How should HER2 fluorescence in‑situ hybridization (FISH) be interpreted when immunohistochemistry (IHC) shows a 2+ result?

HER2 FISH Interpretation in IHC 2+ Cases

When IHC shows 2+ (equivocal) results, FISH testing must be performed and interpreted according to the 2023 ASCO/CAP guidelines, which provide specific algorithms based on the FISH results combined with IHC findings to render a definitive HER2 status—eliminating the "equivocal" category entirely. 1

Algorithmic Approach to Interpretation

The interpretation depends on your specific FISH result pattern. Here's how to proceed:

Scenario 1: HER2/CEP17 ratio ≥ 2.0 AND average HER2 signals ≥ 4.0/cell (Group 1)

• Result: HER2 POSITIVE 1, 2
• This is the classic amplification pattern
• No additional workup needed
• Patient qualifies for HER2-targeted therapy

Scenario 2: HER2/CEP17 ratio ≥ 2.0 BUT average HER2 signals < 4.0/cell (Group 2)

Since you already have IHC 2+, follow this pathway:

• Have a second observer (blinded to initial results) recount at least 20 cells in the IHC 2+ staining area 2
• If recount changes the ISH category → adjudicate per institutional protocol
• If ratio remains ≥ 2.0 with < 4.0 signals/cell → HER2 NEGATIVE with comment 2
• Rationale: These patients showed no benefit from trastuzumab in pivotal trials due to low absolute HER2 copy number despite elevated ratio 2

Scenario 3: HER2/CEP17 ratio < 2.0 BUT average HER2 signals ≥ 6.0/cell (Group 3)

Since you have IHC 2+:

• Have a second observer recount at least 20 cells (blinded) in the IHC 2+ area 2
• If recount changes the category → adjudicate
• If ratio remains < 2.0 with ≥ 6.0 signals/cell → HER2 POSITIVE 2
• Key point: Despite low ratio, the high absolute HER2 copy number indicates amplification (often with CEP17 co-amplification) 2

Scenario 4: HER2/CEP17 ratio < 2.0 AND average HER2 signals 4.0-5.9/cell (Group 4)

This was the "equivocal" category in 2013 guidelines—now resolved:

Since you have IHC 2+:

• Have a second observer recount at least 20 cells (blinded) in the IHC 2+ area 2
• If recount changes the category → adjudicate
• If remains 4.0-5.9 signals/cell with ratio < 2.0 → HER2 NEGATIVE with comment 2
• Do NOT perform additional testing on the same specimen 2
• Rationale: Approximately 5% of cases fall here; most are ER/PR positive and lack protein overexpression. Clinical benefit from HER2 therapy is uncertain without IHC 3+ 2

Scenario 5: HER2/CEP17 ratio < 2.0 AND average HER2 signals < 4.0/cell (Group 5)

• Result: HER2 NEGATIVE 1
• Classic non-amplified pattern
• No additional workup needed

Critical Technical Requirements

Cell counting specifications 1:

• Count at least 20 non-overlapping invasive tumor cells
• Scan entire slide before counting to identify areas of potential amplification
• If a second population with increased HER2 signals comprises >10% of tumor cells, count that population separately (at least 20 cells)
• The 2026 study confirms 20 cells provides adequate interobserver agreement (Fleiss' κ = 0.81-0.87), with no meaningful improvement at higher counts 3

Rejection criteria—repeat test if 1:

• Controls not as expected
• Cannot find/count at least two areas of invasive tumor

• 25% signals unscorable (weak signals)

• 10% signals over cytoplasm

• Poor nuclear resolution or strong autofluorescence

Common Pitfalls to Avoid

1. Don't use alternative chromosome 17 probes as primary strategy: The 2018 update eliminated this approach. One study showed 70% of cases reclassified as HER2-negative had been designated positive only after alternative probe use 4

2. Don't repeat testing on the same specimen for borderline cases: When IHC is not 3+ and FISH is near thresholds, repeated testing will yield different results by chance alone 2

3. Don't ignore the IHC result: The 2018/2023 guidelines mandate integrating IHC and FISH results. Review both slides together to guide area selection 2

4. Don't assume IHC 2+ means 50% will be FISH positive: Studies show only 22.5-29% of IHC 2+ cases are FISH amplified 5, 6. The majority (65.5%) are FISH negative 6

Clinical Context

The 2018 guideline revision reclassified approximately 10.7% of cases, primarily eliminating the equivocal category 4. Most newly HER2-negative cases are ER+ (90%), PR+ (80%), stage 1 (60.9%), and grade 1-2 (59.4%) 4. This prevents overtreatment with HER2-targeted therapy in patients unlikely to benefit.

The required comment for Groups 2-4 when negative: State that evidence is limited/insufficient regarding HER2-targeted therapy benefit in these specific patterns without protein overexpression, and that the specimen is considered HER2-negative due to lack of both high-level amplification and protein overexpression 2.