Optimizing and Validating Systemic DNA Damage Response Profiling to Predict Neoadjuvant Chemoradiation Response in Rectal Cancer
Authors: Demidova Elena V. , Czyzewicz Philip , Hasan Adria , Avkshtol Vladimir , Lesh Randy W. , Handorf Elizabeth , Devarajan Karthik , Schultz Bryant M. , James Johanna D. , Connolly Denise C. , Einarson Margret B. , Baldwin Donald , Golemis Erica A. , Meyer Joshua E. , Arora Sanjeevani 
Published: November 2024

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 (γH2AXS139foci), and by Luminex multi-analyte platform (xMAP) assay providing semi-quantitative assessment of phosphorylated Chk1S345, Chk2T68, γH2AXS139, p53S15and 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 γH2AXS139foci 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, Chk1S345, Chk2T68, γH2AXS139, p53S15) significantly differentiating CRs from PoRs (p ≤ 1e-5). 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.

Translational Relevance.

Although neoadjuvant chemoradiation therapy followed by surgery is the standard of care for patients with locally advanced rectal cancer (LARC), many patients do not benefit from this treatment and suffer from its side effects. The motivation for this study was to reliably identify patients with LARC who will or will not respond to treatment, thereby permitting more effective direction of therapy only to likely responders. In this report, we describe identification and optimization of a novel multianalyte assay for patients diagnosed with LARC. This assay uses a Luminex xMAP platform to detect DNA damage response (DDR) signaling proteins in peripheral blood monocytes of pre-treatment patients. This assay, detecting the DDR proteins, effectively segregates responders from non-responders (p ≤ 1e-5), supporting optimization of treatment efficacy and reduction of unnecessary toxicity, thus advancing personalized medicine in oncology.