Automated Quantification of Extranuclear ERα Using Phosphor-Integrated Dots for Predicting Endocrine Therapy Resistance in HR+/HER2− Breast Cancer

Abstract

In addition to genomic signaling, Estrogen receptor alpha (ERα) is associated with cell proliferation and survival through extranuclear signaling contributing to endocrine therapy (ET) resistance. However, the relationship between extranuclear ERα and ET resistance has not been extensively studied. We sought to measure extranuclear ERα expression by immunohistochemistry using phosphor-integrated dots (IHC-PIDs) and to assess its predictive value for ET resistance. After quantitative detection of ERα by IHC-PIDs in vitro, we developed “the nearest-neighbor method” to calculate the extranuclear ERα. Furthermore, tissue sections from 65 patients with HR+/HER2- BC were examined by IHC-PIDs, and the total ERα, nuclear ERα, extranuclear ERα PIDs score, and ratio of extranuclear-to-nuclear ERα (ENR) were measured using the novel method. We demonstrate that quantification of ERα using IHC-PIDs exhibited strong correlations to real-time qRT-PCR (r2 = 0.94) and flow cytometry (r2 = 0.98). High ERα ENR was significantly associated with poor overall survival (p = 0.048) and disease-free survival (DFS) (p = 0.007). Multivariate analysis revealed that the ERα ENR was an independent prognostic factor for DFS [hazard ratio, 3.8; 95% CI, 1.4–11.8; p = 0.006]. Our automated measurement has high accuracy to localize and assess extranuclear ERα. A high ERα ENR in HR+/HER2− BC indicates decreased likelihood of benefiting from ET.

Novel quantitative immunohistochemical analysis for evaluating PD-L1 expression with phosphor-integrated dots for predicting the efficacy of patients with cancer treated with immune checkpoint inhibitors

“…Guo et al. showed that a high ratio of extranuclear-to-nuclear estrogen receptor alpha (ERα) in patients with hormone receptor-positive and HER2-negative breast cancer indicates a decreased likelihood of benefiting from hormone therapy ( 30 ). Similar to our study, the PID score for PD-L1 expression showed a higher prognostic value than protein detection using IHC-DAB ( 23 ).…”

Immunolocalization of Cytoplasmic ER in ER-negative Breast Carcinoma as a Potent Favorable Prognostic Predictor

“…Welsh et al showed that the incidence of cytoplasmic ER staining only averaged 1.5% (ranging from 0–3.2%) in more over 3,000 breast carcinoma tissues although no information is available in the nuclear ER status of these cases [ 26 ]. On the other hand, Guo et al reported that high ratio of extranuclear-to-nuclear ER in ER-positive breast carcinoma was associated with poor overall survival and disease-free survival by immunohistochemistry using phosphor-integrated dots [ 9 ].…”

Clinical Significance of ABCG2/BCRP Quantified by Fluorescent Nanoparticles in Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy

“…The fluorescence signals of PIDs were observed using fluorescence microscopy (BX53; Olympus Corp., Tokyo, Japan) with a UPLSAPO 40 × 2 (Olympus) objective lens and charge-coupled device camera (DP73; Olympus) in five microscopic fields (with ~1000 cells investigated in each sample). The PID score (PIDs per cell) was thereafter determined ( Figure 1 ) [ 13 , 14 ].…”

“…Our laboratory recently developed nanoparticles suitable for the quantitative immunohistochemical (IHC) method called phosphor-integrated dots (PIDs). Using PID-based IHC (IHC-PID), many biomarker proteins can be visualized and quantitatively analyzed at the single-particle level in paraffine-embedded, formalin-fixed tumor samples [ 13 , 14 ]. The IHC-PID method is a versatile optical technique that can accurately estimate protein expression independent of the optical system.…”