HER2 Low, Ultra-low, and Novel Complementary Biomarkers: Expanding the Spectrum of HER2 Positivity in Breast Cancer

Abstract

HER2 status in breast cancer is assessed to select patients eligible for targeted therapy with anti-HER2 therapies. According to the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP), the HER2 test positivity is defined by protein overexpression (score 3+) at immunohistochemistry (IHC) and/or gene amplification at in situ hybridization (ISH). The introduction of novel anti-HER2 compounds, however, is changing this paradigm because some breast cancers with lower levels of protein expression (i.e. score 1+/2+ with no gene amplification) benefited from HER2 antibody-drug conjugates (ADC). Recently, a potential for HER2 targeting in HER2 "ultra-low" (i.e. score 0 with incomplete and faint staining in ≤10% of tumor cells) and MutL-deficient estrogen receptor (estrogen receptor)-positive/HER2-negative breast cancers has been highlighted. All these novel findings are transforming the traditional dichotomy of HER2 status and have dramatically raised the expectations in this field. Still, a more aware HER2 status assessment coupled with the comprehensive characterization of the clinical and molecular features of these tumors is required. Here, we seek to provide an overview of the current state of HER2 targeting in breast cancers beyond the canonical HER2 positivity and to discuss the practical implications for pathologists and oncologists.

Keywords: HER2 low expression; HER2 ultra low; antibody-drug conjugate; biomarkers; breast cancer; fluorescence in situ hybrdization; immunohistochemistry; targeted therapy.

FIGURE 1

Algorithm for defining HER2 spectrum of expression according to ASCO/CAP guidelines. HER2-low diseases are identified by IHC score 2+ with negative ISH or IHC score 1+. Breast cancer is scored 2+ in case of weak-moderate complete membrane staining in >10% of tumor cells or if the membrane staining is intense but in ≤10% of tumor cells. Score 1+ is defined by faint or barely perceptible incomplete membrane staining in >10% of tumor cells. Gene amplification by ISH is assessed as detailed in the text boxes, according to the types of probes employed. Among the IHC score 0 category, two different types of expression are present, namely a complete lack of expression and the faint or barely perceptible incomplete membrane staining in ≤10% of tumor cells. Albeit HER2-negative, this latter type of tumor indeed shows the expression of the protein and can be described as HER2 “ultra-low”. The continuous versus the discontinued line depicts the different levels of evidence in the current clinical practice. Abbreviations, IHC, immunohistochemistry; ISH, in situ hybridization; HER2, human epidermal growth factor receptor 2. *, dual-probe ISH should be performed for final result. Source: Breast Biomarker Reporting, CAP Cancer Protocol Templates, v1.4.1.1, November 2021 update, available at: https://documents.cap.org/protocols/Breast.Bmk_1.4.1.1.REL_CAPCP.pdf. Created with biorender.com.

FIGURE 2

Possible targeting strategies in HER2-low and HER2-negative breast cancers. 1) Cancer vaccines: most of the research efforts related to cancer vaccines against HER2 regard targeting HER2-related peptides, such as E75 (ECD), GP2 (TMD), and AE37 (ICD); Interestingly, while a phase II trial testing AE37-based vaccine in the advanced stage (in the study defined as stage IIB or greater) did not demonstrate statistically significant survival benefit (5-years DFS) in the overall population, it showed a 5-years DFS of 83% in subjects with HER2-low disease, compared with 62.5% in the GM-CSF-only arm (HR, 0.375; CI: 0.142–0.988; p = 0.039). Similarly, in a subgroup with stage IIB or greater TNBC, a trend toward improved DFS with vaccination was found. The growing body of evidence coupled with subset analyses of this phase II trial led to another phase II clinical trial investigating AE37 in combination with pembrolizumab (NSABP FB-14) in TNBC patients with metastatic disease (NCT04024800). 2) Antibody-drug conjugates: several trials are currently testing ADC in HER2-low disease, with interesting results, particularly regarding those equipped with diffusable cytotoxic moiety as well as cleavable linkers, possibly accounting for bystander killing effect (skull symbol); 3) Bispecific antibodies (bsAbs) and trifunctional antibodies are characterized by the ability to target (at least) two different epitopes, enabling either inhibition of multiple oncogenic pathways or the forced connection between immune cells and cancer tissue. Several anti-HER2 bsAbs are under development, although only a minority is specifically investigated in HER2-low disease; 4) Additional molecular characterization is a possible new prospective, in the light of recent findings describing the predictive value of mismatch repair deficiency related to response to HER2 blockade in HER2-negative breast cancer. Abbreviations: T-DM1, trastuzumab emtansine; T-DXd, trastuzumab deruxtecan; HER2, human epidermal growth factor receptor 2; ECD, extracellular domain; TMD, transmembrane domain; ICD, intracellular domain; aa, amino acid; MHC, major histocompatibility complex; HLA, human leukocyte antigen; ADC, antibody-drug conjugate; D, diffusible cytotoxic moiety; MSH6, MutS Homolog 6; PMS2, PMS1 Homolog 2; MSH2, MutS Homolog 2; MLH1, MutL homolog 1; DFS, disease-free survival; bsAbs, bispecific antibody; CD, cluster of differentiation; ATP, Adenosine triphosphate; ADP, adenosine diphosphate; Fab, antigen-binding fragment; Fc, fragment crystallizable; FcR, Fc receptor; dMMR, mismatch repair deficient; ADCC, Antibody-dependent cellular cytotoxicity; RFC, replication factor C; PCNA, Proliferating cell nuclear antigen. Created with biorender.com.