What is the role of a fish test in breast health assessment?

The Role of FISH Test in Breast Cancer Assessment

Fluorescence in situ hybridization (FISH) testing is a critical method for determining HER2 status in breast cancer, serving as either a confirmatory test for equivocal immunohistochemistry (IHC) results or as part of a dual testing strategy to ensure accurate HER2 assessment.

HER2 Testing Overview

• HER2 status determination is essential for all newly diagnosed invasive breast cancers as it provides important prognostic information and predicts response to HER2-targeted therapies 1
• Two primary methods are used for HER2 testing:
  • Immunohistochemistry (IHC): Detects HER2 protein overexpression 1
  • Fluorescence in situ hybridization (FISH): Measures HER2 gene amplification 1

FISH Testing Indications

• FISH testing is indicated in the following scenarios:
  • To confirm equivocal (2+) IHC results 1
  • As part of a dual testing strategy (both IHC and FISH) for all breast cancers 2
  • When IHC testing shows technical issues or artifacts 1
  • For cases with heterogeneous HER2 expression 1

FISH Testing Methodology

• FISH is performed by counting at least 20 cells within the invasive component of the tumor 1
• The pathologist should scan the entire slide before counting or use IHC to define areas of potential HER2 amplification 1
• If a second population of cells with increased HER2 signals is present and comprises >10% of tumor cells, separate counting must be performed 1
• FISH results are reported as positive (amplified), negative (not amplified), or equivocal 1

Interpretation of FISH Results

• Positive HER2 amplification: HER2/CEP17 ratio ≥2.0 or average HER2 copy number ≥6.0 signals/cell 1
• Negative HER2 amplification: HER2/CEP17 ratio <2.0 with average HER2 copy number <4.0 signals/cell 1
• Equivocal results: HER2/CEP17 ratio <2.0 with average HER2 copy number ≥4.0 but <6.0 signals/cell 1

Non-Classical FISH Results

• Several patterns of "non-classical" HER2 FISH results have been identified:
  • 'Monosomy' (ratio ≥2.0, mean HER2/cell <4.0) 3
  • 'Co-amplified' (ratio <2.0, mean HER2/cell ≥6.0) 3
  • 'Low amplified' (ratio ≥2.0, mean HER2/cell 4.0-5.9) 3
  • Heterogeneous cases with clustered HER2-positive cells 3

Advantages of FISH Testing

• FISH has higher sensitivity compared to IHC (IHC has comparable specificity but lower sensitivity) 4
• FISH results are more objective and quantifiable 1
• FISH can detect gene amplification even when protein overexpression is not detected by IHC 4

FISH vs. IHC: Concordance and Discordance

• Most studies show high concordance between IHC and FISH results, especially for IHC 0/1+ (negative) and IHC 3+ (positive) 5
• Discordance occurs in approximately 5-10% of cases, particularly in the IHC 2+ category 5, 6
• Studies have shown that FISH-positive/IHC-negative cases may have poorer prognosis compared to FISH-negative/IHC-negative cases 4

Alternative Testing for Equivocal Cases

• For cases with equivocal HER2 FISH results, alternative probes (TP53, RAI1, RARA) may be used for further evaluation 6
• Approximately 50% of FISH-equivocal cases may be reclassified as HER2-amplified using alternative probes 6

Quality Assurance in FISH Testing

• FISH testing should only be performed in laboratories accredited to perform such testing 1
• Laboratories should have standardized HER2 testing procedures and programs to periodically evaluate personnel proficiency 1
• External quality assessment participation is recommended to improve reproducibility of results 1
• Validation of tests must be performed before offering the test clinically 1

Cost-Effectiveness Considerations

• Two main testing strategies exist:
  • Reflex testing: IHC first, with FISH only for equivocal (2+) cases 2
  • Dual testing: Both IHC and FISH performed on all cases 2
• Dual testing strategy has been found to be more effective but more costly, with an incremental cost-effectiveness ratio of $70,051.55/QALY 2

Pitfalls and Caveats

• Pre-analytical factors can affect FISH results, including:
  • Fixation time (should be 6-72 hours in 10% neutral buffered formalin) 1
  • Tissue processing and storage 1
  • Section thickness and age of cut sections 1
• FISH tests should be rejected and repeated if:
  • Controls are not as expected 1
  • More than 25% of signals are unscorable due to weak signals 1
  • More than 10% of signals occur over cytoplasm 1
  • Nuclear resolution is poor or autofluorescence is strong 1