Abstract A003: Hsp70 gene expression is enriched in lymphocytes in renal cell carcinoma patients that respond to immune checkpoint inhibitors

Abstract We sought to identify immune features that predict response to immune checkpoint inhibitors (ICI) in renal cell carcinoma (RCC), a disease where only a subset of patients respond due to currently undefined mechanisms. To address this question, we used a single-cell RNA sequencing (scRNA-seq) atlas of the tumors of 70 patients with RCC. 49 of these patients received ICI (either anti-PD-1 alone or in combination), with most samples being taken before the initiation of treatment. Importantly, after treatment, patients were annotated as having benefit (complete or partial response) or non-benefit (progressive disease), providing us with the opportunity to associate immunophenotypes with clinical responses in human patients. One factor that strongly predicted whether each patient responded to ICI was the frequency of CD4+ T cells that fell into a cluster marked by heat shock protein genes, particularly Hsp70 genes such as HSPA6, HSPA1A, and HSPA1B (p = 0.0015). Our group has previously shown that similar genes in RCC also predict benefit from ICI when highly expressed in CD8+ T cells, so we hypothesized that heat shock genes may predict benefit from ICI across lymphocyte subsets (Kashima, ASCO, 2024). After creating a heat shock signature derived from genes upregulated in the CD4+ heat shock cluster, we observed that the score was significantly elevated across all lymphocytes in patients who responded to ICI (p = 0.0026). Similarly, we observed the same pattern when only the most upregulated heat shock gene, HSPA6, was used (p = 0.001). We next performed unbiased hierarchical clustering based on the Spearman correlations of the frequencies of each cluster present in the atlas, revealing that the levels of the heat shock enriched subclusters of NK cells, CD4+ T cells, and CD8+ T cells largely moved together. As validation, we turned to a scRNA-seq dataset of patients who received ICI in the HCRN GU16-260 trial (Hugaboom et al., Cancer Discovery, 2025). This revealed greater progression-free survival in patients with high lymphocyte HSPA6 expression (p = 0.0058, mean of 14.3 months vs. 3.9 months), supporting the link between heat shock expression and benefit from ICI. We also observed a similar result in a smaller cohort of 5 patients where HSPA6 levels trended higher in patients that experienced benefit from ICI (Bi et al, Cancer Cell, 2021). Our findings reveal that high expression of Hsp70 genes in lymphocytes predicts subsequent benefit from ICI in RCC. Future studies will mechanistically test whether this is purely a correlation or a causative link. Our results also suggest that a stress present in the tumor microenvironment of RCC may induce heat shock expression, given that the levels of heat shock CD4+ T cells, CD8+ T cells, and NK cells are all correlated across patients. The identification of this stress would further aid in the prediction of ICI response in RCC and could help elucidate future treatments that may synergize with ICI. Citation Format: Ethan Burns, Soki Kashima, Rishabh Rout, Miya Hugaboom, Zhaochen Ye, Nicholas Schindler, Anasuya Dighe, Maxine Sun, Gwo-Shu Mary Lee, Wenxin Xu, Michael Atkins, Sabina Signoretti, Bradley McGregor, Rana McKay, Toni Choueiri, David Braun. Hsp70 gene expression is enriched in lymphocytes in renal cell carcinoma patients that respond to immune checkpoint inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr A003.