High-Plex Digital Spatial Profiling Identified Prolactin-Induced Protein mRNA Associated With Response and Survival of Everolimus and Letrozole Treatment for Hormone Receptor-Positive/Human Epidermal Growth Factor Receptor 2-Negative Advanced Breast Cancer.

Everolimus (EVE) combined with letrozole is an approved treatment for hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) advanced breast cancer (ABC). However, predictive biomarkers for EVE efficacy remain undefined. In the phase 2 MIRACLE trial, we performed digital spatial profiling (DSP) on pretreatment tumor samples from 21 patients receiving EVE plus letrozole. Patients were divided into resistant and sensitive groups based on their best response to EVE. A total of 119 regions across three compartments-tumor, leukocytes, and stroma-were profiled for immune and transcriptomic markers. Responders had significantly higher fibroblast infiltration in PANCK+ (p = 0.011) and CD45-/PANCK- (p = 0.043) regions, whereas non-responders exhibited increased neutrophils in CD45+ (p = 0.0061) and PANCK+ (p = 0.03) regions. Prolactin-induced protein (PIP) mRNA expression was significantly elevated in non-responders in both PANCK+ (p < 0.0001) and CD45-/PANCK- (p = 0.0006) regions. PIP mRNA expression was found to be associated with EVE resistance and unfavorable progression-free survival (PFS). PIP mRNA expression and specific immune-stromal features are associated with resistance to EVE. These findings suggest the potential of PIP as a spatially resolved predictive biomarker for patient stratification in HR+/HER2- ABC.