HER2 Testing

HER2 (also called ERBB2) is a growth factor receptor on the cell surface. In some cancers the gene is amplified or the protein is over expressed, which drives growth and predicts response to drugs that target HER2. Testing is done on tumour tissue to find which patients should receive these treatments.

Two tests are used together. Immunohistochemistry measures protein on the cell membrane, and scores it from 0 to 3+. In situ hybridisation (fluorescence or chromogenic) counts the HER2 gene copies in the nuclei, and compares them with a control probe for chromosome 17 (CEP17).

Tissue is fixed in 10% neutral buffered formalin for 6 to 72 hours, and cold ischaemia time is kept under 1 hour. Immunohistochemistry takes 1 to 2 days, and in situ hybridisation 2 to 5 days. Automated platforms and validated antibodies are used, with controls in every run.

About 15 to 20% of invasive breast cancers are HER2-positive. HER2 is also over expressed or amplified in 10 to 20% of gastric and gastro oesophageal junction cancers, and in smaller proportions of colorectal, endometrial (serous), salivary duct, biliary, bladder and lung cancers. A newer category, "HER2-low" (immunohistochemistry 1+ or 2+ with a negative in situ test), is found in about half of breast cancers and can be treated with antibody drug conjugates.

Indications.

• All newly diagnosed invasive breast cancers, and recurrences or metastases when treatment depends on it.
• Gastric and gastro oesophageal junction adenocarcinoma at the advanced stage.
• Metastatic colorectal cancer (RAS and BRAF wild type) to select anti-HER2 combinations.
• Serous and other aggressive endometrial carcinomas, and uterine carcinosarcoma.
• Salivary duct carcinoma, biliary tract cancer, urothelial and other solid tumours in selected settings.
• Breast cancer with low HER2 expression, to find patients who might benefit from antibody drug conjugates.

Not indicated for ductal carcinoma in situ for treatment decisions alone, or for tumours where no treatment depends on HER2 status.

Specimen requirements.

• Formalin fixed, paraffin embedded tumour, with adequate fixation (6 to 72 hours in 10% neutral buffered formalin) and short cold ischaemia (under 1 hour).
• Core biopsy or resection, with the invasive tumour clearly identified. The block with the most tumour is chosen.
• Cytology cell blocks fixed in the same way are acceptable.
• Bone metastases: EDTA decalcification, since acid decalcification damages DNA and antigens.
• Clinical details, tumour type, and any previous HER2 result.

Normal appearances.

Normal breast epithelium shows no or very faint membrane staining, and a HER2 to CEP17 ratio of 1 with 1 to 2 copies of the gene per nucleus.

Interpretation.

• Breast, immunohistochemistry 3+ (positive): complete, intense, circumferential membrane staining in more than 10% of invasive tumour cells.
• Breast, immunohistochemistry 2+ (equivocal): weak to moderate complete membrane staining in more than 10% of cells, which needs in situ hybridisation.
• Breast, immunohistochemistry 1+ (negative, HER2-low): faint, incomplete staining in more than 10% of cells.
• Breast, immunohistochemistry 0: no staining, or faint staining in 10% or fewer of cells.
• In situ hybridisation: a ratio of HER2 to CEP17 of 2.0 or more with an average HER2 copy number of 4.0 or more per cell is positive. Other combinations of ratio and copy number are classed in groups that are re-assessed with additional counting or immunohistochemistry before a final call.
• Gastric and gastro oesophageal junction: a positive result is immunohistochemistry 3+, or 2+ with positive in situ hybridisation. Staining is often incomplete (basolateral), and a cluster of at least 5 tumour cells is assessed in a biopsy.
• Intratumoural heterogeneity: a minor positive component (over 10% of cells) is noted, since it can predict response.
• Positive results lead to treatment with anti-HER2 antibodies, tyrosine kinase inhibitors or antibody drug conjugates, combined with chemotherapy.
• Negative results exclude anti-HER2 antibody treatment, and the HER2-low group is flagged for the oncologist.

Critical values and findings needing immediate action.

• A HER2-positive result in metastatic or high risk early cancer: urgent referral to the oncology team for anti-HER2 treatment, with baseline cardiac function (echocardiography), since these drugs can cause cardiomyopathy.
• Equivocal or discordant results: repeat testing on another block, or in a second laboratory.
• Result that differs between primary and metastasis: the more recent specimen is treated as relevant when the metastatic tumour is tested.

Limitations and artefacts.

• Under fixation (fixation under 6 hours), over fixation and long cold ischaemia lower staining, giving false negative results, and tissue from cold or delayed fixation is re-tested or interpreted with caution.
• Edge artefact, necrosis, ductal carcinoma in situ and areas of crushed tissue are not scored.
• Subjectivity in distinguishing 1+, 2+ and 3+, so scoring uses defined criteria, with quality assurance and external review.
• Tumour heterogeneity: the tissue sampled may not represent all of the tumour, and a small core can miss a positive area.
• HER2 status can change with therapy, so metastases are re-tested.
• Chromosome 17 polysomy and deletion distort the ratio, so both ratio and copy number are used.
• Decalcified bone metastases often give unreliable results.
• The HER2-low and HER2-ultralow categories are newer, and the discrimination of 0 from 1+ is difficult and varies between observers.
• Results are dependent on a validated assay and local quality control.

Special groups.

• Women with early breast cancer: HER2 positivity directs adjuvant and neoadjuvant treatment, with cardiac monitoring.
• Men with breast cancer: tested in the same way.
• Patients with gastric cancer: biopsies from several sites (at least 5) for heterogeneity.
• Patients with metastatic disease: re-testing of a new biopsy.
• Pregnant women: some anti-HER2 drugs cause foetal harm, so counselling and contraception are important.
• Regions: HER2 testing is available in few laboratories, and fixation quality is a major problem. A cost effective approach is immunohistochemistry first, with in situ hybridisation for equivocal results, in a reference laboratory with quality assurance. Cost of anti-HER2 treatment limits use, so access and affordability decide the benefit of testing.

Follow up tests.

• In situ hybridisation for immunohistochemistry 2+ results.
• Hormone receptor (oestrogen and progesterone) testing in breast cancer.
• Ki-67 and grade, and genomic tests where used.
• Echocardiography or cardiac MRI for baseline and monitoring of ejection fraction.
• Next generation sequencing for HER2 mutations (lung cancer) and other targets.
• Staging imaging.
• Multidisciplinary meeting for treatment planning.
• Repeat HER2 testing on metastases and at progression.