What is the recommended procedure for conducting the Fluorescence In Situ Hybridization (FISH) test and Human Epidermal growth factor Receptor 2 (HER2)/neu test?

Recommended Procedure for FISH and HER2/neu Testing in Breast Cancer

The recommended procedure for conducting FISH and HER2/neu testing requires standardized protocols in CAP-accredited laboratories with strict quality control measures to ensure accurate determination of HER2 status for appropriate treatment selection. 1

Testing Algorithm

• HER2 status should be determined for all invasive breast cancer cases using a validated testing algorithm 1
• The recommended approach is a two-phase testing algorithm:
  • Initial screening with immunohistochemistry (IHC) 1
  • Reflex to fluorescence in situ hybridization (FISH) for equivocal IHC cases 1
• Alternatively, FISH can be used as the primary testing method 1

Specimen Handling Requirements

• Tissue must be fixed in neutral buffered formalin 1
• Core needle biopsies must be fixed for at least 1 hour 1
• Excisional biopsies must be fixed for 6-48 hours 1
• Specimens fixed in other fixatives should be excluded from testing 1
• Proper tissue handling is critical as poor fixation can lead to false-negative IHC results 1

FISH Testing Procedure

• FISH testing detects gene amplification rather than protein expression 1
• A positive HER2 FISH result is defined as:
  • More than six HER2 gene copies per nucleus, OR
  • A FISH ratio (HER2 gene signals to chromosome 17 signals) of more than 2.2 1
• A negative HER2 FISH result is defined as:
  • Less than 4.0 HER2 gene copies per nucleus, OR
  • FISH ratio of less than 1.8 1
• Equivocal results fall between positive and negative parameters and require additional testing 1

IHC Testing Procedure

• IHC detects HER2 protein overexpression 1
• A positive HER2 IHC result is defined as:
  • 3+ staining (uniform, intense membrane staining of ≥30% of invasive tumor cells) 1
• A negative HER2 IHC result is defined as:
  • 0 or 1+ staining 1
• Equivocal (2+) results require reflex testing with FISH 1

Quality Assurance Requirements

• Laboratories must demonstrate 95% concordance with another validated test for positive and negative assay values 1
• Participation in external proficiency testing with at least two testing events per year is mandatory 1
• Satisfactory performance requires at least 90% correct responses on graded challenges 1
• Ongoing quality control must include review of external and internal controls with each test batch 1
• Laboratories should maintain standardized operating procedures and documentation of controls 1
• Onsite inspection should occur every other year with annual self-inspection 1

Laboratory Accreditation and Volume Requirements

• HER2 testing should be performed in CAP-accredited laboratories or laboratories meeting equivalent accreditation standards 1
• Some guidelines recommend restricting IHC testing to laboratories with a minimum annual caseload of 250 cases 1
• FISH centers should test a minimum of 100 cases per year, preferably at least 150 1
• Higher volume laboratories tend to have better performance, particularly for FISH testing 1

Common Pitfalls and Considerations

• Concordance between local and central laboratory testing is highest for IHC 3+ samples (77%) and lowest for IHC 2+ samples (26%) 2
• Only 33% of samples testing IHC 2+ at local laboratories are confirmed FISH-positive at central laboratories 2
• Discordance rates between FISH and IHC for 3+ cases can be up to 22.22%, indicating that even strong IHC positivity should be confirmed by FISH in some cases 3
• Pre-analytical factors (fixation, processing) significantly impact test accuracy 1
• Tumor heterogeneity may lead to false-negative results if sampling is inadequate 4
• FISH has comparable specificity but higher sensitivity compared to IHC for detecting HER2 status 5

By following these standardized procedures and quality control measures, laboratories can ensure accurate HER2 testing to appropriately identify patients who will benefit from HER2-targeted therapies such as trastuzumab, which significantly improves survival outcomes in HER2-positive breast cancer patients 1.