2727 - Inferring Intratumoral Hypoxia with Targeted Cell-Free Bisulfite Sequencing among Patients with Head and Neck Squamous Cell Carcinoma
Presenter(s)
P. E. Clark1, N. Karasik2, S. R. Campbell3, N. M. Woody4, T. A. Sussman2, J. L. Geiger5, K. Burkitt1, H. Wang6, T. Watson7, T. Austin7, N. Silver8, T. A. Chan9, S. A. Koyfman4, and J. A. Miller1; 1Cleveland Clinic, Cleveland, OH, 2Department of Hematology/Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, 3Department of Radiation Oncology, Cleveland Clinic, Cleveland, OH, 4Department of Radiation Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, 5Department of Hematology and Medical Oncology, Taussig Cancer Center, Cleveland Clinic, Cleveland, OH, 6Department of Laboratory Medicine, Diagnostics Institute, Cleveland Clinic, Cleveland, OH, 7Cleveland Clinic Foundation, Cleveland, OH, 8Department of Otolaryngology, Head and Neck Institute, Cleveland Clinic, Cleveland, OH, 9Department of Radiation Oncology, Cleveland Clinic Foundation, Cleveland, OH
Purpose/Objective(s):
Pre-treatment intratumoral hypoxia is a prognostic and predictive biomarker for response to chemoradiotherapy among patients with head and neck squamous cell carcinomas (HNSCC). Dynamic reversion from hypoxia to normoxia, measurable by 18F-FMISO PET, facilitates selection of patients for treatment de-escalation but is not widely available and does not inform molecular mechanisms. Tissue-based gene expression profiling is limited by tumor heterogeneity, mRNA degradation, and inability to dynamically measure hypoxia. We hypothesized that intratumoral hypoxia could be inferred noninvasively using plasma targeted methylation sequencing.Materials/Methods:
We designed a custom plasma targeted bisulfite sequencing assay to capture tumor-associated cfDNA among patients with HNSCC. This targeted panel included 257 differentially-methylated regions (DMRs) from the validated Hypoxia-M methylome classifier (31.1 kb), 790 human DMRs in 13 additional HPV dysplasia- or hypoxia-associated genes (95.6 kb), and seven high-risk HPV genomes (16, 18, 31, 33, 35, 45, 52, 47.4 kb) spanning a total of ~12,100 CpG sites. Four hybridization probes were included per target region for target enrichment of both DNA strands and methylated or unmethylated bisulfite-converted adapter-ligated cfDNA fragments. Alignment and methylation extraction of trimmed paired-end reads was performed using Bismark (V0.24.2).Results:
Pre-treatment plasma from 12 patients with HPV16-positive (n=7) or HPV-negative (n=5) HNSCC were tested with targeted cell-free methylation sequencing. All patients underwent definitive radiotherapy with concurrent platinum (n=10), cetuximab (n=1), or no chemotherapy (n=1). 75% (9/12) had AJCC8 cT3-4 or cN2-3 tumors and median plasma HPV16 DNA concentration was 30.5 cp/mL among HPV+ tumors. A median 15% (range, 11-21%) of hypoxia DMRs were hypomethylated and 20% (19-25%) were hypermethylated. Concordant with prior analyses, hypoxia-associated promoters such as STC2 (100%) were frequently hypermethylated pre-treatment. Promoter hypomethylation within VEGFA, HIF1A, and CA9 were observed among three patients who developed disease recurrence.Conclusion:
Hypoxia-associated cfDNA methylation signatures were detectable in pre-treatment plasma among patients with HPV-positive and HPV-negative HNSCC. Promoter hypomethylation was observed in hypoxia-responsive genes. Targeted bisulfite sequencing could provide a noninvasive approach to infer bulk intratumoral gene expression for mechanistic and dynamic insights.