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HER2 (HER-2/neu; ERBB2) Testing

HER2 (HER-2/neu; ERBB2) Testing

Clinical Focus

HER2 (HER-2/neu; ERBB2) Testing



Clinical Background

Individuals Suitable for Testing

Test Selection - Figure - Table

Test Interpretation


Clinical Background [return to contents]

The HER2 (also known as HER-2/neu or ERBB2) proto-oncogene, 1 of a family of 4 closely related epidermal growth factor receptor genes, encodes a 185-kd tyrosine kinase. HER2 gene amplification can lead to overproduction of the HER2 protein and to tumor development through enhanced cell proliferation, survival, motility, and adhesion. HER2 amplification and overexpression are observed in approximately 20% of invasive breast cancers and are associated with an aggressive disease course and decreased disease-free and overall survival.1,2

HER2 status is most often used to determine patient eligibility for trastuzumab immunotherapy. Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of HER2, inhibits proliferation of human tumor cells that overexpress HER2. Documentation of HER2 overexpression, either directly with immunohistochemistry (IHC) or indirectly with fluorescence in situ hybridization (FISH), is therefore recommended before prescribing trastuzumab therapy.3 Patients whose breast tumors amplify the HER2 gene and/or overexpress the HER2 protein are suitable candidates for this treatment.4-6

HER2 amplification or overexpression status can also be helpful when considering other types of therapy. HER2-positive tumors have been associated with increased sensitivity to anthracycline (eg, doxorubicin [Adriamycin ®]).7 Conversely, such patients do not benefit as much from cyclophosphamide/methotrexate/5-fluorouracil (CMF) regimens as do those with HER2-negative tumors.8-11 HER2-positive patients also tend to have diminished sensitivity to endocrine therapy (eg, tamoxifen); however, the data are somewhat conflicting, and decisions regarding endocrine treatment should not be based on the results of HER2 testing.12 Similarly, the data regarding HER2 status and taxanes are insufficient for clinical use, although the tendency is for HER2-positive tumors to respond well to treatment.12 For example, an article reported benefit from the addition of paclitaxel after adjuvant doxorubicin/cyclophosphamide therapy in patients with node-positive, HER2-positive breast cancer, while patients with node-positive, HER2-negative breast cancer did not benefit.13

Individuals Suitable for Testing [return to contents]

  • All patients with invasive breast cancer

Test Selection [return to contents]

There are several approaches for detecting HER2 overexpression or amplification. Currently, the only FDA-cleared methods for assessing HER2 status are IHC and FISH. Quest Diagnostics offers both methods and follows the testing and reporting recommendations of the expert American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) Panel.1 The Panel expressed no preference for one method over the other. The figure portrays an algorithm based on the ASCO/CAP Panel recommendations for use and interpretation of IHC and FISH methods.1

Figure. Use and Interpretation of HER2 (ERBB2) IHC and FISH Methods

Immunohistochemistry Method

The IHC method determines the protein overexpression status of the invasive component of the cancer by using an anti-human HER2 antibody. The stained tissue is visually examined by a board-certified pathologist and graded using a 4-point scale. Protein overexpression is indicated by a score of 3+. Staining variations between the in situ and invasive components may occur.

Fluorescence In Situ Hybridization Method

The FISH assay determines gene amplification status of the invasive component of the cancer by using probes specific for the HER2 locus and D17Z1 (centromeric region of chromosome 17; CEP 17, included as an internal control and to account for aneusomy of chromosome 17). Results are reported as the ratio of the HER2 signal to the D17Z1 signal, average of HER2 signals per cell, and average of D17Z1 signals per cell. Cutoff points recommended by the ASCO/CAP Panel and the National Comprehensive Cancer Network to indicate gene amplification status are shown in the Table.1,14

In addition to HER2, IHC, without Interpretation (test code 19214X); HER2, IHC, with Interpretation (test code 30316); and FISH, HER2/neu, Paraffin Block (test code 14620X), Quest Diagnostics also offers a test starting with each method and reflexing to the other: HER2, IHC with Reflex to HER2, FISH (test code 15547) and FISH, HER2/neu with Reflex to IHC (test code 19859) (Table).

Table. Technology Guide to HER2 Tests Used by Quest Diagnostics


Immunohistochemistry (IHC)

Fluorescence In Situ Hybridization (FISH)

Test code 15547 19859
Test name HER2, IHC with Reflex to HER2, FISH FISH, HER2/neu with Reflex to IHC
Clinical use
(FDA cleared)
Evaluate HER2 protein overexpression in breast tumor tissue

Assess eligibility for trastuzumab (Herceptin) treatment

Detect HER2 oncogene amplification

Assess eligibility for trastuzumab (Herceptin) treatment

Assesses Protein overexpression Gene amplification
Method IHC with reflex to FISH (see below) FISH with reflex to IHC (see below)
Primary reagent Polyclonal anti-human HER2 antibody DNA probe specific for the HER2 gene locus (17q11.2-q12) plus an internal control
Test interpretation

Cell membrane stain intensity:

  • 0 = negative
  • 1+ = negative
  • 2+ = equivocal; reflexed to FISH
    at additional charge
  • 3+ = strongly positive for presence of HER2 overexpression
  • Negative for HER2 amplification
    • A ratio of <2.0 and an average HER2 copy number/cell <4.0a
  • Equivocalb; reflexed to IHC at additional charge
    • A ratio of <2.0 and an average HER2 copy number/cell ≥4.0 and <6.0a,c
  • Positive for HER2 amplification
    • A ratio of ≥2.0a,d, or
    • Average HER2 copy number/cell ≥6.0a,d
Analysis time 1 to 3 days 2 to 4 days


By counting at least 20 cells within the area.


Verified by a second observer, counting additional cells, and repeating the FISH test.


Observed in a homogeneous and contiguous population.


Observed in a homogeneous and contiguous population within ˃10% of the invasive tumor cells.

Test Interpretation [return to contents]

The results of HER2 testing are reported as shown in the Table. HER2 overexpression or amplification suggests patient eligibility for trastuzumab treatment (Figure).

Lack of HER2 protein overexpression or gene amplification suggests the patient will not respond to trastuzumab therapy. Similarly, trastuzumab response, or lack thereof, in patients with confirmed equivocal HER2 test results is uncertain. Trastuzumab is clinically indicated only for patients in whom HER2 protein overexpression or gene amplification can be confirmed.

References [return to contents]

  1. Wolff AC, Hammond MEH, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med. 2014;138:241-256.

  2. Nunes RA, Harris LN. The HER2 extracellular domain as a prognostic and predictive factor in breast cancer. Clin Breast Cancer. 2002;3:125-135; discussion 136-137.

  3. Herceptin® (trastuzumab) package insert. South San Francisco, CA: Genentech, Inc. https://www.gene.com/download/pdf/herceptin_prescribing.pdf. 2016 Accessed January 24, 2017.

  4. Baselga J, Tripathy D, Mendelsohn J, et al. Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. J Clin Oncol. 1996;14:737-744.

  5. Cobleigh MA, Vogel CL, Tripathy D, et al. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol. 1999:17:2639-2648.

  6. Gonzales-Angulo AN, Hortobagyi GN, Esteva FJ. Adjuvant therapy with trastuzumab for HER-2/neu–positive breast cancer. Oncologist. 2006;11:857-867.

  7. Paik S, Bryant J, Park C, et al. erbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer. J Natl Cancer Inst. 1998;90:1361-1370.

  8. Allred DC, Clark GM, Tandon AK, et al. HER-2/neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma. J Clin Oncol. 1992;10:599-605.

  9. Gusterson BA, Gelber RD, Goldhirsch A, et al. Prognostic importance of c-erbB-2 expression in breast cancer. International (Ludwig) Breast Cancer Study Group. J Clin Oncol. 1992;10:1049-1056.

  10. Menard S, Valagussa P, Pilotti S, et al. Response to cyclophosphamide, methotrexate, and fluorouracil in lymph node-positive breast cancer according to HER2 overexpression and other tumor biologic variables. J Clin Oncol. 2001;19:329-335.

  11. Kostopoulos I, Arapantoni-Dadioti P, Gogas H, et al. Evaluation of the prognostic value of HER-2 and VEGF in breast cancer patients participating in a randomized study with dose-dense sequential adjuvant chemotherapy. Breast Cancer Res Treat. 2006;96:251-261.

  12. Yamauchi H, Stearns V, Hayes DF. When is a tumor marker ready for prime time? A case study of c-erbB-2 as a predictive factor in breast cancer. J Clin Oncol. 2001;19:2334-2356.

  13. Hayes DF, Thor AD, Dressler LG, et al for the Cancer and Leukemia Group B (CALGB) Investigators. HER2 and response to paclitaxel in node-positive breast cancer. N Engl J Med. 2007;357:1496-1506.

  14. National comprehensive cancer network clinical practice guidelines in oncology (NCCN Guidelines®). Breast Cancer version 2.2016. https://www.nccn.org/. Accessed January 25, 2017.

Content reviewed 04/2017

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