Abstract PS2-09-09: Early on-treatment PD-L1 expression and stromal tumor infiltrating lymphocytes (sTILs) refine the prediction of EFS in the NeoTRIP trial

Abstract Background We previously showed that on-treatment high sTILs were significantly associated with improved EFS and were independent of pathological complete response (pCR) in absence or upon addition of atezolizumab to neoadjuvant chemotherapy. The same is true for PD-L1 positivity but only for patients receiving immunotherapy (Bianchini G ASCO 2025). We hypothesized that on-treatment up-regulation of PD-L1 may be a new marker of benefit from neoadjuvant atezolizumab. Here we analyzed pre-treatment RNA-Seq and imaging mass cytometry (IMC) data to identify baseline features that could predict PD-L1 up-regulation during treatment. We next assessed whether combining on-treatment sTILs and PD-L1 improves prediction of EFS. Methods NeoTRIP randomized 280 patients to nab-paclitaxel/carbo for 8 cycles, alone (CT) or with atezolizumab (CT/A). As Per-Protocol Population, 258 patients were evaluable for EFS, the primary endpoint of the study. We collected samples at baseline (n=258/258; 100%) and on Day 1 Cycle 2 (D1C2) (n=230/258; 89.2%). We centrally assessed sTILs (≥30% to be considered high) and PD-L1 expression (SP142) on immune cells (IC) (IC≥1% considered positive). Association with EFS was evaluated by Kaplan-Meier curves and log-rank test. Pre-treatment IMC and RNA-seq data of tumours PDL1- at baseline and converting to PDL1+ at D1C2 were compared with those remaining PDL1- at D1C2. P-values were corrected by false discovery rate (FDR). Results No differentially expressed genes were observed after FDR correction between tumors that up-regulate versus those that do not up-regulate PD-L1 during CT/A treatment. In the CT arm, up-regulation of PD-L1 was associated with significantly higher baseline expression (FDR < 0.05) of two immunoglobulin genes (IGHV3-64D and IGKVD2-28). Gene set enrichment analysis showed that baseline tumors up-regulating PD-L1 at D1C2 were enriched for interferon gene sets in both arms. Samples from CT arm also showed an enrichment for macrophage-related gene sets, while samples from CT/A arm were enriched for hypoxia and cell cycle gene sets. Immunoglobulin and B cell gene sets were positively and negatively enriched in tumors up-regulating PD-L1 in CT and CT/A arms, respectively. IMC showed no significant differences in baseline cell phenotype densities or cell-cell interactions between the two groups.In the CT arm, combined D1C2 PD-L1 and sTILs did not significantly improve EFS prediction in comparison to sTILs alone (likelihood ratio test p-value = 0.89). In CT/A arm instead, the combination of D1C2 PD-L1 and sTILs was more informative of EFS prediction than sTILs alone (likelihood ratio test p-value = 0.01). PD-L1-/sTILs-Low patients (N=19) had the worst outcome (5-years EFS = 0.37%, 95% C.I. = 0.20%-0.66%]). Conversely, patients with PD-L1+/sTILs-High (N=25) had the best EFS (5-years EFS = 0.86%; 95% C.I. = 0.72%-1.00%). Compared to the latter group, patients with PD-L1+/sTILs-Low tumors (N=30) had less favorable outcome (5-years EFS = 0.71%; 95% C.I. = 0.56%-0.90%). Only one patient was classified as PD-L1-/sTILs-High.The association of PD-L1/sTILs groups with EFS was maintained in the subset of patients with residual disease after surgery (log-rank p-value = 0.001). Conclusions Specific biology might be underlying the up-regulation of PD-L1 during neoadjuvant atezolizumab treatment, although we have not identified statistically robust baseline biomarkers that can predict this behavior. By combining the two clinically established biomarkers PD-L1 and sTILs evaluated on-treatment, we improved outcome prediction indicating an independent predictive-prognostic value of these markers. This association was independent of pCR status. Whether these findings may be translated to neoadjuvant pembrolizumab treatment remains to be defined. Citation Format: M. Dugo, G. Viale, H. Chiun-Sheng, D. Egle, M. Callari, R. H. Ali, B. Bermejo, C. Zamagni, X. Wang, M. Thill, A. Antón-Torres, S. Russo, E. Ciruelos, R. Greil, V. Semiglazov, M. Colleoni, L. Del Mastro, G. Viale, L. Gianni, G. Bianchini. Early on-treatment PD-L1 expression and stromal tumor infiltrating lymphocytes (sTILs) refine the prediction of EFS in the NeoTRIP trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-09-09.