How should HER2 (human epidermal growth factor receptor‑2) expression be tested and managed in breast cancer?

HER2 Testing in Breast Cancer

All patients with invasive breast cancer must undergo HER2 testing using validated immunohistochemistry (IHC) or in situ hybridization (ISH) assays, with results reported as positive, equivocal, or negative according to standardized criteria that directly determine eligibility for HER2-targeted therapies. 1

Testing Requirements and Methods

Mandatory Testing

• Every primary invasive breast cancer requires HER2 testing 1
• Testing should be performed on metastatic sites when available in stage IV disease 1
• Retesting is indicated when a previously HER2-negative primary tumor recurs with clinical behavior suggesting HER2-positive or triple-negative disease 1

Validated Testing Methods

The ASCO/CAP guidelines preferentially recommend FDA-approved assays, though CLIA-certified laboratories may use laboratory-developed tests with documented analytic validity 1. Two primary testing approaches exist:

IHC for protein expression:

• Screens for HER2 protein overexpression on tumor cell membranes
• More susceptible to pre-analytical variables (fixation time, tissue handling) 2

ISH (FISH or other platforms) for gene amplification:

• Detects HER2 gene copy number
• More reproducible across laboratories due to less dependence on fixation methods 2
• Some experts argue FISH should be the primary testing modality given superior accuracy and reproducibility 2

Result Interpretation and Reporting

HER2-Positive (Treatment Indicated)

IHC criteria: 3+ (uniform intense complete membrane staining in >30% of invasive tumor cells) 1, 3

ISH criteria (2013 update):

• Single-probe ISH: HER2/CEP17 ratio ≥2.0 OR average HER2 copy number ≥6.0 signals/cell
• Dual-probe ISH: HER2/CEP17 ratio ≥2.0 OR average HER2 copy number ≥6.0 signals/cell 1

Clinical action: HER2-targeted therapy should be recommended if no histopathologic discordance exists 1

HER2-Equivocal (Reflex Testing Required)

IHC criteria: 2+ (weak to moderate complete membrane staining in >10% of tumor cells) 1, 3

ISH criteria:

• HER2/CEP17 ratio 1.8-2.2 OR
• Average HER2 copy number 4.0-6.0 signals/cell 3

Critical action: Must perform reflex testing on the same specimen using the alternative test (if IHC equivocal, perform ISH; if ISH equivocal, perform IHC) 1. Decision to recommend HER2-targeted therapy must be delayed until reflex testing is complete 1.

HER2-Negative (No Standard HER2-Targeted Therapy)

IHC criteria: 0 or 1+ 1

ISH criteria:

• HER2/CEP17 ratio <2.0 AND average HER2 copy number <4.0 signals/cell 1, 3

Clinical action: HER2-targeted therapy must not be recommended unless histopathologic discordance is observed 1

Critical Pitfalls and Special Scenarios

Uncommon ISH Patterns Requiring Combined IHC/ISH Interpretation

The 2018 ASCO/CAP update addressed approximately 5% of cases with discordant ISH results 4:

• Group 2: HER2/CEP17 ratio ≥2.0 but HER2 copy <4.0 - requires IHC correlation
• Group 3: HER2/CEP17 ratio <2.0 but HER2 copy ≥6.0 - requires IHC correlation
• Group 4: HER2/CEP17 ratio <2.0 with HER2 copy 4.0-6.0 - requires IHC correlation

For these scenarios, concomitant IHC review is mandatory to arrive at accurate HER2 status 4

Histopathologic Discordance

When pathologists or oncologists observe apparent discordance between tumor histology and HER2 results, additional testing should be discussed 1. Rare breast cancers (gland-forming tumors, micropapillary carcinomas) may show IHC 1+ with intense but incomplete (basolateral/U-shaped) staining and be HER2 amplified—these should be reported as equivocal with reflex testing 1

Testing Must Be on Invasive Component

HER2 is frequently increased in in situ breast cancer for unclear reasons, but clinical implications are uncertain 3. All testing must be performed on the invasive component only 3

Persistently Equivocal Results

If HER2 status remains equivocal after reflex testing with alternative assays, the oncologist may consider HER2-targeted therapy on an individualized basis considering patient comorbidities, prognosis, and preferences after discussing available evidence 1. Testing another tumor specimen should be considered to definitively establish HER2 status 1

Emerging Clinical Relevance: HER2-Low Expression

New Therapeutic Landscape

The 2023 ASCO-CAP guideline update acknowledges that trastuzumab deruxtecan (an antibody-drug conjugate) has activity in breast cancers with IHC 1+ or IHC 2+/ISH-negative (termed "HER2-low") 5. This represents a paradigm shift from the binary HER2-positive/negative classification.

Current Reporting Recommendations

While it is premature to create new result categories like "HER2-Low," pathology laboratories should include a footnote in HER2 reports stating 5:

"Patients with breast cancers that are HER2 IHC 3+ or IHC 2+/ISH amplified may be eligible for several therapies that disrupt HER2 signaling pathways. Invasive breast cancers that are HER2 IHC 1+ or IHC 2+/ISH non-amplified may be eligible for specific HER2-targeted antibody-drug conjugates."

Distinguishing IHC 0 from IHC 1+

Best practices to distinguish these subtle differences are now clinically relevant, as DESTINY-Breast04 trial excluded IHC 0 tumors 5. However, evidence is limited that IHC 0 cancers behave differently or don't respond to newer HER2 ADCs 5.

Quality Control Requirements

Laboratories must demonstrate ≥95% concordance with validated FISH assays for samples classified as positive or negative 3. If this criterion cannot be met, specimens should be sent to a reference laboratory 3. Annual concordance testing confirmation is required 3.