy.

5 Cytogenetic Ideogram CEN-17 HER HER2 gene, amplified copy number, ratio HER2/CEN CEN-17 PNA probe directly labeled with fluorescein (FITC) targets the centromeric region of the chromosome (green signals). HER2 DNA probe directly labeled with Texas Red fluorochrome targets the HER2 amplicon (red signals). Results are expressed as a ratio of HER2 gene copies (red signals) per number of chromosome 17 copies (green signals). HER2 FISH pharmdx Technique Probe DNA labeled with fluorescent dye Denaturation of probe and target nucleic acid Labeled probe hybridizes to HER2 gene on chromosome 17 Denature and hybridize Fluorescence In Situ Hybridization Hybridization signals observed using fluorescent microscope

6 The Solution You ve Been Looking For Set your sights on great counting accuracy and productivity with the HER2 FISH pharmdx kit. Featuring a distinct, enhanced signal and improved assay robustness for results you can count on. HER2 FISH pharmdx Demonstrates Proven Results Amplified Accuracy In a multicenter comparison of FFPE breast cancer samples reflecting the natural range of amplification (ratios ; ; and ) tested in three separate runs, the sites involved were in complete agreement with the classification of HER2 status as HER2 negative or positive. 12 Specimen Non-amplified Altered but non-amplified Low-level amplification High-level amplification HER2 Negative HER2 Positive HER2 Testing. 100% accurate in the classification of HER2 status.

7 Clinical Confidence The clinical utility of the Dako HER2 FISH pharmdx kit was investigated using specimens obtained from cases enrolled in the Danish Breast Cancer Cooperative Group (DBCG) (89-D) study. The study included 200 archived specimens from patients diagnosed with grade II-III breast cancer. Evaluable specimens (190) were successfully hybridized and scored. Number of Observations HER2 FISH pharmdx (-) Amplified HER2 FISH pharmdx (+) Amplified Total PathVysion (-) amplified PathVysion (+) amplified Total Concordance = 93.68% (CI95: 90-97%) Concordance of HER2 FISH pharmdx to Vysis PathVysion Test results included 12 discrepancies between HER2 FISH pharmdx kit and Vysis PathVysion HER2 test. The tested population was enriched with HercepTest 2+ specimens. 12 Alternative Interpretation Saves Time and Maintains Concordance Extensive microscopy makes FISH time consuming. Therefore, timesaving counting methods were used in a comparison of HER2 FISH pharmdx kit to Vysis PathVysion HER-2 DNA Probe kit. Concordance remained unchanged for all investigated counting methods. 12 HER2 FISH pharmdx Results based on counting 10 nuclei 20 nuclei* 30 nuclei 60 nuclei PathVysion (-) Amplified n = 97 (-) (+) Amplified Amplified PathVysion (+) Amplified n = 48 Concordance 95% CI (-) (+) Amplified Amplified % % % % *Number of nuclei recommended

8 Noticeable Advantages at Every Step Timesaving Protocol for Optimal Results The HER2 FISH pharmdx kit protocol is simply a better choice compared to other commercially available kits. HER2 FISH pharmdx has fewer procedural steps, so it s faster and easier to use with a lower probability of error. It also uses fewer toxic materials such as formalin and formamide decreasing risk to laboratory personnel. Three simple steps for clear results with HER2 FISH pharmdx: Specimen pre-treatment Pre-treat specimens with Pre-Treatment Solution. Apply Ready-to-Use Pepsin enzyme to prepare slides for hybridization. Hybridization Apply Ready-to-Use, dual-color, HER2/CEN-17 Probe Mix to the specimen. Denature the target and probe nucleic acid. Allow the probe and target nucleic acid to hybridize. Use the Hybridizer for automated procedure. Wash the slides in Stringent Wash Buffer to remove incompletely hybridized nucleic acid hybrids. Interpretation Using a fluorescence microscope and appropriate filters, enumerate the probe signals and calculate ratio. Simplified Procedure Compared to Other HER2 FISH Assays Total assay time is 1.2 hours less than conventional FISH. Fewer steps No post-fixation step following pre-treatment. Only one phase for denaturation. Room temperature incubation of RTU Pepsin For In Vitro Diagnostic Use. FDA approved as an aid in the assessment of patients for whom Herceptin (trastuzumab) treatment is being considered.

9 Fast and Accurate Quantitative Interpretation Filter requirements Count signals with a fluorescence microscope equipped with a 100-watt mercury lamp. Use a specific DAPI filter and either a high quality Texas Red/FITC double filter or specific Texas Red and FITC single filters. Adequate filters are crucial for correct interpretation. Fluorochrome FITC Texas Red Excitation Wavelength 495 nm 596 nm Emission Wavelength 520 nm 615 nm HER2 FISH pharmdx signals Enhanced signals are bright, distinct and easy to evaluate. The centromeric probe (CEN-17) serves as a control for proper hybridization and allows differentiation of chromosome 17 aneusomy from true amplification. Scan path for signal counting. Non-amplified result breast carcinoma stained with HER2 FISH pharmdx. Amplified result breast carcinoma stained with HER2 FISH pharmdx.

10 Scoring Guide Assessable Tissue Score only invasive component of carcinoma. Avoid necrotic areas and cells with ambiguous borders. Disregard nuclei with weak signal intensity and non-specific or high background staining. Signal Location Locate the tumor within the context of an H&E stained slide and evaluate the same area on the FISH-stained slide. Scan several areas of the tumor to account for possible heterogeneity. Select distinct tumor areas for assessment. Begin analysis in upper left quadrant of selected area. Scan from left to right, counting signals in each tumor nucleus. Adjust microscope focus to locate all signals in individual nuclei. Signal Enumeration Count HER2 (red) and CEN-17 (green) signals in 20 nuclei in representative tumor areas. Calculate the HER2/CEN-17 ratio by dividing the total number of HER2 signals by the total number of CEN-17 signals. Two signals of the same size, separated by a distance equal or less than the diameter of one signal, are counted as one signal. Nuclei with high levels of HER2 gene amplification (red) may exhibit formation of signal clusters. Estimate the HER2 signal. HER2 clusters may obscure CEN-17 (green) signals; check using specific FITC filter. Nuclei exhibiting signals of only one color should not be scored. Do not score nuclei demonstrating over-digestion. Ratio of HER2/ CEN-17 Signals <2 2 HER2 Gene Status Non-amplified Amplified Result Negative Positive Results at or near the cut-off ( ) should be interpreted with caution. In those cases, count an additional 20 nuclei and recalculate. To download a PDF of the Scoring Sheet go to:

11 Counting Guide One green signal (split) indicates the presence of one copy of chromosome 17.* One red signal indicates the presence of one copy of the HER2 gene. The ratio of HER2 to CEN-17 is 1/1 = 1; non-amplified. Three green signals (one out of focus) indicate the presence of three copies of chromosome 17. Three red signals indicate the presence of three copies of the HER2 gene. The ratio of HER2 to CEN-17 is 3/3 = 1; non-amplified. Two green signals indicate the presence of two copies of chromosome 17. Three red signals (two split signals) indicate the presence of three copies of the HER2 gene.* The ratio of HER2 to CEN-17 is 3/2 = 1.5; non-amplified. One green signal indicates the presence of one copy of chromosome 17. Five red signals indicate the presence of five copies of the HER2 gene. The ratio of HER2 to CEN-17 is 5/1 = 5; amplified. Three green signals indicate the presence of three copies of chromosome 17. Approximately 12 red signals indicate the presence of 12 copies of the HER2 gene (cluster estimation). The ratio of HER2 to CEN-17 is 12/3 = 4; amplified. Do not score (nuclei are overlapping, not all areas of nuclei are visible). Do not score nuclei with signals of only one color (two green signals). Do not score (over-digested nuclei). Cluster of red signals hiding green signals. Check the green signals with a specific FITC filter, or do not score. * Two signals of the same size, separated by a distance equal to or less than the diameter of one signal, are counted as one signal.

12 HER2 FISH pharmdx Complete Kit Includes: Pre-treatment solution (20x concentrated) Pepsin, ready-to-use HER2/CEN-17 probe mix Stringent wash buffer (20x concentrated) Fluorescence mounting medium, containing DAPI Wash buffer (20x concentrated) Coverslip sealant HER2 FISH pharmdx Product Name Size Code HER2 FISH pharmdx Kit 20 tests K5331 HercepTest (for manual use) 35 tests K5204 HercepTest for the Dako Autostainer/Autostainer Plus 50 tests K5207 HercepTest for Automated Link Platforms 50 tests SK001 Hybridizer 120V S2450 Hybridizer 240V S2451 Supporting Literature For information about supporting literature, contact your local Dako representative or visit 1. Bilous M, Dowsett M, Hanna W, Isola J, Lebeau A, Moreno A, et al. Current perspectives on HER2 testing: a review of national testing guidelines. Mod Pathol 2003;16: Slamon DJ Study # H0648g- Response Rate (%) Newly defined population. Correlating HER2 Testing. Methodologies with Response: IHC versus FISH. ASCO presentation at 36th Annual Meeting. May 19, Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344: Herceptin (Trastuzumab) Full Prescribing Information. Genentech, Inc., USA. November Wolff A, Hammond E, et al. American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer. Arch Pathol Lab Med Jan; 131: Bilous M, Ades C, Armes J, Bishop J, Brown R, Cooke B, et al. Predicting the HER2 status of breast cancer from basic histopathology data: an analysis of 1500 breast cancers as part of the HER2000 International Study. The Breast 2003;12: Zarbo, R, Hammond, E. Conference Summary, Strategic Science Symposium, Her2/neu of Breast Cancer Patients in Clinical Practice, Archives of Pathology & Laboratory Medicine, Arch Pathol Lab Med, 2003;127: Dowsett M, Bartlett J, Ellis IO, Salter J, Hills M, Mallon E, et al. Correlation between immunohistochemistry (HercepTest) and fluorescence in situ hybridization (FISH) for HER2 in 426 breast carcinomas from 37 centres. J Pathol 2003;199: Di Leo A, Dowsett M, Horten B, Penault-Llorca F. Current status of HER2 testing. Oncology 2002;63 Suppl 1:S Bell R. What can we learn from Herceptin trials in metastatic breast cancer. Oncology 2002;63 Suppl 1:S39-S Ellis IO, Dowsett M, Bartlett J, Walker R, Cooke T, Gullick W, et al. Recommendations for HER2 testing in the UK. J Clin Pathol 2000;53: HER2 FISH pharmdx Kit Package Insert. Dako Denmark A/S May 2005 For In Vitro Diagnostic Use. FDA approved as an aid in the assessment of patients for whom Herceptin (trastuzumab) treatment is being considered. Corporate Headquarters Denmark Distributors in more than 60 countries Australia Austria Belgium +32 (0) Brazil Canada China Denmark Finland France Germany Ireland Italy Japan The Netherlands Norway Poland Spain Sweden Switzerland United Kingdom +44 (0) United States of America MAY10