What is the role of HER2/neu (Human Epidermal growth factor Receptor 2) and FISH (Fluorescence In Situ Hybridization) tests in guiding treatment decisions for breast cancer?

HER2/neu and FISH Testing in Breast Cancer

Direct Recommendation

FISH (Fluorescence In Situ Hybridization) is the preferred primary testing method for determining HER2 status in breast cancer patients who are candidates for HER2-targeted therapies, due to its superior accuracy, reproducibility, and stronger correlation with treatment response compared to immunohistochemistry (IHC). 1

Understanding HER2 Biology

• HER2 gene amplification is the primary mechanism driving HER2 protein overexpression in breast cancer, occurring in approximately 15-20% of cases 1, 2
• When amplified, HER2 expression increases dramatically from normal levels (25,000-185,000 receptors/cell) to pathologic levels (500,000-2,000 receptors/cell), creating a clear dichotomous separation rather than a continuous spectrum 1, 2
• HER2-positive breast cancer is associated with worse prognosis when untreated but responds to targeted therapies including trastuzumab, pertuzumab, and lapatinib 1, 2

Testing Methods and Definitions

FISH Testing Criteria

• Positive: HER2/CEP17 ratio ≥2.2 OR average HER2 gene copy number >6.0 per nucleus 1, 3
• Equivocal: HER2/CEP17 ratio 1.8-2.2 OR average gene copy number 4.0-6.0 1, 3
• Negative: HER2/CEP17 ratio <1.8 OR average gene copy number <4.0 per nucleus 1, 3

IHC Testing Criteria

• Positive (3+): Uniform intense membrane staining in >30% of invasive tumor cells 1
• Equivocal (2+): Complete membrane staining that is either nonuniform or weak in intensity but with circumferential distribution in ≥10% of cells 1
• Negative (0/1+): No staining or incomplete/faint membrane staining in ≤10% of cells 1

Why FISH is Superior to IHC

Technical Advantages

• DNA is less affected by tissue processing artifacts than protein, resulting in fewer experimental errors with FISH compared to IHC 1
• FISH demonstrates 97-98% reproducibility between central and peripheral laboratories, compared to significantly lower concordance with IHC 1
• FISH interpretation is more objective (counting gene copies) versus the subjective assessment of membrane staining intensity required for IHC 1

Clinical Predictive Value

• Only FISH-positive patients demonstrate significant improvement in outcomes with trastuzumab or lapatinib-based regimens 1
• FISH-negative metastatic breast cancer is associated with lack of responsiveness to HER2-targeted therapies 1, 3
• IHC is associated with both false-negative (2-8% of IHC 0/1+ cases have gene amplification) and false-positive results (5-22% of IHC 3+ cases lack amplification) 1

Fixation Independence

• IHC protein analysis is highly dependent on formalin fixation time and methods, which are not standardized across institutions 1
• FISH is relatively independent of tissue fixation methods, making results more consistent across different laboratories 1

Recommended Testing Algorithm

Primary FISH Approach (Preferred)

1. Perform FISH as initial test on all invasive breast cancer specimens 1
2. If FISH positive (ratio ≥2.2 or >6 copies): Patient is eligible for HER2-targeted therapy 1, 4
3. If FISH equivocal (ratio 1.8-2.2): Count additional cells or retest 1
4. If FISH negative (ratio <1.8): Patient is not a candidate for HER2-targeted therapy 3

Alternative IHC-First Approach (If FISH unavailable initially)

1. Perform IHC as initial screening test 1, 5
2. IHC 3+: Confirm with reflex FISH (91.6% positive predictive value, but 5-22% may lack amplification) 1, 5
3. IHC 2+: Mandatory reflex FISH testing (15-48% will have gene amplification) 1
4. IHC 0/1+: Consider FISH if high clinical suspicion (2-8% may have occult amplification) 1

Critical Clinical Considerations

Treatment Eligibility Based on Testing

• All major adjuvant trastuzumab trials (NSABP B31, NCCTG N9831, HERA, BCIRG006) enrolled patients with either IHC 3+ or FISH-positive disease 1, 4
• Patients with HER2/CEP17 ratio ≥2.0 were eligible for adjuvant trastuzumab trials, meaning ratios between 2.0-2.2 (technically "equivocal" by current definitions) should not be excluded from therapy 1, 4
• The cost of diagnostic testing is minimal compared to the substantial cost of trastuzumab therapy (~$100,000 for 52 weeks), making accurate testing critical 1

Common Pitfalls to Avoid

• Do not rely solely on IHC 3+ without considering FISH confirmation, as 5-22% of IHC 3+ cases lack gene amplification and will not respond to HER2-targeted therapy 1
• Do not dismiss IHC 2+ cases without FISH testing, as 15-48% harbor gene amplification and would benefit from treatment 1
• Avoid using IHC assays without proper quality control, including standardized fixation protocols (6-48 hours in formalin), automated immunostaining, and trained pathologist interpretation 1
• Do not interpret polysomy 17 (increased CEP17 copies without HER2 amplification) as HER2-positive, as these patients have low probability of responding to HER2-targeted therapy 1

Laboratory Quality Requirements

• HER2 testing must be performed in CAP-accredited laboratories or those meeting specific accreditation requirements 6
• Laboratories should demonstrate 95% concordance with validated tests for positive and negative results 6
• FISH has higher failure rate (5%) compared to IHC (0.08%), requiring adequate tissue and technical expertise 5

Special Populations

Hormone Receptor Status Interaction

• In hormone receptor-positive disease (ER+ and/or PR+), the magnitude of benefit from HER2-targeted therapy may be lower than in hormone receptor-negative disease 4, 7
• However, HER2 testing and treatment decisions should not be altered based on hormone receptor status alone 4

Node-Negative Disease

• HER2 amplification by FISH is an independent predictor of poor clinical outcome in node-negative breast cancer, even in small tumors 8
• Node-negative patients with high-risk features (tumor >2 cm, grade 2-3, age <35, or ER/PR-negative) should undergo HER2 testing 4

Cost and Practical Considerations

• FISH reagent cost ($140) is higher than IHC ($10), but this is negligible compared to treatment costs 5
• FISH requires longer testing time (36 hours vs 4 hours) and interpretation time (7 minutes vs 45 seconds) 5
• The superior accuracy and predictive value of FISH justify these additional costs and time requirements 1, 5