Test Catalog

Test ID: H2BR    
HER2 Amplification Associated with Breast Cancer, FISH, Tissue

Useful For Suggests clinical disorders or settings where the test may be helpful

A predictive marker for patients with both node-positive or node-negative primary and metastatic breast cancer


Patients with HER2 amplification that may be candidates for therapies targeting the human epidermal growth factor receptor 2 (HER2) protein (eg, trastuzumab [Herceptin], pertuzumab, lapatinib)


Confirming the presence of HER2 amplification in cases with 2+ (low level) or 3+ (high level) HER2 overexpression by immunohistochemistry, and for certain histologic subtypes with aberrant patterns of HER2 expression seen by immunohistochemistry (eg, micropapillary carcinoma)

Testing Algorithm Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

Reflex testing will be performed using immunohistochemistry (IHC) when the FISH result falls within certain ranges as defined by the 2018 focused update to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines.(1) For FISH results in ASCO/CAP categories Group 2, 3, and 4 (formerly called "equivocal"), the IHC testing will be added, charged, and reported separately. An integrated interpretation of the IHC and FISH results will be provided (see Interpretation).


A charge and CPT code is applied for each probe set hybridized, analyzed, and reported.

Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

HER2 (ERBB2: c-erb-b2) is an oncogene on the long arm of chromosome 17 that is amplified in approximately 15% to 20% of breast cancers. Amplification or overexpression of HER2 has been shown to be associated with shorter disease-free survival and poorer overall survival in breast cancers. Patients with HER2 gene amplification or overexpression are candidates for treatment with the drugs that target the human epidermal growth factor receptor 2 (HER2) protein or its downstream pathways (eg, trastuzumab [Herceptin], pertuzumab).


FISH with labeled DNA probes to the pericentromeric region of chromosome 17 and to the HER2 locus can be used to determine if a patient's breast cancer has HER2 gene amplification. Immunohistochemical analysis is used to determine if a tumor exhibits HER2 overexpression.

Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.

An interpretive report will be provided.

Interpretation Provides information to assist in interpretation of the test results

An interpretive report will be provided. Results are interpreted utilizing the current American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines.(1)


Under the 2018 Focused Update to the ASCO/CAP Guidelines, reflex immunohistochemistry (IHC) is performed for certain categories of results, known as Groups 2, 3, and 4. These categories are shown in the table below (Group 4 is the category formerly referred to as FISH "equivocal"). If reflex IHC is performed and is either negative (0, 1+) or positive (3+), the result of the FISH assay is considered resolved by IHC as either negative or positive. If the IHC assay shows an equivocal (2+) result, then the FISH slide is re-scored within the areas showing the most intense membranous (2+) staining and the final FISH result is used to determine whether the result is negative or positive.


ASCO/CAP Result Category

HER2:D17Z1 ratio;

average HER2 copies per cell

Reporting approach per 2018 ASCO/CAP guidelines

Group 1

HER2:D17Z1=2.00; HER2/cell  > or =4.0


Group 2

HER2:D17Z1=2.00; HER2/cell <4.0

Reflex IHC; FISH re-analysis if 2+

Group 3

HER2:D17Z1<2.00; HER2/cell > or =6.0

Reflex IHC; FISH re-analysis if 2+

Group 4

HER2:D17Z1<2.00; HER2/cell > or =4.0 <6.0

Reflex IHC; FISH re-analysis if 2+

Group 5

HER2:D17Z1<2.00; HER2/cell <4.0



The degree of HER2 amplification varies in tumors. Some exhibit high levels of amplification (HER2:D17Z1 ratio >4.0), whereas others exhibit low-level amplification (HER2:D17Z1 ratio of 2.0-4.0). It is not currently known if patients with different levels of amplification have the same prognosis and response to therapy.


Reports also interpret the HER2 copy number changes relative to chromosome 17 copy number (aneusomy) or potential structural genomic abnormalities that increase HER2 copy number.


Rare cases may not show HER2 amplification but still have human epidermal growth factor receptor 2 (HER2) protein overexpression demonstrated by immunohistochemistry. The clinical significance of HER2 protein overexpression in the absence of HER2 gene amplification is unclear. However, these patients may have a worse prognosis and be candidates for treatments that target the HER2 protein or its downstream pathways.

Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

Optimum fixation should be between 6 and 72 hours in 10% neutral buffered formalin. Other types of fixatives should not be used.


The prognostic information provided by the HER2 status of a patient's tumor should not be interpreted in isolation because other prognostic features (eg, lymph node status, tumor size, estrogen/progesterone receptor status) may be of equal or greater importance in determining the patient's prognosis.

Supportive Data

The probe was independently validated in a blinded study on 1,156 paraffin-embedded breast tissue samples. The results of the FISH testing was correlated to the immunohistochemical (IHC) analysis, which was interpreted on a scale ranging from 0 to 3+ according to FDA-approved guidelines.

Clinical Reference Recommendations for in-depth reading of a clinical nature

1. Wolff AC, Hammond MEH, Allison KH, et al: Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update. J Clin Oncol 2018 Jul 10;36(20):2105-2122 doi:10.1200/JCO.2018.77.8738

2. College of American Pathologists Cytogenetics Checklist item CYG.48932. 09/2019. p 25

3. Wolff AC, Hammond ME, Hicks DG, et al: Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society for Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Onc 2013 Nov 1;31(31):3997-4013

4. Perez EA, Roche PC, Jenkins RB, et al: HER2 testing in patients with breast cancer: poor correlation between weak positively by immunohistochemistry and gene amplification by fluorescence in situ hybridization. Mayo Clin Proc 2002 Feb;77(2):148-154

5. Romond EH, Perez EA, Bryant J, et al: Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005 Oct 20;353(16):1673-1684

6. Perez EA, Romond EH, Suman VJ, et al: Four-year follow-up of trastuzumab plus adjuvant chemotherapy for operable human epidermal growth factor receptor 2-positive breast cancer: joint analysis of data from NCCTG N9831 and NSABP B-31. J Clin Oncol 2011 Sep 1;29(25):3366-3373

7. Blumenthal GM, Scher NS, Cortazar P, et al: First FDA approval of dual anti-HER2 regimen: pertuzumab in combination with trastuzumab and docetaxel for HER2-positive metastatic breast cancer. Clin Cancer Res 2013 Sep 15;19(18):4911-4916

8. Robidoux A, Tang G, Rastogi P: Lapatinib as a component of neoadjuvant therapy for HER2-positive operable breast cancer (NSABP protocol B-41): an open-label, randomized phase 3 trial. Lancet Oncol 2013 Nov;14(12):1183-1192