Background: SG is a Trop-2–directed antibody-drug conjugate approved in multiple countries for patients with metastatic triple-negative breast cancer who received at least 1 prior systemic therapy and in the US for patients with pretreated HR+/HER2– mBC. In the TROPiCS-02 study, SG significantly improved progression-free survival (PFS; median, 5.5 vs 4.0 mo; HR, 0.66 [95% CI, 0.53-0.83]; P=0.0003) and overall survival (OS; median, 14.4 vs 11.2 mo; HR, 0.79 [95% CI, 0.53-0.83]; P=0.020) over treatment of physician’s choice (TPC) in patients with pretreated, endocrine-resistant HR+/HER2– mBC. In TROPiCS-02 post hoc analyses, SG improved OS regardless of Trop-2 expression by immunohistochemistry (IHC; Rugo HS, et al. SABCS 2022), but association using more sensitive assays are lacking. Here, we examine efficacy outcomes for SG versus TPC by Trop-2 gene (TACSTD2) mRNA expression and the correlation of TACSTD2 expression with other genes. Methods: Patients with HR+/HER2– mBC who received ≥1 prior taxane, endocrine therapy and a CDK4/6 inhibitor, and 2-4 prior chemotherapy regimens for mBC were randomized 1:1 to SG or TPC. The primary endpoint was PFS by central review per RECIST v1.1. RNA was isolated from archival tumor tissue samples using AllPrep DNA/RNA kits and libraries prepared with TruSeq RNA Exome Prep Kit. Sequencing was via NovaSeq (2x150bp). Gene expression quantitation was performed using Salmon. TACSTD2 expression was defined as high or low via median cut. Results: In the mRNA analytical cohort (N=197), 49% had TACSTD2 high expression (SG, n=51; TPC, n=46), and 51% had TACSTD2 low expression (SG, n=47; TPC, n=53). Baseline characteristics were generally consistent between TACSTD2 expression subsets and the intention-to-treat population. A positive concordance between TACSTD2expression and Trop-2 IHC (H-score) was observed (categorical concordance 71%; Cohen’s kappa = 0.41). TACSTD2 expression was similar across HER2 IHC0 and HER2-Low subgroups (log2 transcripts per million [TPM] of 3.36 [IQR, 2.76-4.22] and log2TPM of 3.44 [IQR, 2.59-4.22], respectively). SG showed a numerically higher median PFS (TACSTD2 high, 7.3 vs 5.6 mo; TACSTD2 low, 5.6 vs 2.8 mo) versus TPC regardless of TACSTD2 expression. Similarly, for OS, outcomes favored SG over TPC regardless of TACSTD2 expression. Conclusions: This mRNA-based biomarker analysis demonstrated that SG improves PFS and OS outcomes in patients with pretreated, endocrine-resistant HR+/HER2– mBC regardless of Trop-2 mRNA expression. There was no correlation between Trop-2 mRNA expression and HER2 status. Trop-2 mRNA expression was not predictive of benefit with SG. Additional studies are needed to determine whether Trop-2 mRNA expression has a prognostic role in mBC. Clinical trial information: NCT03901339.