medRxiv. 2026 Mar 30. pii: 2026.03.24.26348354. [Epub ahead of print]
Ravi Bandaru,
Hailu Fu,
Haizi Zheng,
Jocelyn Liang,
Li Wang,
Shuchi Gulati,
Benjamin H Hinrichs,
Mingxiang Teng,
Bin Zhang,
Marsha Kocherginsky,
Dechen Lin,
David A Hildeman,
Francis P Worden,
Matthew O Old,
Neal E Dunlap,
John M Kaczmar,
Maura Gillison,
Dalia El-Gamal,
Trisha Wise-Draper,
Yaping Liu.
Reliable, minimally invasive biomarkers for predicting immunotherapy response in head and neck squamous cell carcinoma (HNSCC) remain an unmet clinical need. Here, using patients from a prospective, multi-institutional phase II clinical trial ( NCT02641093 ), we performed whole-genome sequencing of 185 plasma cell-free DNA (cfDNA) samples collected longitudinally from 68 patients with locally advanced, surgically resectable HNSCC undergoing neoadjuvant and adjuvant pembrolizumab treatment. We developed the regional motif diversity score (rMDS), a novel fragmentomic metric quantifying the entropy of cfDNA 5' end motifs across genomic regions. Remarkably, unsupervised analysis revealed that rMDS robustly distinguished immunotherapy responders from non-responders, outperforming established cfDNA fragmentomic metrics and copy number alterations, while demonstrating independence from technical confounders. Longitudinal analysis revealed dynamic rMDS changes in genomic regions enriched for immune-, lectin-, and keratinization-related genes-hallmarks of squamous cell carcinoma-reflecting the interplay between tumor and peripheral immunity during the immunotherapy treatment. Interestingly, the regions with the most dynamic rMDS changes were highly enriched in telomere-proximal loci, suggesting a novel link between telomere biology and cfDNA fragmentation. A machine learning classifier based on rMDS achieved robust predictive performance across multiple validation settings (AUC 0.89-0.99), with the highest accuracy at post-treatment timepoints and superior to PD-L1 expression and tumor fraction in the same sample. Predicted responders demonstrated significant trends toward improved disease-free survival (log rank test p=0.035, hazard ratio: 2.67, 95% confidence interval: 1.03-6.92), underscoring the clinical utility of rMDS-based stratification. These findings position rMDS as a biologically meaningful and clinically actionable biomarker for immunotherapy response in HNSCC, supporting its integration into future risk assessment frameworks and broader cancer care.