Abstract A032: Low non-immune stromal cell levels and high hypoxic tumor microenvironments are associated with pathologic complete response in the neoadjuvant I-SPY2 TRIAL
Authors: Dubinsky Kailey , Tsopurashvili Elene , Wolf Denise M. , Xu Zheyun , Alkhafaji Silver Al Khafaji , van Montfort Anna , Yau Christina , Heditsian Diane , Fiscalini Allison , Esserman Laura J. , van ‘t Veer Laura , Sayaman Rosalyn W. 
Published: February 2024

Abstract
Background: Non-immune components of the tumor microenvironment (TME) play important roles in cancer. We explore two important components of the TME: non-immune stromal cell populations including fibroblasts, adipocytes, and endothelial cells; and hypoxic conditions. We leverage published gene expression-based biomarkers of stromal cell types and hypoxia, and molecular and clinical data from the I-SPY2 TRIAL (PMID: 35623341) – an adaptive neoadjuvant trial for locally advanced breast cancers. We assess the contribution of stromal cell populations and oxygen-poor environments at pre-treatment in shaping the landscape of immune enrichment and therapeutic response in breast cancers. Methods: We curated 14 stromal and 11 hypoxia gene sets, in addition to 35 immune gene sets from literature. Signatures scores of stromal, hypoxic, and immune TME were calculated from gene expression profiles of pre-treatment tumor samples from I-SPY2 patients (n=987) in the first 9 treatment arms of the trial plus control arms. Correlation of stromal and hypoxia signatures with immune signatures were assessed, and association of TME features with HER2/HR receptor status, I-SPY response-predictive subtype (RPS), and pathological complete response (pCR) were evaluated using non-parametric tests. All p-values were adjusted for multiple testing. Significance levels were defined at adj. p&lt;0.05. Results: Both stromal and hypoxia signatures showed significant differential enrichment across HR/HER2 and RPS subtypes, and association with pCR. Several stromal signatures including adipocytes, endothelial cells, mesenchymal stem cells, and overall stromal score had the lowest enrichment in HR-/HER2- breast cancers, and in the RPS HER2-/immune+ subtype. Moreover, most stromal signatures showed anti-correlation with CD8+ and CD4+ T cell enrichment. Stromal signatures including endothelial cells, smooth muscle cells, and overall score were significantly lower in patients who achieved pCR. Two hypoxia gene sets demonstrated highly significant enrichment in HR- versus HR+ samples regardless of HER2 status. All hypoxia signatures varied significantly across RPS subtypes, with the highest scores in HER2-/Immune-/DRD+, especially relative to HER2-/Immune-/DRD- and HER2+/BP-Luminal subtypes. Hypoxia signatures strongly linked to hypoxia inducible factor (HIF) and angiogenesis via VEGF were positively associated with pCR. These hypoxia signatures showed treatment-specific associations with pCR, specifically in agents targeting mechanisms with known interactions with hypoxia-induced biology: trebananib (angiogenesis inhibitor), ganetespib (HSP90 inhibitor) and veliparib/carboplatin (DNA damage repair inhibitor). Conclusions: Hypoxia and stromal cell gene signatures show breast cancer subtype-specific biology. Relative absence of non-immune stromal cell types is associated with higher immune infiltration and higher pCR rates, while specific hypoxia pathways were associated with response to therapeutic agents targeting hypoxia-related pathways.
Citation Format: Kailey Dubinsky, Elene Tsopurashvili, Denise M. Wolf, Zheyun Xu, Silver Al Khafaji Alkhafaji, Anna van Montfort, Christina Yau, Diane Heditsian, Allison Fiscalini, Laura J. Esserman, Laura van ‘t Veer, Rosalyn W. Sayaman. Low non-immune stromal cell levels and high hypoxic tumor microenvironments are associated with pathologic complete response in the neoadjuvant I-SPY2 TRIAL [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A032.