Identification of potential immune evasion mechanisms in aggressive small renal cell carcinomas through single-cell and bulk RNA sequencing.

574 Background: Small renal cell carcinoma (RCC) is generally nonaggressive but in some cases, small RCCs exhibit distant metastasis. The biology and biomarker of small aggressive RCCs remain unclear, necessitating appropriate evaluation to avoid undertreatment. Previous biomarker studies via genetic profiling of tumor tissues were hindered by RCC heterogeneity. We utilized bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) of blood samples to identify genetic biomarkers and characterize the tumor immune microenvironment in patients with aggressive small RCC. Methods: We identified four T1a (≤4 cm) clear cell RCCs (ccRCC) with synchronous distant metastasis as aggressive, and four age-, sex-, and tumor size-matched ccRCC patients without metastasis as non-aggressive, from a prospective cohort (ClinicalTrials.gov Identifier: NCT03694912). Two healthy kidney donors served as controls. Using the 10X Genomics platform, we performed scRNA-seq and bulk analysis on ten peripheral blood mononuclear cell (PBMC) samples. Samples were demultiplexed with Souporcell, and sequencing reads were normalized and analyzed using R/Seurat. Cellular components were identified with marker genes, and bulk analysis included differential gene expression and pathway enrichment. Results: The mean tumor size and age of the eight ccRCC patients were 2.5 cm and 63 years, respectively. Among the four aggressive ccRCCs, two had lung metastasis and two had bone metastasis. We analyzed 32,417 cells, identifying 41 subpopulations. Single-cell analysis revealed variations in T and natural killer (NK) cell proportions among aggressive, non-aggressive, and normal groups, with significant differences. The aggressive group showed increased MYOM2, OVCH1-AS1, and HLA-DQA2 expression. Despite an increased proportion of CD4+ cytotoxic T lymphocytes (CTLs) with disease severity, key cytotoxic and immune regulatory genes like GNLY, CD247, CCL4, NKG7, and TGFB1 were decreased, suggesting compromised antitumor function in aggressive states. Conclusions: This study offers insights into the molecular and cellular characteristics of aggressive small RCCs via PBMC analysis using single-cell and bulk RNA sequencing. The increased proportion of CD4+ CTLs with decreased expression of critical cytotoxic and immune regulatory genes in aggressive RCCs suggests a mechanism for immune evasion. These findings enhance our understanding of aggressive small RCC biology and highlight potential biomarkers and therapeutic targets for adjuvant treatment. Clinical trial information: NCT03694912 .