Abstract 1980: The significance of circulating tumor cells and circulating tumor DNA as prognostic markers in human papillomavirus-positive head and neck squamous cell carcinoma

Abstract Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) respond favorably to treatment; however, some are resistant to treatment. Liquid biopsy is a potential prognostic biomarker for various types of cancer. In this study, we comprehensively investigated circulating tumor cells (CTCs), cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA) and compared them to determine their potential as predictive biomarkers for HPV-positive HNSCC. First, we isolated CTCs and cfDNA from the peripheral blood of 42 HPV-positive HNSCC patients before treatment. CTCs were obtained using a size-based separation method with a microfilter or the CD45 depletion method. The expression of three epithelial-related genes (EPCAM, MET, and EGFR) in the isolated CTCs was determined by RT-qPCR. When at least one of the three epithelial-related genes was detected, the sample was defined as positive for CTCs. cfDNA was isolated from serum samples using the QIAamp Circulating Nucleic Acid Kit (QIAGEN) and quantified using an Agilent 4200 TapeStation. We then determined the level of cfDNA per 1ml of serum. Next, we examined ctDNA to detect E7 genes specific to HPV in cfDNA as ctDNA using droplet digital PCR. Finally, we analyzed the clinical significance of CTCs, cfDNA and ctDNA. CTCs were isolated in 21patients (50.0%) and the HPV E7sequence was detected in 17 patients (40.1%) of 42 HPV-positive HNSCC patients. Among the clinical factors, only N stage and ctDNA positivity were significantly associated(p=0.03). There were no associations between the presence of CTCs and clinical factors. We further investigated whether CTCs and ctDNA correlated with prognosis using multivariate analysis. CTC positivity was most strongly associated with progression-free survival (PFS) (Hazard Ratio=5.549, p=0.037); however, the presence of ctDNA was not correlated with PFS. The presence of CTCs was found to be the most prognostic factor in HPV-positive HNSCC patients compared to the presence of ctDNA. Moreover, the CTC-positive/ctDNA-negative group showed the most unfavorable PFS(p=0.02). The cfDNA levels were significantly lower in the CTC-positive/ctDNA-negative group (p=0.01). Previous studies have reported that cfDNA is primarily released by tumor apoptosis. The low levels of cfDNA in CTC-positive/ ctDNA-negative patients suggest that CTCs may be shed into the peripheral bloodstream in a state of survival.Thus, our study provides new insights into predictive biomarkers of treatment resistance in patients with HPV-positive HNSCC. Citation Format: Hiroe Tada, Hideyuki Takahashi, Kazuaki Chikamatsu. The significance of circulating tumor cells and circulating tumor DNA as prognostic markers in human papillomavirus-positive head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1980.