HER2 Fluorescence in Situ Hybridization (FISH) 

Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescence-tagged probes to detect specific DNA sequences in tissue samples; the sequences may then be localized and quantified on fluorescence microscopy. Determination of human epidermal receptor type 2 (HER2) status by FISH assay is based on the gene copy number and the ratio between the numbers of HER2 and CEP17 sequences.

Category

Fluorescence in situ hybridization (FISH) assay, HER2

Device details

Test kits that have been used include the following:

• Abbott Molecular - PathVysion HER-2 DNA Probe Kit
• Dako A/S -HER2 pharmDx Kit (see the image below)Breast adenocarcinoma tissue stained with Dako HER2 pharmDx Kit. Tumor cells show HER2 gene amplification. Image courtesy of Dako A/S.
• Ventana - INFORM HER2 FISH DNA Probe (discontinued)

Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescence-tagged probes to detect specific DNA sequences in tissue samples; the sequences may then be localized and quantified on fluorescence microscopy.

Human epidermal receptor type 2 gene (HER2) FISH assays are performed in formalin-fixed, paraffin-embedded tissue specimens with DNA probes that hybridize directly with HER2 sequences. The kits also include a probe for CEP (chromosome enumeration probe) 17. Determination of HER2 status by FISH assay is based on the gene copy number and the ratio between the numbers of HER2 and CEP17 sequences.

Approximately 18-20% of malignant breast cancers show amplification of the gene for human epidermal receptor type 2 (HER2), resulting in overexpression of the HER2 receptor, a transmembrane tyrosine kinase receptor within the epidermal growth factor receptor (EGFR) family.^[1] Activation of this class of cellular receptors is known to increase activity in a variety of molecular pathways associated with tumor growth and progression.

Before the use of adjuvant therapy with trastuzumab (a humanized recombinant monoclonal antibody directed specifically against the HER2 receptor) became routine, extensive preclinical and clinical data showed that HER2 overexpression is associated with a more aggressive tumor phenotype and a worse prognosis (ie, higher recurrence rate and mortality), independent of other clinical features (eg, age, stage, and tumor grade).^[2]

HER2-positive status has been shown to predict improved response to certain chemotherapy agents (ie, doxorubicin, trastuzumab, and lapatinib, a tyrosine kinase inhibitor). Retrospective analysis of clinical trials has shown that HER2-positive patients benefit from anthracycline-based regimens, secondary to associated amplification of the topoisomerase II-alpha (TOP2A) gene, which is the direct target of these agents. Preliminary data also suggest that HER2 may predict response to and benefit from paclitaxel in the adjuvant setting.

Two types of HER2 tests are generally available: immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Newer methodologies for establishing HER2 status, such as real-time polymerase chain reaction (RT-PCR) and CISH (chromogenic in situ hybridization), have not yet been validated. A FISH assay for TOP2A is also available but has not been validated.

Breast cancer specimens should first undergo HER2 testing by means of a validated IHC assay for HER2 expression.^[1] IHC is a semiquantitative method that measures overexpression of the HER2 receptor, with the degree of staining graded on a scale from 0 to 3+.

Patients with breast cancers scored 3+ (uniform, intense membrane staining of more than 30% of invasive tumor cells) should definitely receive anti-HER2 therapy. Specimens with a score of 0 or 1+ are considered HER2-negative; a score of 2+ is interpreted as equivocal or inconclusive. Equivocal IHC results may be seen in 15% of invasive breast cancers.

Breast cancer specimens with equivocal IHC results should undergo further testing by means of a FISH assay.^[1] FISH assays are far more quantitative and reproducible in this setting because they directly measure the number of copies of the HER2 gene. In general, FISH testing is thought to be more reliable, but it is more expensive and more time-consuming than IHC. Nevertheless, more centers are relying on FISH alone for determining HER2 status.

In addition to its use in breast cancer, the Dako HER2 pharmDx FISH assay has been approved by the US Food and Drug Administration (FDA) for the evaluation of patients with metastatic gastric cancer whose tumor characteristics indicate that they might be candidates for trastuzumab therapy.

Press et al, comparing 2 commercial fluorescence in situ hybridization (FISH) assays with 4 immunohistochemistry (IHC) assays in 117 breast cancer specimens with known human epidermal receptor type 2 gene (HER2) amplification and HER2 receptor overexpression status, determined the 2 FISH assays to have accuracies of 97.4% (PathVysion; Abbott Molecular) and 95.7% (INFORM; Ventana) and found the IHC assays to have accuracies ranging from 88.9% (HercepTest; Dako) to 96.6% (obtained with R60, a rabbit polyclonal antibody).^[3]

Because approximately 20% of current testing for human epidermal receptor type 2 (HER2) may be inaccurate, the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) have recommended guidelines for such testing to ensure accuracy.^[1] Any laboratory providing clinical testing for HER2 must have considerable experience with the measurement of this molecular marker. Assay procedures must be validated by the laboratory before the test is offered clinically.

Optimal fluorescence in situ hybridization (FISH) testing requirements include the following^[1] :

• Tissue fixation for fewer than 6 hours or longer than 48 hours is not recommended
• The test is rejected and repeated if (1) controls are not as expected, (2) the observer cannot find and count at least 2 areas of invasive tumor, (3) 25% of signals are unscorable because of weak signals, (4) 10% of signals occur over cytoplasm, (5) nuclear resolution is poor, or (6) autofluorescence is strong

FISH testing is interpreted in terms of HER2/chromosome enumeration probe (CEP) 17 ratio and gene copy number as follows^[1] :

• Positive HER2 amplification -HER2/CEP17 ratio > 2.2 or HER2 gene copy number > 6.0
• Equivocal HER2 amplification -HER2/CEP17 ratio 1.8-2.2 or HER2 gene copy number 4.0-6.0
• Negative HER2 amplification -HER2/CEP17 < 1.8 or HER2 gene copy number < 4.0

Interpretation is based on counting at least 20 nonoverlapping cells in 2 separate areas of invasive cancer; a pathologist must confirm that the counting involved invasive tumor. If results are equivocal, the sample is subjected to increased counting, counting is repeated, or both.

For information on the management of patients with human epidermal receptor type 2 (HER2)-positive breast cancer, see Breast Cancer (Oncology).

Discordant results (eg, an immunohistochemistry [IHC] score of 3+ with a negative fluorescence in situ hybridization [FISH] result or an IHC score lower than 3+ with a positive FISH result) have been observed in approximately 4% of invasive breast cancer specimens. Equivocal FISH results are seen in fewer than 3% of invasive breast cancer specimens, who had previously been considered HER2-positive. Currently, no data support excluding this group from treatment with trastuzumab.