Abstract 3623: Quantifying pharmacodynamic markers of radioligand therapies in tumor by multiplex immunofluorescence and automated quantitative analysis (AQUA) algorithms

Abstract

Abstract Purpose: Due to profound clinical successes observed with radioligand therapies (RLT) across multiple cancers coupled with advances in drug handling and delivery logistics recently, clinical trials investigating RLTs are on the rise. Ionizing radiation produced by radioisotopes linked to antibodies causes double-stranded breaks either directly or via generation of reactive oxygen species. This type of DNA damage is associated with an increase in proteins mediating cell cycle arrest (e.g., p21/CIP1) and repairing damaged DNA (e.g., CHK2 and γH2Ax). Additionally, proliferating cells (identifiable by Ki67) are most susceptible to ionizing radiation as DNA is exposed during the mitotic phase of the cell cycle. We incorporated these biologically verified markers into a multiplexed fluorescence immunohistochemistry (mFIHC) assay for robust quantitation of the pharmacodynamic activity of RLTs and gain insights into the DNA damage response in on-treatment tumor core biopsies. Study Design: We designed a novel mFIHC assay incorporating antibodies to p21, pCHK2, γH2Ax, Ki67, CK to quantitatively characterize DNA damage across five tumor indications of commercially available ovarian cancer, colorectal cancer, gastric cancer, endometrial (uterine) cancer, and breast cancer formalin-fixed paraffin-embedded (FFPE) tissue blocks. We successfully validated this mFIHC assay using automated staining (Bond RX), imaging (PhenoImager HT), and combined with hypothesis-driven spatial profiling algorithms (e.g., AQUA Technology). Results: Sensitivity and specificity were confirmed on known positive and negative controls. Reproducibility and precision were observed across instruments, operators, and independent experiments for all markers The frequency of p21 ranged from 5% - 8%, pCHK2 from 3% - 36%, pH2Ax from 0% - 8%, Ki67 from 4% - 61%, and CK cells ranged from 25% to 87%. Over 56 unique parameters were evaluated. The prevalence of p21, pCHK2, and γH2Ax ranged from 0% to >30% across five tumor indications tested and were highly concordant. Conclusion: The validated mFIHC assay examines the expression of proteins mediating cell cycle arrest and DNA damage in tumor and stromal cells. This panel may be used to further understand the complexity of DNA damage response pathways in tumor and non-tumor areas in the context of RLT clinical trials. Citation Format: James Santos, Virginia Nyanchama, Vincent Romanet, Ernesta Dammassa, Lisa Kattenhorn, Ying Huang, Jimmie Lim, Xun Li, James Deeds, Lori Iaconis, Emmanuel Pacia, Margaret McLaughlin, Naveen Dakappagari, Jennifer Bordeaux. Quantifying pharmacodynamic markers of radioligand therapies in tumor by multiplex immunofluorescence and automated quantitative analysis (AQUA) algorithms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3623.