Pathological precision diagnosis and recent advances in HER2 low and ultralow in breast cancer: a narrative review

Introduction

Background

The emergence of new antibody-drug conjugate (ADC) drugs disrupts the reliance of conventional anti-human epidermal growth factor receptor 2 (HER2) targeted therapy on HER2 receptor expression or gene amplification, thereby establishing HER2 low as a novel type of breast cancer treatment and reshaping the landscape of breast cancer treatment. While patients with HER2 low expression benefit from innovative ADC therapies, the assessment of HER2 immunohistochemistry (IHC) in clinical practice is based on the original negative definition of HER2. On the basis of the original definition of HER2 negative in clinical practice, patients with HER2 IHC 1+ or 2+ and fluorescence in situ hybridization (FISH) negative are classified as HER2 low-level expression, while the category for HER2 IHC 0 includes infiltrating cancer cells with a result of 0 and ≤10% showing incomplete and weak cell membrane staining (1).

Rationale and knowledge gap

In 2023, the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) revised their guidelines for HER2 testing in breast cancer following the release of results from the Phase III DESTINY-Breast04 trial in 2022. The updated ASCO/CAP guidelines affirm the reliability and efficacy of current HER2 testing methods, while emphasizing the importance of accurately identifying patients with “HER2 low” to ensure optimal selection for anti-HER2-targeted therapy (1). However, there is limited research on the clinical pathological characteristics and prognosis of HER2 low and ultralow breast cancer. The precise detection of HER2 low expression and ultralow expression, especially the detection limit, is currently unclear.

Objective

The purpose is to provide some suggestions on how to correctly understand HER2 low and ultralow breast cancer. Further standardization of the definitions of HER2 low expression and ultralow expression is needed to ensure consistency and accuracy in clinical practice. At the same time, we seek to further improve and optimize detection techniques and methods, reduce false negatives and false positives, and enhance the sensitivity and specificity of HER2 detection. We present this article in accordance with the Narrative Review reporting checklist (available at https://tbcr.amegroups.org/article/view/10.21037/tbcr-24-33/rc).

Methods

This paper explores the clinicopathological features of HER2 low and ultralow expression in breast cancer, immunohistochemical staining, and its heterogeneity through literature search and other means. As of May 2024, a comprehensive literature search, compilation, and analysis were conducted across PubMed, Baidu Scholar, ClinicalTrials.gov, and relevant academic conferences (Table 1).

Background and current status of HER2 low and ultralow expression in breast cancer

Recently, the pivotal Phase III clinical trial, DESTINY-Breast06, of the antibody drug conjugate trastuzumab deruxtecan (T-DXd), presented detailed data for the first time at the ASCO conference on June 2, 2024. The results demonstrated that T-DXd led to a statistically and clinically significant improvement in progression-free survival (PFS) in hormone receptors (HR)-positive/HER2-low and HER2-ultralow populations with prior first-line endocrine therapy progression. This global, randomized, open-label Phase III clinical trial was designed to compare the efficacy and safety of T-DXd (5.4 mg/kg, q3w) with investigator-selected chemotherapy regimens [treatment of physician’s choice (TPC): capecitabine, paclitaxel or albumin-bound paclitaxel] in patients with advanced or metastatic breast cancer expressing HR-positive/HER2 low [IHC 1+ or IHC 2+/in situ hybridization (ISH)−] or HER2-ultralow (0< IHC <1+).

The DESTINY-Breast04 study included the HER2-low population, making it a new, targetable therapeutic subtype. The inclusion criteria of the DESTINY-Breast06 study now further broaden the boundaries of HER2 expression to include patients with HER2-ultralow, defined as HER2 IHC 0 with membrane staining, i.e., IHC >0 and <1+. Therefore, the further refinement of the concept of HER2 expression in this study is expected to bring more accurate treatment plans for HER2-ultralow group and expand the benefit population of T-DXd. As for the most important HER2 ultralow in the result of DESTINY-Breast06, its accurate interpretation is a certain challenge for pathologists, and the inclusion criteria in the test of DESTINY-Breast06 is based on a confirmation from the central laboratory. Therefore, the inclusion conditions of HER2 ultralow put forward higher requirements for HER2 IHC detection.

Doubts and thoughts on precise pathological detection of HER2 low and ultralow

For the precise pathological detection of HER2 ultralow, several questions and reflections are raised: (I) is there a difference in clinicopathological features and prognosis between low and ultralow expression of HER2 IHC? (II) Whether IHC is suitable for screening for HER2 ultralow and no staining of HER2; (III) heterogeneity of HER2 low and ultralow; (IV) influencing factors of HER2 low and ultralow expression in the pre-testing process.

(I) Is there a difference in clinicopathological features and prognosis between low and ultralow expression of HER2 IHC?

In the study of DESTINY-Breast06, the classification of HER2 IHC is made according to ASCO/CAP guidelines (1), and patients with IHC 0 are further divided into HER2 ultralow and HER2 IHC 0 (no staining of the cell membrane). The HER2-ultralow population is “10% of tumor cells with weak, incomplete membrane staining” from previous IHC 0. This study to date of ultralow HER2 expression breast cancer enrolled 1,363 patients, including 86 (6.3%) HER2-0, 395 (29.0%) HER2 ultralow, and 882 (64.7%) HER2 low patients.

Currently, there is very limited research on whether patients with HER2 ultralow breast cancer exhibit clinicopathological features that differ from HER2-0 and HER2-low tumors. Studies have shown significant differences in histological type and postoperative endocrine therapy between HER2 ultralow and HER2-0 patients (2). In patients with HER2 ultralow, HR-positive (81.0%) tumors were more common than HR-negative (19.0%). HER2 status (0, ultralow, or low) had no prognostic value in these HER2-negative breast cancer populations. HR status is an independent prognostic factor for DFS in patients with HER2-negative breast cancer (2). In HR-negative breast cancer, patients with HER2-0 breast cancer had lower levels of Ki67 expression compared to HER2 ultralow and HER2 low breast cancer. Among HR positive breast cancer patients, the overall survival (OS) of HER2-0 is worse than that of HER2 ultralow breast cancer patients (3). Research has shown that a sharp increase in HER2 may trigger the cell’s intrinsic defense system, therefore the sustained low expression of HER2 can transform tumor cells into more aggressive ones through chromatin remodeling (4).

(II) Whether IHC is suitable for screening for HER2 ultralow and no staining of HER2

The results of preclinical studies suggest that there is a lower limit of HER2 expression, below which T-DXd therapy will not be beneficial (5). Until the clinical evidence of HER2 IHC-0 non-staining population is available, the identification and differentiation of HER2 low and HER2 ultralow still need to be focused at this stage.

The DESTINY-Breast06 study not only further widens the boundaries of HER2 expression to include patients with HER2 ultralow, but also points out that current IHC tests are relatively poor at distinguishing between low or ultralow HER2 expression and zero HER2 expression. The initial purpose of the HER2 IHC assay is to distinguish IHC 3+ from other types, not to distinguish very low levels of HER2 expression from no expression. Although some emerging technologies can improve the consistency and accuracy of HER2 detection, these new technologies must be fully validated in clinical studies to ensure their effectiveness before clinical application.

RNA levels measured by RNAScope were strongly correlated with protein levels measured by the automated quantitative analysis (AQUA) method and H scores for IHC 1+, 2+, and 3+ cases, suggesting that RNAScope is a reliable way to assess RNA levels (6). However, there were no significant differences in both protein levels and RNA levels between cases with HER2 scores of 0, ultralow expression, and 1+. The RNA level of T-DXd responders was significantly higher than that of non-responders, and the response rate of patients to T-DXd scored by RNAScope may be better than that classified by IHC score (6). On the other hand, next-generation sequencing (NGS) detection of 35 cases of invasive breast cancer whose HER2 status had been determined by FISH showed that the HER2 status determined by NGS was 97% accurate compared with that determined by FISH (7). In addition, NGS can detect HER2 mutations in cancers that lack HER2 amplification. In data from eight breast cancer genome sequencing projects, 25 patients with HER2 somatic mutations were found in cancers lacking HER2 gene amplification, most of which were activation mutations (8). For HER2 low expression and HER2-0 group, TP53, PIK3CA, CDH1, ESR1 and GATA3 were the five genes with the highest mutation frequency. Amplification of CCND1, FGFR1, and MYC was the most common alteration (9).

(III) Heterogeneity of HER2 low and ultralow in breast cancer

With the approval of T-DXd for the treatment of patients with unresectable/metastatic HER2 low breast cancer and the results of the DESTINY-Breast06 clinical trial suggesting that T-DXd may also be clinically beneficial for HER2 ultralow breast cancer, the diagnosis of HER2-low and HER2 ultralow can directly affect the screening of patients who benefit from ADC drugs.

However, at present, the overall agreement among pathologists on the interpretation of HER2 low and HER2 ultralow is poor, mainly due to the large heterogeneity of HER2 expression in breast cancer: (I) spatial heterogeneity. In Na et al. (10), when a four-group classification system (IHC 0; IHC 1+; IHC 2+/silver-enhanced in situ hybridization (SISH) negative, IHC 2+/SISH positive; or IHC 3+) based on HER2 IHC score and SISH results was used. The overall agreement rate between core needle biopsy (CNB) and surgically excised specimens was only 60.6%, with the largest inconsistency occurring in CNB-HER2-0 cases, of which 42.8% were reclassified as HER2 low in surgically excised specimens. (II) Temporal heterogeneity. In terms of the evolution of HER2 before and after neoadjuvant therapy, Tarantino et al. (11) showed that compared with HER2-positive tumors, HER2-0 or HER2-low tumors were more likely to have changes in HER2 expression after neoadjuvant therapy (32.3% vs. 21.3%; P<0.001). In addition, HER2-low tumors also showed greater heterogeneity and worse stability between primary and recurrent/metastatic tumors. Lin et al. (12) showed that 31.7% of primary tumors with HER2-0 were transformed into HER2-low and 2.9% into HER2-positive tumors. For primary tumors with HER2-low, 32.7% converted to HER2-0 and 5.0% to HER2-positive, with HER2-positive tumors having the highest stability. (III) Intratumoral heterogeneity, manifested by uneven distribution and different intensity of HER2 expression in different regions of the same tumor. Study by Filho et al. (13) showed that 10% of breast cancers had intratumoral heterogeneity of HER2, which had a significant impact on patients’ accurate diagnosis and therapeutic efficacy.

In HER2 IHC 0 (without staining), HER2 ultralow and HER2 IHC 1+, Wu et al. (14) summarized the heterogeneity types as cluster type, mixed type and dispersed type according to the HER2 expression pattern. The results showed that 28% of cases with ultralow expression of HER2 showed heterogeneity, and the main types were dispersed type and cluster type. Eighty-six percentage of HER2 IHC 1+ cases showed heterogeneity, with the main type being mixed type, followed by cluster type and dispersed type. For heterogeneous cases, the accuracy of pathologist visual assessment was significantly lower than that of homogeneous cases (0.68 vs. 0.91), and artificial intelligence (AI) assisted assessment could significantly improve the interpretation accuracy of heterogeneous cases, among which the most obvious improvement was in dispersed cases (14).

HER2 is an important biomarker of breast cancer. HER2 low and HER2 ultralow account for a large proportion of breast cancer patients, and the heterogeneity of HER2 expression is particularly prominent in these patients. It is mainly reflected that the status of HER2 often changes in the same tumor lesion, different tumor locations and the entire treatment process, which will affect the therapeutic response and drug resistance of tumors against HER2-targeted drugs, and also put forward higher requirements for the accurate evaluation of HER2 status.

(IV) Influencing factors of HER2 low and ultralow expression in the pre-testing process

At present, HER2 detection still mainly depends on IHC, but various key factors in the routine IHC detection process may affect the final result, including sample type, sampling and treatment before detection, storage time of wax blocks and white pieces, antibody selection and detection conditions during detection, quality control, etc. These factors may have a greater effect on HER2 low and ultralow expression. In clinical diagnosis and treatment, metastatic samples are often small biopsy specimens. Due to the significant spatio-temporal heterogeneity of breast cancers with low HER2 expression, the HER2 status in small biopsy specimens may not represent the HER2 status of the whole tumor. In patients with initial HER2 IHC interpretation of 0 and no cell membrane staining, it is possible to increase the chances of detection results of HER2 ultralow or even low HER2 expression by reinterpreting the status of the primary lesion, replacing the wax block, and re-biopsy. In addition, the use of different HER2 antibodies during the detection process will affect the final staining result. Systematic evaluation of antibodies will be necessary in the future to identify well-validated immunohistochemical agents that can sensitively and accurately identify HER2 ultralow populations.

Because it is not clear whether HER2-0 (without staining) patients can also benefit from T-DXd treatment, the search for the lower limit of the population that can benefit from T-DXd has not stopped. Jung et al. (15) developed an AI-based IHC analysis tool based on the annotations of 153 pathologists and using 6,188 whole slice image (WSI) stained by 19 different IHC methods (including HER2), which could generate a continuous score of HER2 expression (HER2ecs, 0–100%). The IHC staining intensity of the membrane, nucleus, cytoplasm of each cell was quantified separately. Taking 401 HER2 IHC WSI from four centers, the results showed that 6.0% of membrane-specific HER2ecs was the optimal threshold for distinguishing HER2 staining free cells from HER2 ultralow expression.

Professor Giuseppe Curigliano, principal investigator of DESTINY-Breast06 from the Faculty of Medicine of the University of Milan, Italy, also pointed out at the 2024 ASCO Congress that further verification of the extremely low cut-off value of HER2 expression will be carried out in the future. In addition, the clinical trial of DESTINY Breast15 (NCT05950945) will further explore the benefits of T-DXd in HER2 IHC-0 non-staining.

Conclusions

Therefore, the accurate diagnosis of HER2 low and ultralow expression mainly focuses on the following challenges: (I) the definition of HER2 low expression needs to be further standardized to ensure consistency and accuracy in clinical practice. Although no new classification of HER2 expression levels has yet been introduced, a clear distinction between IHC 0 (unstained and stained) and IHC 1+ is important for clinical treatment decisions. (II) Dynamic change of HER2 state. HER2 status may change over the course of the disease, and this dynamic change may influence treatment decisions. Therefore, it is recommended to reassess HER2 status at the time of disease recurrence or metastasis, even if the previous test results were HER2-0. (III) Further improve and optimize detection techniques and methods, reduce false negatives and false positives, and improve the sensitivity and specificity of detection.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://tbcr.amegroups.org/article/view/10.21037/tbcr-24-33/rc

Peer Review File: Available at https://tbcr.amegroups.org/article/view/10.21037/tbcr-24-33/prf

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://tbcr.amegroups.org/article/view/10.21037/tbcr-24-33/coif). Y.L. serves as an unpaid editorial board member of Translational Breast Cancer Research from May 2023 to April 2025. The other author has no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Wolff AC, Somerfield MR, Dowsett M, et al. Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: ASCO-College of American Pathologists Guideline Update. J Clin Oncol 2023;41:3867-72. [Crossref] [PubMed]
2. Chen Z, Jia H, Zhang H, et al. Is HER2 ultra-low breast cancer different from HER2 null or HER2 low breast cancer? A study of 1363 patients. Breast Cancer Res Treat 2023;202:313-23. [Crossref] [PubMed]
3. Tuluhong D, Li X, Gao H, et al. Molecular characteristics and prognosis of breast cancer patients with different level of HER2 positivity after adjuvant and neoadjuvant chemotherapy. Eur J Cancer Prev 2023;32:377-87. [Crossref] [PubMed]
4. Hayat A, Carter EP, King HW, et al. Low HER2 expression in normal breast epithelium enables dedifferentiation and malignant transformation via chromatin opening. Dis Model Mech 2023;16:dmm049894. [Crossref] [PubMed]
5. Ogitani Y, Aida T, Hagihara K, et al. DS-8201a, A Novel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor Efficacy with Differentiation from T-DM1. Clin Cancer Res 2016;22:5097-108. [Crossref] [PubMed]
6. Li X, Lee JH, Gao Y, et al. Correlation of HER2 Protein Level With mRNA Level Quantified by RNAscope in Breast Cancer. Mod Pathol 2024;37:100408. [Crossref] [PubMed]
7. Lipson D, He J, Yelensky R, et al. Next-generation sequencing of FFPE breast cancers demonstrates high concordance with FISH in calling HER2 amplifications and commonly detects other clinically relevant genomic alterations (poster abstract). Cancer Res 2012;72:Abstract nr PD02-07.
8. Bose R, Kavuri SM, Searleman AC, et al. Activating HER2 mutations in HER2 gene amplification negative breast cancer. Cancer Discov 2013;3:224-37. [Crossref] [PubMed]
9. Tarantino P, Gupta H, Hughes ME, et al. Comprehensive genomic characterization of HER2-low and HER2-0 breast cancer. Nat Commun 2023;14:7496. [Crossref] [PubMed]
10. Na S, Kim M, Park Y, et al. Concordance of HER2 status between core needle biopsy and surgical resection specimens of breast cancer: an analysis focusing on the HER2-low status. Breast Cancer 2024;31:705-16. [Crossref] [PubMed]
11. Tarantino P, Ajari O, Graham N, et al. Evolution of HER2 expression between pre-treatment biopsy and residual disease after neoadjuvant therapy for breast cancer. Eur J Cancer 2024;201:113920. [Crossref] [PubMed]
12. Lin M, Luo T, Jin Y, et al. HER2-low heterogeneity between primary and paired recurrent/metastatic breast cancer: Implications in treatment and prognosis. Cancer 2024;130:851-62. [Crossref] [PubMed]
13. Filho OM, Viale G, Stein S, et al. Impact of HER2 Heterogeneity on Treatment Response of Early-Stage HER2-Positive Breast Cancer: Phase II Neoadjuvant Clinical Trial of T-DM1 Combined with Pertuzumab. Cancer Discov 2021;11:2474-87. [Crossref] [PubMed]
14. Wu S, Yue M, Zhang J, et al. The Role of Artificial Intelligence in Accurate Interpretation of HER2 Immunohistochemical Scores 0 and 1+ in Breast Cancer. Mod Pathol 2023;36:100054. [Crossref] [PubMed]
15. Jung W, Lee T, Shin S, et al. Identification of HER2 ultra-low based on an artificial intelligence (AI)-powered HER2 subcellular quantification from HER2 immunohistochemistry images. J Clin Oncol 2024;42:abstr1115. [Crossref]