Abstract 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 p0.05 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. Citation Format: Bill Diplas, Xin Pei, Yingjie Zhu, Shu Yazaki, Luc G.T. Morris, Richard J. Wong, Sean M. McBride, Heiko Schoder, Yao Yu, Alan L. Ho, Eric Sherman, Nora Katabi, Nancy Lee, Nadeem Riaz. Hypoxia selects for early PI3K pathway alterations and drives clonal complexity in HPV-positive oropharyngeal cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7291.
Diplas et al. (2026) studied this question.