9Characterizing carbonic anhydrase 9 and HIF-2α RNA expression in clear cell renal cell carcinoma (ccRCC)

Abstract Background ccRCC is frequently driven by VHL loss, leading to HIF-2α stabilization and upregulation of downstream targets such as carbonic anhydrase 9 (CA9). While CA9 is a well-established diagnostic marker of HIF pathway activation, its prognostic and predictive roles remain unclear. Emerging evidence suggests that high CA9 and HIF-2α expression may be associated with improved outcomes in ccRCC. With the recent approval of the HIF-2α inhibitor belzutifan, understanding whether these markers predict treatment response is critical for guiding biomarker-driven therapy. Methods Next-generation sequencing of DNA (592-gene panel or whole exome) and RNA (whole transcriptome) were performed on ccRCC specimens through Caris Life Sciences to comprehensively characterize genomic and transcriptomic alterations. Expression levels of CA9 and HIF-2α were quantified based on RNA transcripts per million (TPM) and categorized as High or Low using the 75th vs 25th percentile cutoff. Overall survival (OS) was defined as the time from initial diagnosis to death or last known follow-up. Time on treatment (TOT) was defined as the duration from the start of systemic therapy to treatment discontinuation. Results A total of 764 ccRCC specimens were analyzed. Of these, 433 (56.7%) were derived from primary kidney tumors, while the remaining samples were from metastatic sites, including lung (n = 71, 9.3%), bone (n = 70, 9.2%), endocrine (n = 43, 5.6%), liver (n = 23, 3%), lymph node (n = 23, 3%), CNS (n = 19, 2.5%), GI (n = 4, 0.5%) and other sites (n = 78, 10.2%). VHL alterations occurred in 82% (626/764) ccRCC tumors. The median age was 63. CA9 and HIF-2α expression levels were relatively higher in primary kidney tumors compared to metastatic sites. Expression levels of CA9 and HIF-2α were comparable across racial and ethnic subgroups. High CA9 expression (Q4 vs Q1) was associated with improved OS (mOS: 95 vs 42 months; HR 0.55, P = .005). Similarly, high HIF-2α expression (Q4 vs Q1) correlated with longer survival (mOS: 54.8 vs 38.3 months; HR 0.56, P .001). Among belzutifan-treated patients (n = 80), CA9 expression was not associated with OS (mOS: NE vs 17.3, p : 0.851) or TOT of belzutifan (mTOT: 3.49 vs 2.34, p: 0.175). HIF-2α expression was not associated with OS (mOS: 16.2 vs 13.4, p : 0.89) or TOT of belzutifan among VHL mutant patients (2.96 vs 2.96; p: 0.91). There were limited VHL wild-type ccRCC patients (n = 9) treated with belzutifan in our cohort. Conclusions This comprehensive analysis of 764 ccRCC specimens demonstrates that VHL alterations are present in the majority of ccRCC tumors. CA9 and HIF-2α expression were consistent across racial and ethnic groups but showed higher expression in primary tumors compared to metastatic sites. High expression of both CA9 and HIF-2α were associated with significantly improved overall survival in the general ccRCC population. However, neither CA9 nor HIF-2α expression levels predicted response to belzutifan therapy. These findings enhance our understanding of HIF pathway biology in ccRCC and provide important context for the potential clinical application of CA9 PET imaging and HIF-2α inhibitor therapy in diverse patient populations.