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Abstract Background: Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is standard of care for locally advanced rectal cancer (LARC). About 20% patients receiving nCRT alone experience a pathologic complete response (pCR), while up to 25% patients exhibit a poor response. Currently, there is no biomarker to predict response to nCRT. We hypothesized that inherited ability to recognize and repair DNA damage, especially lethal double-strand breaks (DSBs), manifest in lymphocytes, would yield a reproducible test predicting therapeutic response to nCRT. Methods: Expression of DNA damage response (DDR) proteins was studied using peripheral blood lymphocytes (PBLs) from LARC patients and healthy controls. PBLs from LARC patients collected either before nCRT or after a standard course of ~5. 5 weeks of nCRT were assessed by immunofluorescence (IF) for phospho-γH2AXSer139 and by Luminex multi-analyte platform (xMAP) approaches. The xMAP assay was modified from a qualitative to a semi-quantitative assay that measures the following DDR-associated proteins- total ATR, phospho-γH2AXSer139, total MDM2, phospho-Chk1S345, phospho-Chk2T68, phospho-p53S15, total p21. The analytical performance of the assay was assessed with vendor-provided reference controls and banked PBLs from healthy controls (n=50). LARC patients were segregated by neoadjuvant rectal (NAR) score to determine if tested biomarkers correlated with nCRT response. We used two independent sets of LARC biospecimens for xMAP analysis (set 1, n=48; set 2, n=44). Results: IF-based assessment: Using PBLs from LARC patients, we found that poor responders (PoR; NAR score14; n=21) had significantly lower γH2AX foci than complete responders (CR; NAR score1; n=21) (P0. 0001, logistic regression). No significant difference was observed in positive γH2AX foci from PBLs drawn pre-nCRT versus post-nCRT from the same patient (p=0. 4961, n=11, Wilcoxon test). xMAP assay performance: The performance assessment showed linear sample curves, precision with acceptable inter- and intra-assay coefficients of variability, and high reproducibility with ~1% outliers identified in replicates. Clinical associations using xMAP assay: levels of six proteins (ATR, p-γH2AXSer139, MDM2, p-Chk1S345, p-Chk2T68, p-p53S15) significantly differentiated CRs from PoRs (p-values and False Discovery Rate ⇐ 1e-5). The strongest associations with nCRT response were observed for: ATR, MDM2 and γH2AX. Conclusion: 1) we standardized an xMAP assay to assess DDRs in human PBLs, and 2) found novel biomarkers that predict response to nCRT in LARC patients using minimally invasive PBL biospecimen. In ongoing work, we are using a larger biospecimen set to establish clinical validity parameters. Citation Format: Adria Hasan, Elena V. Demidova, Philip Czyzewicz, Karthik Devarajan, Margret B. Einarson, Donald Baldwin, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arora. Novel biomarkers that predict response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer 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 (6Suppl): Abstract nr 2506.
Hasan et al. (Fri,) studied this question.