BlueCross and BlueShield of Montana Medical Policy/Codes
Quantitative Assay for Measurement of HER2 Total Protein Expression and HER2 Dimers
Chapter: Medicine: Tests
Current Effective Date: November 26, 2013
Original Effective Date: November 26, 2013
Publish Date: August 26, 2013
Description

Novel assays that quantitatively measure total HER2 protein expression and homodimers have been developed in an effort to improve the accuracy and consistency of HER2 testing.

Background

The HER-family of receptor tyrosine kinases (EGFR/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4) plays a major role in the pathogenesis of many solid tumors. In approximately 25-30% of breast cancers, overexpression of HER2 has been linked to shorter disease-free (DFS) and overall survival (OS), 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 DFS and OS 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. (1)

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+, and 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 relationship 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. (2) 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 [CISH]).

Normally, 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 homodimerize 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, Monogram Biosciences, South San Francisco, CA) was developed to quantify total HER2 expression and HER2 homodimers in formalin-fixed, paraffin-embedded tissue samples.

Regulatory Status

U.S. Food and Drug Administration (FDA) does not regulate in-house or “home brew” tests for HER2, tests developed and used at unique or individual laboratory sites.

The HERmark assay has been validated according to the specifications prescribed by the Clinical Laboratory Improvement Amendments (CLIA) and is performed in a College of American Pathologists-certified clinical reference laboratory at Monogram Biosciences.

Policy

Each benefit plan, summary plan description or contract defines which services are covered, which services are excluded, and which services are subject to dollar caps or other limitations, conditions or exclusions. Members and their providers have the responsibility for consulting the member's benefit plan, summary plan description or contract to determine if there are any exclusions or other benefit limitations applicable to this service or supply.  If there is a discrepancy between a Medical Policy and a member's benefit plan, summary plan description or contract, the benefit plan, summary plan description or contract will govern.

Investigational

Blue Cross and Blue Shield of Montana (BCBSMT) considers the assessment of HER2 status by quantitative total HER2 protein expression and HER2 homodimer measurement experimental, investigational and unproven.

Rationale

Technical performance of the assay

The HERmark assay was based on a proprietary technology platform that enables quantification of proteins and protein-protein complexes through the release of a fluorescent tag (VeraTag reporter, Monogram Biosciences) conjugated to specific HER antibodies, requiring proximity to a second HER antibody (so-called proximity-based technology).

The HERmark assay is currently commercially available only for quantification of HER2 total protein expression (H2T) and HER2 homodimers (H2D). The company plans to validate use of the test to measure HER heterodimers.

HER2 protein quantification was normalized to tumor area and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting, and with HER2 immunohistochemistry (IHC) test categories and histoscores in cell lines and in 170 human breast tumors. (2) In contrast to conventional IHC test categories, the HER2 protein levels determined by VeraTag assay represent a continuous measurement over a dynamic range greater than 2 log10, and HER2 homodimer levels were consistent with crosslinking and immunoprecipitation results.

Huang and colleagues compared results of the HERmark assay with those of IHC and fluorescence in situ hybridization (FISH) centrally performed at the Mayo Clinic in 237 archived formalin-fixed, paraffin-embedded (FFPE) breast cancers. (3) IHC had already been performed at the time of initial diagnosis in all of the cases but was repeated for the purpose of this validation, and interpreted by one reviewer and scored as negative, equivocal, or positive according to the American Society of Clinical Oncologists/College of American Pathologists (ASCO/CAP) guidelines. (4) Reflex FISH for HER2 gene amplification had also been performed at the time of initial diagnosis on all 94 of the IHC 2+ cases. Repeat FISH was performed at the same laboratory and an overall evaluation performed by one pathologist. Of the 84 cases in the immunohistochemically negative subgroup, 80 (95%), 2 (2%) and 2 (2%) were classified as negative, equivocal, and positive by HERmark, respectively. Of the 101 cases in the immunohistochemically equivocal subgroup, 33 (32.7%), 31 (30.7%), and 37 (36.6%) were classified as negative, equivocal, and positive by HERmark, respectively. Of the 52 cases in the immunohistochemically positive subgroup, 1 (2%), 3 (6%), and 48 (92%) were classified as negative, equivocal, and positive by HERmark, respectively. The overall concordance was 67%, with a weighted κ of 63% (95% confidence interval [CI]: 55-70%) calculated using the κ statistic. When the equivocal cases were excluded from the HERmark and immunohistochemical results, the positive and negative concordance between HERmark and central immunohistochemical testing was 98%. The overall concordance was 98%, with a κ of 95% (95% CI: 89-100%).

Reflex FISH was performed on 94 breast cancers that had been determined as 2+ immunohistochemically at the time of initial diagnosis. Variable H2T and H2D levels were correlated to corresponding results for the HER2/centromere 17 (HER2/CEP17) ratio. Of the 94 cases that were 2+ immunohistochemically, 62 (66%), 5 (5%), and 27 (29%) were determined at the same central laboratory as negative, equivocal, and positive by FISH, respectively. Of the 62 FISH-negative cases, 24 (39%), 21 (34%), and 17 (27%) were determined as negative, equivocal, and positive by HERmark, respectively. Of the 5 FISH-equivocal cases, 1 (20%), 2 (40%), and 2 (40%) were determined as negative, equivocal, and positive by HERmark, respectively. Of the 27 FISH-positive cases, 3 (11%), 6 (22%), and 18 (67%) were determined as negative, equivocal, and positive by HERmark, respectively.

How well does HERmark predict subpopulation response to trastuzumab?

Bates and colleagues measured HER2 protein expression (H2T) in formalin-fixed, paraffin-embedded primary breast tumors from 98 women treated with trastuzumab-based therapy for metastatic breast cancer. (5) Using subpopulation treatment effect pattern plots, the population was divided into H2T low (H2T <13.8), H2T high (H2T ≥68.5), and H2T intermediate (13.8 ≤H2T<68.5) subgroups. Kaplan–Meier (KM) analyses were carried out comparing the groups for time-to-progression (TTP) and overall survival (OS). Cox multivariate analyses were carried out to identify correlates of clinical outcome. Bootstrapping analyses were carried out to test the robustness of the results. TTP improved with increasing H2T until, at the highest levels of H2T, an abrupt decrease in the TTP was observed. KM analyses demonstrated that patients with H2T low tumors (median TTP: 4.2 months; hazard ratio [HR]: 3.7; p<0.0001) or H2T high tumors (median TTP: 4.6 months; HR: 2.7; p=0.008) had significantly shorter TTP than patients whose tumors were H2T intermediate (median TTP: 12 months). OS analyses yielded similar results. The authors concluded that patients with very high levels of H2T may represent a subgroup with de novo resistance to trastuzumab but that these results were preliminary and require confirmation in larger controlled clinical cohorts.

Joensuu and colleagues reported the results of measurement of total HER2 content (H2T) using HERmark from formalin-fixed tumor tissue of 899 women who participated in the FinHer trial (ISRCTN76560285) to determine if very high tumor total HER2 content influences outcome in early breast cancer treated with adjuvant trastuzumab plus chemotherapy. (6) In a chromogenic in situ hybridization (CISH) test, 197 (21.9%) patients had HER2-positive cancer and were randomly assigned to receive trastuzumab or control. Tumor H2T levels varied greatly, by 1,808-fold. High H2T levels strongly correlated with a positive HER2 status by CISH (P < 0.0001). Patients with very high H2T (defined by ≥22-fold the median of HER2-negative cancers; 13% of CISH-positive cancers) did not benefit from adjuvant trastuzumab (HR: 1.23; 95% CI: 0.33-4.62; p=0.75), whereas the rest of the patients with HER2-positive disease by CISH (87%) did benefit (HR: 0.52; 95% CI: 0.28-1.00; p=0.050). The authors concluded that patients with HER2-positive breast cancer with very high tumor HER2 content may benefit less from adjuvant trastuzumab compared with those whose cancer has more moderate HER2 content.

Toi and colleagues investigated the relationship between quantitative measurements of HER2 expression (H2T) or HER2 homodimers (H2D) and OS in a clinic-based population of 72 patients drawn from 6 oncology clinics in Japan who had metastatic breast cancer and had been treated with at least one chemotherapy regimen prior to receiving trastuzumab. (7) Patients were originally selected for treatment with trastuzumab by IHC (88%) or FISH (12%). HERmark assay results were correlated with OS using univariate Kaplan-Meier, hazard function plots and multivariate Cox regression analyses. Clinical outcome data were drawn from medical chart review. Measurements of H2T and H2D were tested for association with OS, defined as the time from start of trastuzumab treatment to cancer-associated death or the end of the follow-up period. The median duration of patient follow-up period was 18.2 months. The median duration of trastuzumab treatment was 14.6 months. As a whole, 2-year survival rate of the cohort was 60.8% (95% CI: 48.4-73.2%). In the univariate analyses, patients were classified into 4 subgroups defined by the 25th, 50th, and 75th percentiles for each of the 3 variables, H2T, H2D, and their ratio H2D/ H2T. Hazard function plots were estimated in the 4 H2T subgroups, and the subgroups with the 25% highest and lowest H2T values had substantially lower risk of death than the middle 2 subgroups. Dividing the cohort into high HER2-expressing (≥the median value of H2T) and low HER2-expressing (<the median value of H2T) sub-groups and using the Cox regression analysis with the continuous H2T value in each of the 2 subgroups, those patients with higher values for HER2 expression lived longer than those with lower values for H2T in the high HER2- expressing group (HR: 0.06; p=0.010; 95% CI: 0.01-0.51). In contrast, in the low HER2-expressing group, the opposite trend (those with lower H2T values were favored) was observed (HR: 16.0; p=0.017; 95% CI: 1.64-155.9). The authors concluded that their data suggest that there are 2 subpopulations of patients in this cohort that behave differently with respect to HER2 expression and OS and that the quantitative amount of HER2 expression measured by HERmark may be a new useful marker to identify a more relevant target population for trastuzumab treatment in patients with metastatic breast cancer.

Lipton and colleagues used the HERmark assay to quantify HER2 expression and examined outcomes in 102 trastuzumab-treated metastatic breast cancer patients previously assessed as IHC 3+ by local but not central IHC, or FISH-positive, and then retested by central FISH. (8) Of 102 metastatic breast cancer patients previously scored as IHC 3+ or 2+/FISH-positive and treated with trastuzumab-containing regimens, 98 had both central FISH and HER2 total expression values. Sixty-six of 76 central FISH-positive patients (87%) had high HER2 total expression levels (concordant positive), and 19 of 22 central FISH-negative patients (86%) were HER2 total expression low (concordant negative). Fourteen percent (3 of 22) of central FISH-negative patients were HER2 total expression high (discordant HER2 total expression high), and 13% (10 of 76) of central FISH-positive patients were HER2 total expression low (discordant HER2 total expression low). The concordant positive group had a significantly longer time to progression (time-to-progression [TTP] median: 11.3 months) compared with the concordant negative group (median TTP: 4.5 months; HR: 0.42; p<0.001), and also compared with the discordant HER2 total expression low group (median TTP: 3.7 months; HR: 0.43; p=0.01). The discordant HER2 total expression low group behaved similarly compared with concordant negatives (HR: 1; p=0.99). In analyses restricted to central FISH-positive patients only (n=77), Cox proportional hazards multivariate regression identified HER2 total expression as an independent predictor of TTP (HR: 0.29; p=0.0015) and overall survival (HR=0.19, p<0.001). The authors concluded that a subset of patients with HER2 gene amplification by FISH express low levels of HER2 protein and have reduced response to trastuzumab-containing therapy, similar to FISH-negative.

Clinical utility

Data on the clinical utility of HERmark are lacking. Clinical trials are needed to understand the relationship between quantitative HER2 expression and homodimer measurements with clinical outcomes in breast cancer patients stratified by the HERmark assay being treated with anti-HER2 therapy in the adjuvant and metastatic settings.

Duchnowska and colleagues investigated the correlation between quantitative HER-2 expression in primary breast cancers and the time-to-brain metastasis (TTBM) in HER-2+ advanced breast cancer patients treated with trastuzumab. (9) The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER-2+ metastatic breast cancer. HER-2/neu gene copy number was quantified as the HER-2/centromeric probe for chromosome 17 (CEP17) ratio by central laboratory FISH. HER-2 protein was quantified as total HER-2 protein expression (H2T) by the HERmark® assay in formalin-fixed, paraffin-embedded tumor samples. HER-2 variables were correlated with clinical features and TTBM was measured from the initiation of trastuzumab-containing therapy. A higher H2T level (continuous variable) was correlated with shorter TTBM, whereas HER-2 amplification by FISH and a continuous HER-2/CEP17 ratio were not predictive (p =0.013, .28, and .25, respectively). In the subset of patients that was centrally determined by FISH to be HER-2+, an above-the-median H2T level was significantly associated with a shorter TTBM (hazard ratio, [HR]: 2.4; p=0.005), whereas this was not true for the median HER-2/CEP17 ratio by FISH (p=0.4). Correlation between a continuous H2T level and TTBM was confirmed on multivariate analysis (HR: 3.3; p=0.024). The authors concluded that their data revealed a strong relationship between the quantitative HER-2 protein expression level and the risk for brain relapse in HER-2+ advanced breast cancer patients and that quantitative assessment of HER-2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this group.

Summary

Retrospective analyses using HERmark have shown that the assay may predict a worse response to trastuzumab in certain populations. The clinical utility of the HERmark assay has not been demonstrated, and clinical trials are needed to determine the impact on the clinical outcomes of patients stratified by the HERmark assay.

Practice Guidelines and Position Statements

National Comprehensive Cancer Network (NCCN) Guidelines on the treatment of breast cancer (v3.2012) do not address the use of HERmark. (10)

National Cancer Institute (NCI) Physician Query Database (PDQ) returned no trials using HERmark.

Coding

Disclaimer for coding information on Medical Policies

Procedure and diagnosis codes on Medical Policy documents are included only as a general reference tool for each policy. They may not be all-inclusive.

The presence or absence of procedure, service, supply, device or diagnosis codes in a Medical Policy document has no relevance for determination of benefit coverage for members or reimbursement for providers. Only the written coverage position in a medical policy should be used for such determinations.

Benefit coverage determinations based on written Medical Policy coverage positions must include review of the member’s benefit contract or Summary Plan Description (SPD) for defined coverage vs. non-coverage, benefit exclusions, and benefit limitations such as dollar or duration caps. 

ICD-9 Codes

Experimental, investigational and unproven for all diagnoses.

ICD-10 Codes

Experimental, investigational and unproven for all diagnoses.

Procedural Codes: 84999
References
  1. 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(2):R44.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  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. National Comprehensive Cancer Network (NCCN). Breast cancer (V.3.2012). Available online at: www.nccn.org Last accessed September, 2012.
  11. Quantitative Assay for Measurement of HER2 Total Protein Expression and HER2 Dimers. Chicago, Illinois: Blue Cross Blue Shield Association Medical Policy Reference Manual (October 2012) Medicine 2.04.76.
History
August 2013  New 2013 BCBSMT medical policy.  The assessment of HER2 status by quantitative total HER2 protein expression and HER2 homodimer measurement is considered experimental, investigational and unproven.  
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Quantitative Assay for Measurement of HER2 Total Protein Expression and HER2 Dimers