• What is HER-2/neu?
• How common is HER-2 positive breast cancer?
• Do I need to know my HER-2 status?
• How will knowing my HER-2 status affect my therapy decisions?
• How will my HER-2 status be identified?
• Does the type of HER-2 test make a difference?
• What if I have already had a biopsy, but don't know if I've been screened for HER-2?
• What are the limitations associated with PathVysion I should be aware of?
• What are the warnings associated with PathVysion I should be aware of?

What is HER-2/neu?

HER-2/neu is also referred to as HER-2 or cerB-2. HER-2 stands for Human Epidermal Growth Factor Receptor 2. The HER-2 gene is one of thousands of genes that comprise every living person's genetic code and is present in the nucleus of all human cells, including breast tissue cells. The HER-2 gene provides the blueprint for the manufacture of the HER-2 protein. This protein, which is attached to the cell membrane, regulates normal cell growth and function.

How common is HER-2 positive breast cancer?

Abnormally high quantities of the HER-2 gene have been associated with rapid tumor cell growth, resistance to therapy, and shorter disease-free periods and overall survival. Approximately 20-25%, or 1 out of every 4, breast cancers have an abnormally high quantity of the HER-2 gene, and are considered HER-2 positive.

Do I need to know my HER-2 status?

Your HER-2 status can be an important factor as you and your doctor decide on your best treatment options. The NCCN (National Comprehensive Cancer Network) and ASCO (The American Society of Clinical Oncologists) recommend HER-2 assessment for all invasive breast cancers.

While it is recommended at initial diagnosis, HER-2 status can be determined at anytime after initial diagnosis as well. There is discussion within the medical community about whether HER-2 status should be assessed with each occurrence or recurrence of invasive breast cancer.

How will knowing my HER-2 status affect my therapy decisions?

Determination of HER-2 status has become an important tool in the routine assessment and management of patients with breast cancer. Abnormally high quantities of the HER-2 gene have been shown to result in more aggressive tumor behavior. HER-2 positive patients may react differently to chemotherapy. Studies have shown HER-2 positive tumors obtain greater benefit from the addition of anthracyclines and from more dose-intensive anthracycline administration than HER-2 negative tumors.

How will my HER-2 status be identified?

The two most widely used technologies are ImmunoHistochemistry (IHC) and Fluorescence in situ Hybridization (FISH). The PathVysion test employs FISH technology to determine HER-2 status.

Does the type of HER-2 test make a difference?

Yes. An accurate HER-2 assessment guides doctors to make the most appropriate therapy decisions based on the patient's own genetic profile.

The IHC test is a protein-based test that is used to determine the total amount of HER-2 protein receptors on the surface of the cell. To do this, the surface of the cell is "stained" with an antibody. The surface protein measured by IHC can be damaged during preparation of the tissue sample and this may cause variability in outcome. A pathologist must judge the degree of color change in the cell to determine a HER-2 protein measurement level of 0, 1+, 2+, or 3+. Because this test is relatively subjective, the results often are confirmed by performing a FISH test.

PathVysion, the FISH HER-2 Test, can help overcome many of the inherent technical and interpretative limitations of other techniques, such as immunohistochemistry. The PathVysion® test yields highly accurate and reliable results at the molecular level. PathVysion "highlights" the HER-2 genes inside the cell, making them appear fluorescent so they may be accurately counted. If a patient has an abnormal number of HER-2 genes, she is considered HER-2 positive. If the test shows a normal gene count, the test results are considered HER-2 negative. PathVysion allows the physician or pathologist to literally count the genes, which can help overcome technical and interpretative limitations helping to positively identify women who are candidates for HER-2-targeted therapies.

What if I have already had a biopsy, but don't know if I've been screened for HER-2?

Your HER-2 results can be obtained from your biopsy tissue, at any time, by using the PathVysion test. Because the test measures the HER-2 gene at the stable DNA molecular level, results can be obtained from stored tissue samples. Ask your doctor for more information. Knowing your HER-2 status could be important for your treatment decisions.

What are the limitations associated with PathVysion I should be aware of?

1. The PathVysion Kit has been optimized only for identifying and quantifying chromosome 17 and the HER-2/neu gene in interphase nuclei from formalin-fixed, paraffin-embedded human breast tissue specimens. Other types of specimens or fixatives should not be used.
2. The performance of the PathVysion Kit was validated using the procedures provided in this package insert only. Modifications to these procedures may alter the performance of the assay.
3. Performance characteristics of the PathVysion Kit have been established only for node positive patients receiving the designated regimens of CAF and for metastatic breast cancer patients being considered for HERCEPTIN therapy. Performance with other treatment regimens has not been established.
4. The clinical interpretation of any test results should be evaluated within the context of the patient’s medical history and other diagnostic laboratory test results.
5. FISH assay results may not be informative if the specimen quality and/or specimen slide preparation is inadequate.
6. Technologists performing the FISH signal enumeration must be capable of visually distinguishing between the orange and green signals.

What are the warnings associated with PathVysion I should be aware of?

1. For In Vitro Diagnostic Use.
2. The PathVysion Kit is intended for use only on formalin-fixed, paraffin-embedded breast cancer tissue; it is not intended for use on fresh or non-breast cancer tissue.
3. All biological specimens should be treated as if capable of transmitting infectious agents. The ProbeChek control slides are manufactured from human cell lines that have been fixed in 10% formalin. Because it is often impossible to know which might be infectious, all human specimens and control slides should be treated with universal precautions. Guidelines for specimen handling are available from the U.S. Centers for Disease Control and Prevention.16
4. Exposure of the specimens to acids, strong bases or extreme heat, should be avoided. Such conditions are known to damage DNA and may result in FISH assay failure.
5. Failure to follow all procedures for slide denaturation, hybridization and detection may cause unacceptable or erroneous results.
6. To identify target areas, H & E staining should be conducted on every 10th slide of the same tissue block.
7. Hybridization conditions may be adversely affected by the use of reagents other than those provided by Abbott Molecular Inc.
8. Proper storage of kit components is essential to ensure the labeled shelf life. Assay results may be adversely affected by kit components stored under other conditions.
9. If stored at low temperatures, 20X SSC may crystallize. If the crystals cannot be redissolved at room temperature, the solution should be discarded.
10. If any other working reagents precipitate or become cloudy, they should be discarded and fresh solutions prepared.
11. The DAPI Counterstain contains DAPI (4,6-diamidino-2-phenylindole) and 1,4-phenylenediamine.
    
    •  DAPI is a possible mutagen based on positive genotoxic effects. Avoid inhalation, ingestion or contact with skin.
    
    •  DAPI is a possible mutagen based on positive genotoxic effects. Avoid inhalation, ingestion or contact with skin.
12. Fluorophores are readily photo bleached by exposure to light. To limit this degradation, handle all solutions containing fluorophores in reduced light. This includes all steps involved in handling the hybridized slide. Carry out all steps, which do not require light for manipulation (incubation periods, washes, etc.) in the dark.
13. LSI HER-2/neu & CEP 17 DNA probe mixture contains formamide, a teratogen. Avoid contact with skin and mucous membranes.
14. Calibrated thermometers are required for measuring temperatures of solutions, water baths and incubators.
15. Always verify the temperature of the pretreatment solution, denaturation solution and wash buffers prior to each use by measuring the temperature of the solution in the coplin jar with a calibrated thermometer.
16. All hazardous materials should be disposed of according to your institution's guidelines for hazardous disposal. The following are European Community (EC) risk and safety information. Restricted to professional users.

• What is HER-2/neu?
• How important is HER-2 amplification in the treatment of breast cancer?
• What is the connection between HER-2 status and the selection of adriamycin-based therapy?
• What is the connection between HER-2 status and Herceptin® therapy selection?
• Why is accurate assessment of HER-2 status essential?
• Should one be assessing HER-2 at the DNA level or the gene product (protein) level?
• What are the most accurate methods available for HER-2 status assessment?
• What is the PathVysion HER-2 DNA Probe Kit?
• What is direct labeled FISH?
• Why does the PathVysion HER-2 DNA kit include a probe for chromosome 17?
• What clinical trials have been conducted on the PathVysion HER-2 assay?
• Why can it be beneficial to specifically request the PathVysion HER-2 DNA direct label FISH when assessing HER-2 status?
• Can the PathVysion HER-2 assay be performed in the average lab?
• How do I explain the benefits of the PathVysion HER-2 assay to my patients?
• Which patients should receive the PathVysion HER-2 assay?
• Where can I obtain more information about the PathVysion HER-2 assay?

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.

References
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