What is HER-2/neu?
HER-2/neu, also known as c-erbB2, is a gene that plays a key role in the regulation of cell growth. The HER-2 gene is amplified in human breast, ovarian, and other cancers, and plays an important role in the progression of many tumors. This amplification is characterized by increased transcription of mRNA and subsequent overexpression of the gene product. Approximately 20-25% of breast cancers have amplification of the HER-2 gene. Detection of HER-2 amplification is important in the diagnosis, prognosis, selection of appropriate therapy and prediction of therapeutic outcome in certain cancers4.
How important is HER-2 amplification in the treatment of breast cancer?
HER-2 amplification and subsequent overexpression of the gene product have emerged as a key prognostic and therapeutic marker in breast cancer. Amplification of this gene is found to be associated with rapid proliferation, shorter disease-free survival and poorer overall survival in both node-negative and node-positive ductal breast cancers. With the advent of monoclonal targeted therapies such as Herceptin® ® (Trastuzumab), accurate HER-2 assessment is a critical component in determining appropriate therapy selection. Both the National Comprehensive Cancer Network (NCCN®) and the American Society of Clinical Oncology (ASCO®) recommend HER-2 assessment for all invasive breast cancers.
What is the connection between HER-2 status and the selection of adriamycin-based therapy?
An interaction between HER-2 amplification and response to adriamycin-based therapies has been demonstrated. Specifically, HER-2 amplification is associated with improved response to intensified adriamycin-based chemotherapies. Clinical trials demonstrate that patients with HER-2 amplification may benefit from dose intensive adriamycin-based therapy based on disease-free and overall survival. Dose intensification of adriamycin did not benefit women without HER-2 amplification.5
What is the connection between HER-2 status and Herceptin® therapy selection?
The Herceptin package insert states that detection of HER-2 status is necessary for selection of patients appropriate for Herceptin therapy. In a single-arm study of Herceptin as a single agent no relapsed metastatic breast cancer, the overall response rate in 173 patients with FISH positive tumors tested was 19%, whereas no objective responses were observed in patients with FISH( -) tumors. All patients' specimens had previously demonstrated over expression of HER-2 at the 2+ or 3+ level by IHC. For more information on HER-2 testing and Herceptin, visit www.herceptin.com.
Why is accurate assessment of HER-2 status essential?
HER-2 status is a strong, independent predictor of prognosis and disease free progression. Accurate assessment of HER-2 status is important in selecting appropriate therapy, including Herceptin therapy.
The risks of incorrect HER-2 status assessment are significant: false positive assessments may lead to inappropriate medical decisions, causing a patient to undergo unnecessary therapy. Conversely, a false negative assessment may deprive a patient of potentially beneficial therapy.
Should one be assessing HER-2 at the DNA level or the gene product (protein) level?
Extra copies of the HER-2 gene drive excess protein on the surface of the cell. HER-2 assessment at a direct molecular level by FISH has been shown to be more accurate than IHC antibody-based tests. Responses in IHC 2+ and 3+ MBC patients treated with single-agent Herceptin were seen exclusively in FISH positive patients.2
What are the most accurate methods available for HER-2 status assessment?
Tests for HER-2 status measure either gene copy number or presence of protein receptors. The two most widely used technologies and the only two approved by the FDA in the United States are Immunohistochemistry (IHC) and Fluorescence in situ Hybridization (FISH). Chromogenic in situ Hybridization (CISH) and Silver in situ Hybridization (SISH) are two other technologies that measure HER-2 gene copy number. All of these techniques are routinely utilized on formalin-fixed, paraffin-embedded breast tissue.
While IHC has been a widely used technique, questions have been raised as to which technique, either FISH or IHC, yields the most reliable and accurate detection of HER-2 status. From the clinical pathology perspective, IHC inherently possesses several key shortcomings that can interfere with the accurate assessment of HER-2 status. Formalin-fixation of tissues is important in stabilizing cellular morphology and it introduces variables for IHC determination that are not within the laboratory's power to control. Specifically, formalin-fixation can cause destruction of the HER-2 epitope, which can lead to false negative IHC results. To help minimize this shortcoming, antigen retrieval techniques have now been employed. However, these antigen retrieval techniques cause a significant number of false positive IHC results.
Direct FISH technology targets stable DNA at a molecular level within formalin-fixed, paraffin-embedded specimens thereby overcoming the misclassifications associated with IHC. Direct FISH is a very sensitive method that yields higly accurate, reliable and reproducible results simply from counting the number of fluorescent signals. From these unambiguous results, the pathologist can make accurate HER-2 assessments, eventually leading oncologists and patients to the most efficacious selection of therapy.
What is the PathVysion HER-2 DNA Probe Kit?
The PathVysion HER-2 DNA Probe Kit utilizes patented, direct labeled Fluorescent in situ Hybridization DNA probes to yield definitive results at the molecular level. FISH is a molecular genetic technique employing a fluorescent DNA probe that produces a bright microscopic signal when it selectively attaches to the gene or chromosome specific complementary DNA. The PathVysion HER-2 DNA Probe Kit contains two probes. One probe is directed to the HER-2 gene and the second DNA probe attaches itself to the chromosome 17 centromere. The signals appear as orange and green fluorescent dots for the HER-2 gene and chromosome 17, respectively. This enables a ratio of HER-2 to chromosome 17 signals to be calculated thereby correcting for aneuploidy of chromosome 17, which may not represent an amplified HER-2 state. Through this direct labeled FISH technology, the PathVysion HER-2 assay provides an accurate, reliable and reproducible means for assessing HER-2 status in breast cancer.
What is direct labeled FISH?
FISH has become a main-stream technology routinely used in diagnostic laboratories. Two kinds of FISH technology are commercially available - - direct labeled and indirect labeled.
Direct labeled FISH uses probes that have been pre-labeled with a specific fluorophore, allowing the fluorescent signal to be bound to the target in a single hybridization step. This method is more advanced, has fewer steps, is easier-to-read and has faster time-to-result. With PathVysion, HER-2 assessment is accomplished with a sequence specific DNA probe for the HER-2 gene and a second, separate DNA probe labeled in a different fluorophore for the repetitive sequences at the centromere of chromosome 17 (the HER-2 gene resides on chromosome 17). Direct FISH is Abbott Molecular's specialty.
Indirect labeled FISH involves probes prelabeled with a hapten, most commonly digoxigenin. After hybridization, fluorochrome-labeled antibodies to the hapten are used for probe detection. As an example, anti-digoxigenin, provides a "sandwiching" assay. Indirect FISH also requires additional blocking reagents, amplification steps, and has been reported to have a higher degree of background fluorescence problems. The need for extra handling post-hybridization can cause disruptions in work-flow within a busy clinical laboratory; therefore, direct label FISH represents a superior technology and offers many advantages.
Why does the PathVysion HER-2 DNA kit include a probe for chromosome 17?
The HER-2 gene is located on chromosome 17. The cut-off between normal and abnormal is set by biology; more than one copy of the HER-2 gene per chromosome 17 is abnormal. Determination of the ratio of HER-2 gene copy number to chromosome 17 copy number is useful in the discrimination of aneusomy of chromosome 17 from true HER-2 gene amplification. In the case of aneusomy, more than the normal number of two copies of chromosome 17 are present in the cell and consequently, more than the normal number of two copies of the HER-2 gene are present. This result does not represent HER-2 amplification. Therefore a ratio of HER-2 to chromosome 17 is used in the cutoff of the PathVysion assay.
The chromosome 17 control probe also serves as a methodologic internal control for hybridization and controls for "nuclear truncation". When dealing with solid tumor tissue, nuclear truncation may occur. Most nuclei are roughly 10 microns thick, with a 4-6 micron section taken to be tested. The chromosome 17 probe is utilized to assess truncation and ensure the appropriate nuclei are evaluated in assessing HER-2 amplification.
What clinical trials have been conducted on the PathVysion HER-2 assay?
The pivotal clinical trial demonstrated that HER-2 amplification as determined by the PathVysion HER-2 DNA Probe could be used to identify those patients with a poor prognosis and furthermore, more likely to benefit from dose intensification of adriamycin-based therapies. The PathVysion HER-2 assay reliably detected HER-2 amplification, demonstrating a significant interaction between CAF (cyclophosphamide, doxorubicin, 5-fluorouracil) and amplified HER-23.
Herceptin package insert data from a retrospective analysis of 660 patients enrolled in the Herceptin clinical studies (all scoring 2+ or 3+ by the Clinical Trial IHC assay) demonstrate that the clinical benefits of Herceptin were greater in patients whose tumors tested FISH(+) than those that were FISH( - ). In the singlearm study of Herceptin as a single agent, the overall response rate in patients whose tumors tested as FISH(+) was 20%, while there were no responses in those who tested as FISH( - ).2
Why can it be beneficial to specifically request the PathVysion HER-2 DNA direct label FISH when assessing HER-2 status?
HER-2 status assessment is now considered the standard of care in the selection of therapy for invasive breast cancer, and some laboratories continue to use IHC to assess HER-2 status. PathVysion is the only method of HER-2 assessment that is FDA approved for use as an aid in determining prognostic factors in patients with stage II, node-positive breast cancer, as an aid to predict disease-free and overall survival in patients with stage II, node-positive breast cancer treated with adriamycin-based chemotherapy, and assisting in Herceptin (Trastuzumab) monoclonal antibody therapy selection.
Can the PathVysion HER-2 assay be performed in the average lab?
Yes. Most major reference labs, cancer centers and many local hospitals now perform the PathVysion HER-2 assay as their primary method of assessing HER-2 status. The protocol for the PathVysion HER-2 assay can be easily performed at any laboratory that currently performs IHC testing.
How do I explain the benefits of the PathVysion HER-2 assay to my patients?
Results of the PathVysion HER-2 asssay can be best explained to patients in terms of how they directly affect the therapy they are most likely to receive. When a breast cancer patient knows her HER-2 status, the appropriate therapy choices become clear.
Which patients should receive the PathVysion HER-2 assay?
Testing for HER-2 plays a key role in the management of invasive breast cancer. ASCO Guidelines and NCCN Practice guidelines recommend HER-2 testing for all patients with invasive breast cancer. NCCN Practice guidelines indicate that patients should be tested by either IHC or FISH, but that all IHC 2+ results should be confirmed by FISH.2
Where can I obtain more information about the PathVysion HER-2 assay?
More information is currently available on PathVysion HER-2 from Abbott Molecular, Inc., including technical information, clinical trial data (package insert), a Patient Q&A brochure, a laboratory brochure. For more information, please contact Abbott Molecular today at 1-800-553-7042.
1. Press, et al. "Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials.", Clinical Cancer Research 2005; 11(18) September 15, 2005.
2. Allison M, The HER2 testing conundrum. Nature Biotechnology 28 (2): 117-119, 2010.
3. PathVysion HER-2 DNA Probe Kit package insert.
4. Sauter G, et al. Guidelines for Human Epidermal Growth Factor Receptor 2 Testing: Biologic and Methodologic Considerations. J Clin Oncol 27:1323-1333, 2009.
5. Dressler L, et al. Comparison of HER2 Status by Fluorescence in Situ Hybridization and Immunohistochemistry to Predict Benefit From Dose Escalation of Adjuvant Doxorubicin-Based Therapy i Node-Positive Breast Cancer Patients. J Clin Oncol. 23:4287-4297, 2005