Abstract Background The identification of biomarkers for optimization of immune checkpoint inhibitors (ICI) treatment is an unmet clinical need. In the Phase III randomized trial, NeoTRIPaPDL1, a post-hoc analysis of whole transcriptome RNA-Seq data, previously showed that the 27-gene IO score is a potential predictive biomarker of increased pathological complete response with the addition of atezolizumab to carboplatin/nab-paclitaxel (Bianchini G ESMO 2021). However, the laboratory implementation of gene-expression signatures measured using RNA-seq is challenging. Therefore, we further assessed the predictive value of the IO score using a twenty-seven gene RT-qPCR assay on NeoTRIP samples, and compared to the previously reported RNA-Seq version of the assay. Methods The NeoTRIP study randomized patients to eight cycles of carboplatin/nab-paclitaxel (CT) with or without atezolizumab (CT/A). 258 patients were evaluable for pCR (breast and nodes) as Per-Protocol Population. We assessed the IO score as binary and continuous variables using the CAP/CLIA validated DetermaIO qPCR test (Saltman et al 2021) on pre-treatment core biopsies (n=220/258; 85.3%), all of which have RNA-Seq data available. We evaluated the association between IO score defined by RT-qPCR and RNA-Seq, and the association of the IO score defined by RT-qPCR test with PD-L1 IHC (Ventana SP142), stromal TILs (sTILs), and pCR. Results Comparison of continuous IO scores between the RT-qPCR assay and the RNA-Seq algorithm had a Pearson’s correlation of 0.94 (p < 0.0001). High agreement between categorical IO scores was also observed (Cohens’ kappa = 0.84; 95% confidence interval [CI] = 0.77-0.91; p < 0.0001). RT-qPCR IO score was balanced in the two arms (p = 0.65) with 44% and 40% positive patients in the CT and CT/A arms, respectively. The RT-qPCR IO score was correlated with both PD-L1 (Pearson’s r = 0.64; p < 0.0001) and sTILs (Pearson’s r = 0.67; p < 0.0001). Continuous IO score was significantly predictive of pCR in CT/A (Odds ratio [OR] = 3.12; 95% CI = 1.20-8.10; p<0.019), but not CT arm (OR = 1.28; 95% CI = 0.54-3.01; p = 0.578). Considering the binary IO score, OR were 2.87 [1.27-6.47] (p = 0.011) and 0.91 [0.43-1.93] (p = 0.812), in CT/A and CT, respectively (interaction test p = 0.043). The pCR rate for CT/A vs CT was 69.8% vs 46.9% in IO score positive [+22.9%, p = 0.046, Chi-squared test] and 44.6% vs 49.2% [-4.6%, p = 0.73] in IO score negative. A significant interaction was found between continuous PD-L1 and continuous IO-score (p = 0.006). Among PD-L1-neg, 9 patients were IO score positive (10.1%). The pCR rate in this group was 3/4 (75%) in the CT/A arm and 1/5 (20%) in CT arm. The predictive value of IO score by RT-qPCR was similar to RNA-Seq. Conclusions We observed a high level of agreement and concordance between IO scores assessed by RT-qPCR and RNA-Seq, indicating that the 27-gene IO assay and algorithm is robust and the choice of platform has limited impact. This finding also demonstrates the high quality of NeoTRIP RNA-Seq data. In this post-hoc analysis, IO score assessment by this CLIA validated RT-qPCR test was confirmed to be predictive of atezolizumab benefit over CT alone in a randomized trial. Citation Format: Matteo Dugo, Chiun-Sheng Huang, Daniel Egle, Begoña Bermejo, Claudio Zamagni, Robert S. Seitz, Tyler J. Nielsen, Marc Thill, Antonio Anton, Stefania Russo, Eva Maria Ciruelos, Brock L. Schweitzer, Douglas T. Ross, Barbara Galbardi, Richard Greil, Vladimir Semiglazov, Balázs Gyorffy, Marco Colleoni, Catherine Kelly, Gabriella Mariani, Lucia Del Mastro, Pinuccia Valagussa, Giuseppe Viale, Maurizio Callari, Luca Gianni, Giampaolo Bianchini. Predictive value of RT-qPCR 27-gene IO score and comparison with RNA-Seq IO score in the NeoTRIPaPDL1 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD10-06.