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HER-2/neu Testing

HER-2/neu (ERBB2) Testing

Clinical Focus

HER-2/neu (ERBB2) Testing



Clinical Use

Clinical Background

Individuals Suitable for Testing

Test Selection - Table - Figure

Test Interpretation


Additional References

Clinical Use [return to contents]

ERBB2 testing is used to predict 5-year disease-free and overall survival in patients with breast cancer, assess eligibility for trastuzumab (Herceptin®) treatment, assist in dose selection for certain drugs, and predict response to drug therapies.

Clinical Background [return to contents]

The ERBB2 (formerly HER-2/neu, human epidermal growth factor receptor 2) proto-oncogene, 1 of a family of 4 closely related growth factor receptor genes, encodes a 185-kd tyrosine kinase. ERBB2 gene amplification can lead to overproduction of the ERBB2 (formerly HER-2) protein and to tumor development through enhanced cell proliferation, survival, motility, and adhesion. ERBB2 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

ERBB2 status is most often used to determine patient eligibility for trastuzumab immunotherapy. Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of ERBB2, inhibits proliferation of human tumors cells that overexpress ERBB2. Documentation of ERBB2 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 ERBB2 gene and/or overexpress the ERBB2 protein are suitable candidates for this treatment.4-6

ERBB2 amplification or overexpression status can also be helpful when considering other types of therapy. ERBB2-positive tumors have been associated with increased sensitivity to anthracycline (eg, doxorubicin [Adriamycin®]) and cyclophosphamide/doxorubicin/5-fluorouracil (CAF) chemotherapy.7-10 Conversely, such patients do not benefit as much from cyclophosphamide/methotrexate/5-fluorouracil (CMF) regimens as do those with ERBB2-negative tumors.11-14 ERBB2-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 ERBB2 testing.15 Similarly, the data regarding ERBB2 status and taxanes are insufficient for clinical use, although the tendency is for ERBB2-positive tumors to respond well to treatment.15 For example, an article reported benefit from the addition of paclitaxel after adjuvant doxorubicin/cyclophosphamide therapy in patients with node-positive, ERBB2-positive breast cancer, while patients with node-positive, ERBB2-negative breast cancer did not benefit.16

Individuals Suitable for Testing [return to contents]

  • Includes all patients with invasive breast cancer1

Test Selection [return to contents]

There are several approaches for detecting ERBB2 overexpression or amplification. Currently, the only FDA-cleared methods for assessing ERBB2 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 of one method over the other. The figure portrays an algorithm for use and interpretation of IHC and FISH methods; the algorithm is based on the ASCO/CAP Panel recommendations.

Figure. Use and Interpretation of ERBB2 (HER-2) IHC and FISH Methods

IHC Method

The IHC method determines protein overexpression status of the invasive component of the cancer by using an anti-human ERBB2 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.

FISH Method

The FISH assay determines gene amplification status of the invasive component of the cancer by using probes specific for the ERBB2 locus and CEP 17; CEP 17 is included as an internal control and to account for aneusomy of chromosome 17. Results are reported as the ratio of ERBB2 signal to CEP 17 signal. A ratio of >2.2 indicates gene amplification. This cut-point is recommended by ASCO/CAP Panel and the NCCN,1,17 even though some clinical trials used a ratio cut-point of ≥2.0.6

Comparative information for these 2 methods can be found in Table. Aliases for these assays include
c-erbB-2, HER-2/neu, human epidermal growth factor receptor-2, and p185 (IHC only).

Table. Technology Guide to HER-2 (ERBB2) Tests Used by Quest Diagnostics


Immunohistochemistry (IHC)

Fluorescence In Situ Hybridization (FISH)

Test code 15547 14620(X)
Clinical use (FDA cleared) Evaluate ERBB2 protein overexpression in breast tumor tissue

Assess eligibility for trastuzumab (Herceptin) treatment

Detect ERBB2 oncogene amplification

Assess eligibility for trastuzumab treatment

Assess prognosis of stage II, node-positive breast cancer patients

Predict disease-free and overall survival in patients with stage II, node-positive breast cancer treated with adjuvant cyclophosphamide, doxorubicin (Adriamycin), 5-fluorouracil (CAF) chemotherapy

Assesses Protein overexpression Gene amplification
Method Immunohistochemistry (IHC)
with reflex to FISH (see below)
Fluorescence in situ hybridization (FISH) with reflex to IHC (see below)
Primary reagent Polyclonal anti-human ERBB2 antibody DNA probe specific for the ERBB2 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 ERBB2 overexpression

Ratio of ERBB2:CEP 17 control:

<1.8 = negative for ERBB2 amplification

1.8 to 2.2 = equivocala; reflexed to
IHC at additional charge

>2.2 = positive for ERBB2 amplification

Analysis time 1 to 3 days 2 to 4 days

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

Test Interpretation [return to contents]

ERBB2 IHC and FISH results are reported as shown in Table. ERBB2 overexpression or amplification suggests patient eligibility for trastuzumab treatment (Figure). In addition, stage II, node-positive breast cancer patients with amplification may benefit from higher doses of CAF.8,9

Lack of ERBB2 protein overexpression or gene amplification suggests the patient will not respond to trastuzumab therapy; however, clinical trials are needed to confirm this presumption. Similarly, trastuzumab response, or lack thereof, in patients with confirmed equivocal ERBB2 test results is uncertain. Trastuzumab is clinically indicated only for patients in whom ERBB2 protein overexpression or gene amplification can be confirmed.

References [return to contents]

  1. Wolff AC, Hammond MEH, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131:18-43.

  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. October, 2010. Available
    at:http://www.gene.com/gene/products/information/oncology/herceptin/insert.jsp. Accessed October 2, 2012.

  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. Muss HB, Thor AD, Berry DA, et al. c-erbB-2 expression and response to adjuvant therapy in women with node-positive early breast cancer. N Engl J Med. 1994;330:1260-1266. Erratum in: N Engl J Med. 1994;331:211.

  9. Thor AD, Berry DA, Budman DR, et al. erbB-2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer. J Natl Cancer Inst. 1998;90:1346-1360.

  10. Dressler LG, Berry DA, Broadwater G, et al. Comparison of HER2 status by fluorescence in situ hybridization and immunohistochemistry to predict benefit from dose escalation of adjuvant doxorubicin-based therapy in node-positive breast cancer patients. J Clin Oncol. 2005;23:4287-4297

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Carlson RW, Moench SJ, Hammond MEH, et al. HER2 testing in breast cancer: NCCN Task Force report and recommendations. J Natl Compr Canc Netw. 2006;4(Suppl 3):S1-S22. Also available at: http://www.nccn.org/professionals/physician_gls/f_guidelines.asp. Accessed October 2, 2012.

Additional References [return to contents]

  1. Press MF, Slamon DF, Klom KJ, et al. Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens. J Clin Oncol. 2002;20:3095-3105.

  2. Hayes DF, Thor AD. c-erbB2 in breast cancer: development of a clinically useful marker. Semin Oncol. 2002;29:231-245.

Content reviewed 05/2013
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