Standard HER2 Assays Do Not Accurately Identify HER2-Low Breast Cancer

Data collected from 1400 global laboratories indicate that standard immunohistochemistry testing may not be the most effective method to identify patients with breast cancer with HER2-low disease.

Standard HER2 assays do not appear to accurately identify patients with breast cancer who have low HER2 immunohistochemistry (IHC) scores ranging from 0 to 1+, according to survey data collected from over 1400 global laboratories that were presented at the 2021 San Antonio Breast Cancer Symposium.1

Results from the College of American Pathologists’ (CAP) Proficiency Testing Surveys showed that 52 of the 80 cores evaluated (65%) had a 90% or greater concordance agreement, and 15 (25%) had a concordance agreement of less than 70%.

Furthermore, results from a separate cohort of the analysis that was done in the Department of Pathology at Yale School of Medicine showed that 92 of 170 cases evaluated were read as a score of 0 by at least 1 pathologist, and of those, 26% (n = 24/92) had a concordance agreement of 90% or higher. Moreover, 45 of the 170 cases were read as a score of 3+ by at least 1 pathologist, and of those, 58% (n = 26/45) had a concordance agreement of 90% or higher. The comparison of 0 or 1+ concordant cases vs 2+ or 3+ concordant cases showed a significant difference x2 = 12.07, P < .0005).

“The scoring accuracy for HER2 IHC in the low range [0 and 1+] is poor, and this inaccuracy in the real world could lead to misassignment of many patients for treatment with fam-trastuzumab deruxtecan-nxki [Enhertu] for [those who are] HER2 low,” lead study author Aileen Fernandez, PhD, a postdoctoral fellow at Yale School of Medicine, and colleagues, wrote in a poster presentation on the data.

Antibody-drug conjugates (ADCs) are developed to effectively deliver cytotoxic agents directly to malignant cells. Trastuzumab deruxtecan, an ADC comprised of the HER2-targeting antibody trastuzumab (Herceptin), an enzyme-cleavable linker, and a cytotoxic topoisomerase I inhibitor, has demonstrated antitumor activity in patients with breast cancer who have low levels of HER2 expression. Current companion diagnostic tests for HER2-targeted therapies, IHC, and fluorescent in situ hybridization, have been optimized for high levels of HER2.2-4

Here, investigators hypothesized that the current standard assays utilized in the clinic do not efficiently differentiate between patients whose cancers have HER2 expression of 0 or 1+. Thus, patients who could potentially derive benefit from treatment with trastuzumab deruxtecan may be missed in practice.

The primary objective of the study was to determine the suitability of the current standard HER2 IHC assays to select patients with low HER2 positivity for treatment with trastuzumab deruxtecan.

Two independent data set were examined for this study. Investigators evaluated 2 years of CAP Proficiency Testing Surveys for HER2 expression in breast cancer, and each dataset covered scores from up to 1452 laboratories, with 20 cores each, and supplemental questions regarding the methodology utilized. Each lab received 2 tissue microarrays (TMAs) of 10 breast cancer cores, and the TMAs were comprised of cores that were HER2 IHC 0, 1, or 3. Notably, no 2+ cores were included in the TMAs. Laboratories then stained for HER2 using the standard IHC assay, and submitted their scores to CAP.

For the Yale cohort, hematoxylin and eosin, as well as HER2 IHC, digitally scanned images of 170 independent archival breast biopsies were collected in 2018. Notably, the selection was enriched in HER2 IHC 2+ and 3+ cases. The datasets were then read by 18 board-certified pathologists, most of whom had more than 5 years of experience. These pathologists proceeded to score the cases as 0, 1+, 2+, or 3+. The percent interrater agreement for each case was then summarized.

“Consensus of the pathologists on the critical 0 and 1+ score cases are insufficient to define a criterion standard and thus, the pre-analytic questions will need to be addressed in a future study,” the study authors concluded.


  1. Fernandez AI, Liu M, Bellizzi A, et al. Examination of low HER2 expression in breast cancer. Presented at: 2021 San Antonio Breast Cancer Symposium; December 7-10, 2021; San Antonio, TX. Abstract P1-02-02.
  2. Doi T, Shitara K, Naito Y, et al. Safety, pharmacokinetics, and antitumour activity of trastuzumab deruxtecan (DS-8201), a HER2-targeting antibody-drug conjugate, in patients with advanced breast and gastric or gastro-oesophageal tumours: a phase 1 dose-escalation study. Lancet Oncol. 2017;18(11):1512-1522. doi:10.1016/S1470-2045(17)30604-6
  3. McCabe A, Dolled-Filhart M, Camp RL, et al. Automated quantitative analysis (AQUA) of in situ protein expression, antibody concentration, and prognosis. J Natl Cancer Inst. 2005;97(24):1808-1815. doi:10.1093/jnci/dji427
  4. Onsum MD, Geretti E, Paragas V, et al. Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2-positive patients. Am J Pathol. 2013;183(5):1446-1460. doi:10.1016/j.ajpath.2013.07.015