Biologic and clinical implications of HPV genome state variation in oropharyngeal cancers.

6076 Background: HPV integration enhances carcinogenesis by ensuring viral DNA retention and increasing E6/E7 expression. Though HPV usually integrates in cervical cancer, DNA episomes persist in ~50% of HPV+ oropharyngeal cancers (OPCs), and prior studies relating integration to clinical outcomes are contradictory. Defining molecular traits and clinical behavior of episomal vs. integrated OPCs is impeded by the complexity of HPV genome states, which defy classification by any one assay. Here we classified HPV states based on concordance between HPV-host fusion mRNA detection (suggesting integration) and E6/E7 levels. Gene expression and clinical outcomes were compared among groups to seek biomarkers allowing therapeutic personalization. Methods: Samples were curated from 851 therapy-naïve HPV+ OPCs receiving robotic surgery at a single institution (2007-2020). RNA sequencing was performed on 50 HPV+ OPCs that later recurred (cases) and 50 that were cured (controls). Groups were matched for stage, smoking, and adjuvant therapy. OPCs were deemed likely episomal if absence of HPV-host fusion mRNA was accompanied by E6/E7 levels in the bottom tertile and likely integrated if fusion mRNA presence coincided with E6/E7 in the top tertile. These two OPC subsets were defined as E6/E7-concordant and the rest as E6/E7-discordant. Molecular traits were analyzed as previously (Sannigrahi MK et.al, JNCI 2025 117:7) using GSEA of Hallmark pathways and by two scores derived by GSVA of host mRNAs that potently stratified recurrence risk across multiple HPV+ OPC cohorts: (1) an immune suppression score (ISS) measuring reduced anti-tumor immunity and (2) a t umor progression score ( TPS ) capturing aggressive tumor cell-intrinsic traits. Results: In the overall cohort (n=100), the fusion (+) OPCs (n=49) had increased risk of recurrence (OR 2.90, 95% CI=1.27-6.64, p=.01). Whereas E6/E7 levels alone did not stratify recurrence risk, combining it with fusion status optimized prediction: the likely integrated OPCs (n=23) had high recurrence risk vs. likely episomal OPCs (n=24) (OR=3.81, 95% CI=1.13-12.82, p=.03) despite similar clinical characteristics in both groups. Time to recurrence was also shorter in likely integrated vs. episomal subsets (p=.02). By contrast, fusion read status did not stratify recurrence risk in the E6/E7-disconcordant OPCs (n=53). Adverse gene expression features were upregulated in likely integrated vs. likely episomal OPCs, as reflected in increased TPS (p=.04) and ISS (p=.04). These differences were absent in fusion (+) tumors of the E6/E7-discordant group. Conclusions: Our findings offer the most compelling evidence to date supporting independent association of HPV integration with adverse tumor biology and recurrence risk in OPCs. Jointly considering HPV-host fusion mRNAs and E6/E7 levels may guide the molecular biomarker development needed to personalize therapy based on HPV genome state.