PATHOLOGY
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 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.
HER2
FISH pharmDx
™Clarity You Can Count On
Herceptin + Chemotherapy (CT) by HER2+ Status2,3 Overall response rate (%)
0 20 FISH + IHC3+ CT Alone CT + Herceptin 40 60 IHC2+/3+ 27 31 32 54 56 50
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 immuno-histochemical (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.
Breast Cancer Specimen IHC Status
0/1+ 2+ 3+ Percentage of All Cases6 74-83 9-15 11-12 Reliability of HER2 Status9 Reliably Negative Weakly Positive (Equivocal), Require Confirmatory Testing
Reliably Positive Concordance IHC/FISH8 (HercepTest/PathVysion) (n = 426 from 37 Centers) 99.3% 50% 94%*
* 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.
HercepTest Highly Concordant to FISH Use of HER2 FISH pharmDx in a HER2
Testing Algorithm 5,6,7
NEGATIVE
Non-amplified (Score <2) Amplified (Score ≥2)POSITIVE
HER2 FISH pharmDx™
HER2 FISH pharmDx
HercepTest™
0
Negative
SCORE
1+
Negative Weakly Positive
2+
(Equivocal)3+
Positive NEGATIVE Non-amplified (Score <2) POSITIVE Amplified (Score ≥2)Patient Tumor Sample
Report to Oncologist for Herceptin® Consideration
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 over-expression, 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
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 the event of chromosome 17 aneusomy.11
Clearly Effective from a Scientific View
Positive result, Score 3+. Breast cancer specimen stained with HercepTest™.
Amplified result, Score >2. Breast cancer specimen stained with HER2 FISH pharmDx.
IHC and FISH Targets for HER2 Testing
Breast Cancer Cell
FISH IHC Ligand Ligand HER2 Receptors DNA mRNA Transcript
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
Hybridization signals observed using fluorescent microscope Fluorescence In Situ Hybridization
Denature and hybridize
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).
13 Cytogenetic Ideogram 12 11.2 11.1 11.1 11.2 12 21.1 21.2 21.3 22 23 24 25 HER2 CEN-17
HER2 gene, amplified copy number, ratio HER2/CEN-17 ≥2.
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 0.9–1.2; 1.4–1.7; 3.0–4.0 and 5.0–8.0) tested in three separate runs, the sites involved were in complete agreement with the classification of HER2 status as HER2 negative or positive.12
The Solution You’ve Been Looking For
Specimen
Non-amplified Altered but non-amplified
Low-level amplification High-level amplification HER2 Negative 15 15 0 0 HER2 Positive 0 0 15 15
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.
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 Concordance = 93.68% (CI95: 90-97%) HER2 FISH pharmDx™ Results based on counting 10 nuclei 20 nuclei* 30 nuclei 60 nuclei (-) (+) Amplified Amplified 95 2 96 1 95 2 95 2 PathVysion (-) Amplified n = 97 PathVysion (+) Amplified n = 48 Concordance 95% CI (-) (+) Amplified Amplified 4 44 5 43 4 44 5 43 96% 93-99 96% 93-99 96% 93-99 95% 92-99
*Number of nuclei recommended Number of Observations
PathVysion (-) amplified PathVysion (+) amplified
Total
HER2 FISH pharmDx (-) Amplified
128 8 136
HER2 FISH pharmDx (+) Amplified 4 50 54 Total 132 58 190
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.
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
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.
Noticeable Advantages at Every Step
For In Vitro Diagnostic Use. FDA approvedas an aid in the assessment of patients for whom Herceptin® (trastuzumab) treatment is being consid-ered.
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.
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.
Fluorochrome FITC Texas Red Excitation Wavelength 495 nm 596 nm Emission Wavelength 520 nm 615 nm
Non-amplified result breast carcinoma stained with HER2 FISH pharmDx.
Amplified result breast carcinoma stained with HER2 FISH pharmDx.
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 (1.8 – 2.2) 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:
* 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.
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.
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).
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.
Cluster of red signals hiding green signals. Check the green signals with a specific FITC filter, or do not score.
Do not score (over-digested nuclei).
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 www.dako.com
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
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;16:173-82. 2. 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, 2000.
3. 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;344:783-92.
4. Herceptin® (Trastuzumab) Full Prescribing Information. Genentech, Inc., USA. November 2006.
5. 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. 2007 Jan; 131:18-43.
6. 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;12:92-8.
7. 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: 549-553.
8. 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;199:418-23.
9. Di Leo A, Dowsett M, Horten B, Penault-Llorca F. Current status of HER2 testing. Oncology 2002;63 Suppl 1:S25-32.
10. Bell R. What can we learn from Herceptin® trials in metastatic breast cancer. Oncology
2002;63 Suppl 1:S39-S46.
11. 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;53:890-2.
12. 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.
00 19 5 04 M AY 10 Australia
+61 2 9502 8700 Canada +1 905 858 8510 France +33 1 30 50 00 50 Japan +81 3 5802 7211 Spain +34 93 499 05 06 United States of America +1 805 566 6655
Austria
+43 1 408 43 34 0 China+86 21 6327 1122 Germany +49 40 69 69 470 The Netherlands +31 20 42 11 100 Sweden +46 8 556 20 600
Belgium
+32 (0) 16 38 72 20 Denmark +45 44 85 97 56 Ireland +353 1 479 0568 Norway+47 23 14 05 40 Switzerland +41 41 760 11 66
Brazil
+55 11 50708300 Finland +358 9 348 73 950 Italy +39 02 58 078 1 Poland +48 58 661 1879 United Kingdom +44 (0)1 353 66 99 11 Distributors in more than 60 countries Corporate Headquarters Denmark +45 44 85 95 00 www.dako.com
