Optimizing and Validating Systemic DNA Damage Response Profiling to Predict Neoadjuvant Chemoradiation Response in Rectal Cancer

Abstract

Purpose. This study aimed to stratify patients with locally advanced rectal cancer (LARC) based on their response to neoadjuvant chemoradiation therapy (nCRT) using DNA damage response (DDR)-related proteins measured in peripheral blood monocytes (PBMCs). We optimized and validated an innovative assay to quantify these proteins, providing a predictive framework for nCRT response. Experimental Design. We used PBMCs collected from LARC patients either before or after standard course of ~5.5 weeks of nCRT, with patients categorized by neoadjuvant rectal (NAR) score. DDR was assessed by immunofluorescence (gammaH2AX foci), and by Luminex multi-analyte platform (xMAP) assay providing semi-quantitative assessment of phosphorylated Chk1, Chk2, gammaH2AX, p53 and total ATR, MDM2, p21. Assay performance was evaluated using reference controls and banked PBMCs from healthy controls (n=50). Results. PBMCs from poor responders (PoR; NAR >14; n=21) had significantly lower gammaH2AX foci than complete responders (CR; NAR <1; n=21) (p<0.0001), with no significant differences between pre- and post-nCRT samples (p=0.4961). The xMAP assay performance assessment showed linear sample curves, precision with acceptable inter- and intra-assay coefficients of variability, and high reproducibility with ~1% outliers in replicates. Clinical associations using the xMAP assay found levels of six proteins (ATR, MDM2, and phospho forms of Chk1, Chk2, gammaH2AX, and p53) significantly differentiating CRs from PoRs. Univariate CART analysis determined thresholds that segregated PoRs from CRs with high precision (p<0.001). Conclusion. We optimized an assay to assess DDR proteins in PBMCs and identified specific proteins, along with their threshold levels, that can accurately predict response to nCRT in patients with LARC.