Human epidermal growth factor receptor 2 (HER2) is a protein found on the surface of breast cells. In some aggressive cancers, the gene that produces HER2 is present in abnormally high numbers, a condition known as gene amplification. This leads to an overabundance of the HER2 protein on the tumor cell surface, which drives rapid cell growth. Physicians use Fluorescence In Situ Hybridization (FISH) to accurately determine if this amplification is present. This test counts the copies of the HER2 gene directly in a sample of tumor tissue, and the interpretation of the results determines the appropriate course of treatment.
The Biological Significance of HER2
HER2 is one of four members of a family of receptors that sit on the cell surface and receive signals that promote growth and division. In a healthy cell, the number of HER2 receptors is carefully regulated to maintain normal function. When the HER2 gene is amplified, the overproduction of the protein creates an excess of these receptors. This continuously stimulates the cancer cell to grow and multiply uncontrollably, driving tumor progression.
Gene amplification is present in approximately 15% to 20% of invasive breast cancers and is associated with more aggressive tumor behavior. Historically, HER2-positive breast cancer was linked to a less favorable outlook and higher rates of recurrence. Testing for HER2 status is necessary because it acts as both a prognostic factor, indicating the likely course of the disease, and a predictive factor, determining the tumor’s response to specific medications. A confirmed HER2-positive diagnosis means the cancer is highly susceptible to targeted drugs.
Understanding the Fluorescence In Situ Hybridization Technique
Fluorescence In Situ Hybridization is a molecular cytogenetic technique used to physically count the number of HER2 gene copies within the nucleus of a tumor cell. The method uses fluorescently labeled DNA probes designed to bind specifically to target regions on the chromosomes. For the HER2 test, a dual-probe system is employed to ensure the most accurate reading.
One probe attaches to the HER2 gene locus on chromosome 17. A second reference probe, known as CEP17 (Chromosome Enumeration Probe 17), binds to the centromere of the same chromosome. The CEP17 probe acts as an internal control, allowing pathologists to account for cells that may have an abnormal number of chromosome 17 copies. By counting the number of signals for both the HER2 gene and the CEP17 control, the precise level of gene amplification can be calculated. The resulting images are viewed under a specialized fluorescent microscope, where the signals appear as distinct, countable dots within the cell nuclei.
Interpreting the HER2/CEP17 Ratio
The final interpretation of the FISH test relies on calculating the ratio of the average number of HER2 gene signals to the average number of CEP17 signals counted across multiple tumor cell nuclei. This ratio provides a reliable measure of gene amplification, which is the defining characteristic of HER2-positive disease. Clinical guidelines categorize results into three main groups: Positive, Negative, and Equivocal.
A test result is classified as HER2-Positive, or Amplified, if the calculated HER2/CEP17 ratio is \(2.0\) or greater. If the ratio is less than \(2.0\), the result can still be considered positive if the average number of HER2 signals is high, typically \(6.0\) or more signals per cell. This finding confirms a significant increase in gene copy number, indicating the tumor is driven by HER2 overexpression.
Conversely, a result is classified as HER2-Negative, or Non-Amplified, if the HER2/CEP17 ratio is less than \(2.0\) and the average number of HER2 signals is less than \(4.0\) per cell. This indicates that the tumor cells have a normal or near-normal number of HER2 gene copies. These tumors will not benefit from specific HER2-targeted drugs.
The most complex category is the Equivocal or Borderline result, which includes patterns that fall outside the clear positive and negative thresholds. For instance, a ratio of less than \(2.0\) but an average HER2 copy number between \(4.0\) and \(6.0\) signals per cell is often considered equivocal. These cases require additional review, which often includes a pathologist re-counting the signals or correlating the FISH result with the protein expression level determined by the Immunohistochemistry (IHC) test. This helps prevent misclassification and ensures the most accurate determination of the tumor’s status.
Clinical Implications of HER2 Status
The final classification of HER2 status has a direct impact on the patient’s treatment strategy. A HER2-Positive result indicates the tumor is susceptible to a class of medications known as anti-HER2 targeted therapies. The drug Trastuzumab, for example, is a monoclonal antibody designed to bind directly to the overexpressed HER2 receptors on the cancer cell surface.
By blocking the receptor, Trastuzumab prevents the growth signals from reaching the cell’s interior, effectively stopping uncontrolled proliferation. Other targeted agents may also be used in combination with chemotherapy to maximize the therapeutic effect.
For tumors classified as HER2-Negative, targeted anti-HER2 drugs are ineffective, and treatment must follow an alternative path. These tumors are instead treated using combinations of traditional chemotherapy, hormone therapy if the tumor is hormone receptor-positive, or other novel therapies. The distinction between HER2-Positive and HER2-Negative guides the selection of the most effective systemic therapy and is an integral part of planning long-term care.