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Quantitative Assay for Measurement of HER2 Total Protein Expression and HER2 Dimers

Policy Number: MP-487

Latest Review Date: December 2020

Category: Laboratory

Policy Grade: B


The assessment of HER2 (human epidermal growth factor receptor 2) status by quantitative total HER2 protein expression and HER2 homodimer measurement is considered not medically necessary and investigational.


Novel assays that quantitatively measure total human epidermal growth factor receptor 2 (HER2) protein expression and homodimers have been developed to improve the accuracy and consistency of HER2 testing.  However, the HERmark Breast Cancer Assay, the sole assay included in this review, was discontinued by the manufacturer in September 2020 (Monogram Biosciences, oral communication, November 2020).

Human Epidermal Growth Factor Receptor 2

The human epidermal growth factor receptor (HER) family of receptor tyrosine kinases (EGFR/HER1, ErbB2/HER2, ErbB3/HER3, ErbB4/HER4) plays a major role in the pathogenesis of many solid tumors. In approximately 25% to 30% of breast cancers, overexpression of HER2 has been linked to shorter disease-free and overall survival, lack of responsiveness to tamoxifen antiestrogen therapy, and altered responsiveness to a variety of cytotoxic chemotherapy regimens.

Trastuzumab, a monoclonal antibody directed at the extracellular domain of HER2, has offered significant shorter disease-free and overall survival advantages in the metastatic and adjuvant settings in HER2-overexpressing patients, although not all patients respond. Fewer than 50% of patients with metastatic HER2-positive breast cancer show initial benefit from trastuzumab treatment, and many of those eventually develop resistance.

Current methodologies for the selection of HER2-positive patients include immunohistochemistry (IHC) to detect HER2 protein overexpression and fluorescence in situ hybridization (FISH) to detect HER2 gene amplification. However, controversy still exists regarding the accuracy, reliability, and interobserver variability of these assay methods. IHC provides a semiquantitative measure of protein levels (scored as 0, 1+, 2+, 3+) and the interpretation may be subjective. FISH is a quantitative measurement of gene amplification, in which the HER2 gene copy number is counted. However, FISH, which is considered to be more quantitative analytically, is not always representative of protein expression, and multiple studies have failed to demonstrate a relation between HER2 gene copy number and response to trastuzumab. Whereas patients who overexpress HER2 protein (IHC) or show evidence of HER2 gene amplification (FISH) have been shown to experience better outcomes on trastuzumab than those scored negative by those assays, differences in the degree of expression or amplification by these methods have generally not been shown to discriminate between groups with different outcomes. IHC and FISH testing may be affected by interlaboratory variability, and neither test provides quantitative data that reflect the activation state of signaling pathways in tumors, which may limit their utility in patient selection. Most laboratories in North America and Europe use IHC to determine HER2 protein status, with equivocal category results (2+) confirmed by FISH (or more recently by chromogenic in situ hybridization).

Typically, HER2 activates signaling pathways by dimerizing with ligand-bound EGFR-family members such as HER1 and HER3.  A HER2 ligand has not been identified, but overexpressed HER2 is constitutively active. When HER2 is pathologically overexpressed, the receptor may homo-dimerize and activate signaling cascades in the absence of the normal regulatory control imposed by the requirement for ligand binding of its heterodimerization partners.

A novel assay (HERmark® Breast Cancer Assay) was developed to quantify total HER2 protein expression and HER2 homodimers in formalin-fixed, paraffin-embedded tissue samples. On the HERmark® website, the manufacturer describes the test as "a novel HER-2 testing alternative to identify candidates for HER2-targeted therapy," and did not clearly target use for any particular breast cancer subpopulations (e.g., those with equivocal and/or discordant IHC/FISH tests). However, the HERmark Breast Cancer Assay was discontinued by the manufacturer (Monogram Biosciences) in September 2020 because "the standard of care no longer warrants anything but the FISH or IHC assays" (Monogram Biosciences, oral communication, November 2020).


The most recent literature search performed was through October 29, 2020.

Summary of Evidence

For individuals who have breast cancer and are undergoing assessment of HER2 status who receive quantitative total HER2 protein expression and HER2 homodimer measurement, the evidence includes validation studies and retrospective analysis of the association between levels and survival outcomes. Relevant outcomes are overall survival, disease-specific survival, test accuracy, and test validity. Retrospective analysis using HERmark have shown that the assay may predict a worse response to trastuzumab in certain populations. However, findings have been inconsistent, and no clear association with clinical outcomes has been shown. Additionally, cut points for defining patient groups varied across studies. Clinical utility of the HERmark assay has not been demonstrated. The evidence is insufficient to determine the effects of the technology on health outcomes.

Practice Guidelines and Position Statements

American Society of Clinical Oncology and College of American Pathologists

In 2018, the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) published a clinical practice guideline focused update on HER2 testing in breast cancer. The guidance updated 2013 ASCO/CAP recommendations. The guideline recommends "All newly diagnosed patients with breast cancer must have a HER2 test performed. Patients who then develop metastatic disease must have a HER2 test performed in a metastatic site, if tissue sample is available." For "acceptable (immunohistochemistry [IHC] and in situ hybridization [ISH]) tests," the guideline states: "Should preferentially use an FDA-approved IHC, brightfield ISH, or fluorescent in situ hybridization (FISH) assay."

National Comprehensive Cancer Network

National Comprehensive Cancer Network (NCCN) Guidelines on the treatment of breast cancer (v.6.2020) do not address the use of HERmark.

U.S. Preventive Services Task Force Recommendations

Not applicable.


HER2 Quantitative Assay, HERmark test, HER-2


Clinical laboratories may develop and validate tests in-house and market them as a laboratory service; laboratory-developed tests must meet the general regulatory standards of the Clinical Laboratory Improvement Amendments. HERmark® Breast Cancer Assay (Monogram Biosciences) is available under the auspices of the Clinical Laboratory Improvement Amendments. Laboratories that offer laboratory-developed tests must be licensed by the Clinical Laboratory Improvement Amendments for high-complexity testing. To date, the U.S. Food and Drug Administration has chosen not to require any regulatory review of this test.


Coverage is subject to member’s specific benefits.  Group specific policy will supersede this policy when applicable.

ITS: Home Policy provisions apply.

FEP:  Special benefit consideration may apply.  Refer to member’s benefit plan.  FEP does not consider investigational if FDA approved and will be reviewed for medical necessity.


CPT Codes:


Unlisted chemistry procedure


  1. Arteaga CL, O'Neill A, Moulder SL, et al. A phase I-II study of combined blockade of the ErbB receptor network with trastuzumab and gefitinib in patients with HER2 (ErbB2)-overexpressing metastatic breast cancer. Clin Cancer Res. Oct 01 2008; 14(19):6277-6283.
  2. Barros FF, Abdel-Fatah TM, Moseley P, et al. Characterisation of HER heterodimers in breast cancer using in situ proximity ligation assay. Breast Cancer Res Treat. Apr 2014; 144(2):273-285.
  3. Bates M, Sperinde J, Köstler WJ et al. Identification of a subpopulation of metastatic breast cancer patients with very high HER2 expression levels and possible resistance to trastuzumab. Ann Oncol 2011; 22(9):2014-20.
  4. Bates M, Sperinde J, Kostler WJ, et al. Identification of a subpopulation of metastatic breast cancer patients with very high HER2 expression levels and possible resistance to trastuzumab. Ann Oncol. Sep 2011; 22(9):2014-2020.
  5. Camp RL, Dolled-Filhart M, Rimm DL. X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res. Nov 1 2004; 10(21):7252-7259.
  6. Chumsri S, Sperinde J, Liu H, et al. High p95HER2/HER2 Ratio Associated With Poor Outcome in Trastuzumab-Treated HER2-Positive Metastatic Breast Cancer NCCTG N0337 and NCCTG 98-32-52 (Alliance). Clin Cancer Res. Jul 1 2018; 24(13):3053-3058.
  7. de Alava E, Ocana A, Abad M, et al. Neuregulin expression modulates clinical response to trastuzumab in patients with metastatic breast cancer. J Clin Oncol. Jul 01 2007; 25(19):2656-2663.
  8. DeFazio-Eli L, Strommen K, Dao-Pick T et al. Quantitative assays for the measurement of HER1-HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action. Breast Cancer Res 2011; 13:R44.
  9. Duchnowska R, Biernat W, Szostakiewicz B et al. Correlation between quantitative HER-2 protein expression and risk for brain metastases in HER-2+ advanced breast cancer patients receiving trastuzumab-containing therapy. Oncologist 2012; 17(1):26-35.
  10. Duchnowska R, Sperinde J, Chenna A, et al. Quantitative measurements of tumoral p95HER2 protein expression in metastatic breast cancer patients treated with trastuzumab: independent validation of the p95HER2 clinical cutoff. Clin Cancer Res. May 15 2014; 20(10):2805-2813.
  11. Duchnowska R, Sperinde J, Czartoryska-Arlukowicz B, et al. Predictive value of quantitative HER2, HER3 and p95HER2 levels in HER2-positive advanced breast cancer patients treated with lapatinib following progression on trastuzumab. Oncotarget. Nov 28 2017; 8(61):104149-104159.
  12. Green AR, Barros FF, Abdel-Fatah TM, et al. HER2/HER3 heterodimers and p21 expression are capable of predicting adjuvant trastuzumab response in HER2+ breast cancer. Breast Cancer Res Treat. May 2014; 145(1):33-44.
  13. Han SW, Cha Y, Paquet A et al. Correlation of HER2, p95HER2 and HER3 expression and treatment outcome of lapatinib plus capecitabine in her2-positive metastatic breast cancer. PloS One 2012; 7(7):e39943.
  14. Huang W, Reinholz M, Weidler J et al. Comparison of central HER2 testing with quantitative total HER2 expression and HER2 homodimer measurements using a novel proximity-based assay. Am J Clin Pathol 2010; 134(2):303-11.
  15. Huang W, Reinholz M, Weidler J, et al. Comparison of central HER2 testing with quantitative total HER2 expression and HER2 homodimer measurements using a novel proximity-based assay. Am J Clin Pathol. Aug 2010; 134(2):303-311.
  16. Joensuu H, Sperinde J, Leinonen M et al. Very high quantitative tumor HER2 content and outcome in early breast cancer. Ann Oncol 2011; 22(9):2007-13.
  17. Larson JS, Goodman LJ, Tan Y et al. Analytical validation of a highly quantitative, sensitive, accurate, and reproducible assay (HERmark®) for the measurement of HER2 total protein and HER2 homodimers in FFPE breast cancer tumor specimens. Patholog Res Int 2010; 2010:814176.
  18. Lipton A, Goodman L, Leitzel K et al. HER3, p95HER2, and HER2 protein expression levels define multiple subtypes of HER2-positive metastatic breast cancer. Breast Cancer Res Treat 2013.
  19. Lipton A, Köstler WJ, Leitzel K et al. Quantitative HER2 protein levels predict outcome in fluorescence in situ hybridization-positive patients with metastatic breast cancer treated with trastuzumab. Cancer 2010; 116(22):5168-78.
  20. Monogram biosciences announces HER2:HER3 heterodimer and HER3/PI3K VeraTag™ assays are now available for use in development of cancer therapeutics: November 11, 2008. Available online at: Last accessed November 12, 2015.
  21. Monogram Biosciences. HERmark Breast Cancer Assay. 2017; Accessed October 24, 2017.
  22. Montemurro F, Prat A, Rossi V, et al. Potential biomarkers of long-term benefit from single-agent trastuzumab or lapatinib in HER2-positive metastatic breast cancer. Mol Oncol. Feb 2014; 8(1):20-26.
  23. National Comprehensive Cancer Network (NCCN). NCCN Clinical practice guidelines in oncology: Breast cancer. Version 6.2020. Accessed November 4, 2020.
  24. Piccart M, Lohrisch C, Di Leo A, et al. The predictive value of HER2 in breast cancer. Oncology. 2001; 61 Suppl 2:73-82.
  25. Scaltriti M, Nuciforo P, Bradbury I, et al. High HER2 expression correlates with response to the combination of lapatinib and trastuzumab. Clin Cancer Res. Feb 1 2015; 21(3):569-576.
  26. Shi Y, Huang W, Tan Y et al. A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue. Diagn Mol Pathol 2009; 18(1):11-21.
  27. Toi M, Sperinde J, Huang W et al. Differential survival following trastuzumab treatment based on quantitative HER2 expression and HER2 homodimers in a clinic-based cohort of patients with metastatic breast cancer. BMC Cancer 2010; 10:56.
  28. Wolff AC, Hammond ME, 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(1):18-43.
  29. Yardley DA, Kaufman PA, Huang W, et al. Quantitative measurement of HER2 expression in breast cancers: comparison with 'real-world' routine HER2 testing in a multicenter Collaborative Biomarker Study and correlation with overall survival. Breast Cancer Res. Mar 18 2015; 17: 41.


Medical Policy Group (3), October 2011

Medical Policy Administration Committee, October 2011

Available for comment November 11 through December 27, 2011

Medical Policy Panel, October 2012

Medical Policy Group, October 2012 (3): 2012 Updates to Key Points and References

Medical Policy Panel, October 2013

Medical Policy Group, October 2013 (1) Update to Key Points and References; no change to policy statement

Medical Policy Panel, October 2014

Medical Policy Panel, December 2015

Medical Policy Group, January 2016 (3):  2014 & 2015 Updates to Key Points & References; no change in policy statement

Medical Policy Panel, December 2017

Medical Policy Group, January 2018 (3): 2017 Updates to Description, Key Points, Approved by Governing Bodies & References; no change in policy statement

Medical Policy Panel, December 2018

Medical Policy Group, January 2019 (9): 2018 Updates to Description, Key Points, References. No change to policy statement.

Medical Policy Panel, December 2019

Medical Policy Group, December 2019 (9): 2019 Updates to Description, Key Points. No change to policy statement.

Medical Policy Panel, December 2020

Medical Policy Group, December 2020 (9): 2020 Updates to Description, Key Points, References. No change to policy statement.

This medical policy is not an authorization, certification, explanation of benefits, or a contract. Eligibility and benefits are determined on a case-by-case basis according to the terms of the member’s plan in effect as of the date services are rendered. All medical policies are based on (i) research of current medical literature and (ii) review of common medical practices in the treatment and diagnosis of disease as of the date hereof. Physicians and other providers are solely responsible for all aspects of medical care and treatment, including the type, quality, and levels of care and treatment.

This policy is intended to be used for adjudication of claims (including pre-admission certification, pre-determinations, and pre-procedure review) in Blue Cross and Blue Shield’s administration of plan contracts.

The plan does not approve or deny procedures, services, testing, or equipment for our members. Our decisions concern coverage only. The decision of whether or not to have a certain test, treatment or procedure is one made between the physician and his/her patient. The plan administers benefits based on the member’s contract and corporate medical policies. Physicians should always exercise their best medical judgment in providing the care they feel is most appropriate for their patients. Needed care should not be delayed or refused because of a coverage determination.

As a general rule, benefits are payable under health plans only in cases of medical necessity and only if services or supplies are not investigational, provided the customer group contracts have such coverage.

The following Association Technology Evaluation Criteria must be met for a service/supply to be considered for coverage:

1. The technology must have final approval from the appropriate government regulatory bodies;

2. The scientific evidence must permit conclusions concerning the effect of the technology on health outcomes;

3. The technology must improve the net health outcome;

4. The technology must be as beneficial as any established alternatives;

5. The improvement must be attainable outside the investigational setting.

Medical Necessity means that health care services (e.g., procedures, treatments, supplies, devices, equipment, facilities or drugs) that a physician, exercising prudent clinical judgment, would provide to a patient for the purpose of preventing, evaluating, diagnosing or treating an illness, injury or disease or its symptoms, and that are:

1. In accordance with generally accepted standards of medical practice; and

2. Clinically appropriate in terms of type, frequency, extent, site and duration and considered effective for the patient’s illness, injury or disease; and

3. Not primarily for the convenience of the patient, physician or other health care provider; and

4. Not more costly than an alternative service or sequence of services at least as likely to produce equivalent therapeutic or diagnostic results as to the diagnosis or treatment of that patient’s illness, injury or disease.