When is FISH Test for HER2 Positive in Breast Cancer
A FISH test is considered positive for HER2 when the HER2/CEP17 ratio is greater than 2.2 OR when the average HER2 gene copy number is 6.0 or more signals per cell. 1
Primary Testing Criteria for HER2 Positivity by FISH
FISH positivity is defined by either of two parameters:
- HER2/CEP17 ratio > 2.2 (for dual-probe FISH assays using a chromosome 17 centromeric control probe) 1
- Average HER2 gene copy number ≥ 6.0 signals per cell (for single-probe FISH assays without internal control) 1
These thresholds were established based on clinical trial data from major trastuzumab studies, where patients meeting these criteria demonstrated significant benefit from HER2-targeted therapy. 2
When FISH Testing Should Be Performed
FISH testing is indicated in the following clinical scenarios:
As reflex testing for equivocal IHC results (2+ staining) - This is the most common indication, where IHC shows complete membrane staining that is either nonuniform or weak in intensity in at least 10% of tumor cells 1
As primary testing method - FISH can be used as the initial HER2 assessment instead of IHC, though this is less common due to higher cost and longer turnaround time 1, 3, 4
For confirmation when IHC 3+ results appear discordant with clinical/histopathologic features - If the pathologist or oncologist observes apparent histopathologic discordance after initial testing 1
When IHC testing fails or is indeterminate due to technical issues, inadequate fixation, or artifacts 1
Equivocal FISH Results Requiring Additional Testing
FISH results are considered equivocal when:
- HER2/CEP17 ratio is 1.8 to 2.2 (dual-probe assay) 1
- Average HER2 gene copy number is 4.0 to 6.0 signals per cell (single-probe assay) 1
When FISH results are equivocal, reflex testing with IHC on the same specimen is mandatory to arrive at a definitive HER2 status designation. 1 This represents a critical update from earlier guidelines and eliminates the equivocal category when both tests are performed. 5
Important Clinical Considerations
A notable caveat exists for patients with HER2/CEP17 ratios between 2.0 and 2.2: These patients were considered HER2-positive and were eligible for treatment in the adjuvant trastuzumab trials (NSABP B31, NCCTG N9831, HERA). 1, 2 Therefore, available efficacy data support including them in HER2-targeted therapy, even though they fall into what would otherwise be an equivocal range. 1
Polysomy 17 creates interpretive challenges: Approximately 8% of specimens show polysomy 17, mostly among cases with 4-6 HER2 gene copies (equivocal range). 1 In these cases, the HER2/CEP17 ratio may be low despite increased absolute HER2 copy numbers, requiring careful interpretation and often reflex IHC testing. 1, 6
Testing Algorithm and Quality Requirements
The recommended testing sequence is:
- Initial IHC testing on all invasive breast cancer specimens 1, 3
- Automatic reflex to FISH for IHC 2+ (equivocal) results 1
- Consider FISH for IHC 3+ if histopathologic features suggest discordance 1
Laboratories performing FISH testing must demonstrate 95% concordance with validated methods and maintain ongoing quality assurance programs. 1, 3 Testing should only be performed in CAP-accredited laboratories or equivalent. 1, 3
Impact of Updated Guidelines
The 2018 ASCO/CAP guideline update significantly changed HER2 classification: In a retrospective study of 2,233 cases, 8.2% of cases were reclassified when reassessed by 2018 guidelines compared to 2013 guidelines. 5 Most notably, 175 previously equivocal cases were definitively classified as either negative (n=173) or positive (n=2) after mandatory reflex IHC testing. 5
Alternative FISH probes (TP53, RAI1, RARA) may be used for persistently equivocal cases: Studies show that approximately 50% of cases equivocal by conventional FISH become classified as HER2-amplified when tested with alternative chromosome 17 probes. 6 However, this approach requires further clinical validation to determine treatment benefit. 6