Inconsistent Results With Different Secondary Reflex Assays for Resolving HER2 Status

Area of Science:

• Oncology
• Molecular Diagnostics
• Genetics

Background:

• Guidelines recommend secondary reflex testing for equivocal HER2 breast cancer results.
• Immunohistochemistry (IHC) and in situ hybridization (ISH) can yield ambiguous HER2 statuses.

Purpose of the Study:

• To compare the concordance of two reflex ISH assays and one PCR assay for resolving equivocal HER2 breast cancer results.
• To evaluate the reliability of secondary reflex testing in breast cancer diagnostics.

Main Methods:

• Twenty-nine breast cancers with equivocal IHC and ISH results were retested.
• Two separate ISH assays used differentially labeled probes for ERBB2 (HER2) and control loci (RAI1 or ORC4).
• Real-time quantitative PCR amplified ERBB2 and a control locus (EIF5B).

Main Results:

• HER2/RAI1 and HER2/ORC4 ISH assays showed concordance in 72% of cases.
• All three secondary reflex assays were concordant in only 62% of cases.
• Discrepancies between ISH readers occurred near the cutoff threshold.

Conclusions:

• Different control loci in ISH assays lead to discordant results.
• PCR assays may classify cases as nonamplified due to potential contamination.
• The clinical correlation of secondary reflex assay results remains unknown, despite the need for resolving equivocal HER2 statuses.