Hypoxia selects for early PI3K pathway alterations and drives clonal complexity in HPV-positive oropharyngeal cancer

Purpose: Tumor hypoxia drives treatment resistance in head and neck cancer, yet its molecular determinants remain unclear. We previously demonstrated that HPV-related oropharyngeal cancer (OPC) exhibiting hypoxia is more aggressive necessitating escalated treatment. Here, we investigated the genetic drivers of tumor hypoxia and their impact on tumor evolution. Methods: We analyzed 152 patients with early-stage HPV-related OPC from a prospective hypoxia-directed dose de-escalation trial (NCT03323463). Comprehensive genomic profiling included WES (n=122) and RNA-seq (n=91). Tumor hypoxia was assessed using 18F-FMISO PET with a standardized hybrid method incorporating qualitative assessment of four image characteristics and quantitative tumor-to-background ratios (TBR >1.3 considered hypoxic). Clonal architecture was reconstructed using PhylogicNDT. We validated molecular correlates of hypoxia in two independent cohorts of HPV-related oropharyngeal cancer from the TCGA and JAVELIN-HN. Hypoxia in validation cohorts was determined using three established mRNA-based hypoxia signatures (Buffa, Winter, Toustrup). Results: Hypoxia as determined by FMISO PET was present in 72% of tumors at baseline, and correlated with well-established RNA-based signatures of hypoxia. Mutational profiling showed that hypoxic tumors were enriched for PI3K pathway alterations, most notably PIK3CA mutations (OR=5.45, adjusted p=0.05). Critically, this association was restricted to clonal PIK3CA mutations-subclonal mutations showed no hypoxia association, suggesting early selection under hypoxic conditions. This hypoxia-PI3K relationship was validated in independent cohorts of HPV-OPC (TCGA, JAVELIN-HN) using expression-based signatures of hypoxia (adjusted p<0.05 for Winter, Buffa and Toustrup hypoxia signatures). Lastly, clonal mapping further revealed that hypoxic tumors exhibited significantly greater clonal complexity (p < 0.05; Shannon diversity). Multi-region sequencing in a subset of 41 tumors confirmed extensive spatial heterogeneity, strongly linked to hypoxic tumors. Conclusions: Our findings show that hypoxia drives early selection for PI3K pathway alterations and promotes clonal complexity, demonstrating how the microenvironment directs tumor evolution in HPV-related OPC.