Up to 30% of patients with locally advanced head and neck squamous cell carcinoma (LA-HNSCC) relapse. Molecular residual disease (MRD) detection using multiple assays after definitive therapy has not been reported. In this study, we included patients with LA-HNSCC (stage III Human Papilloma virus (HPV)-positive, III-IVB HPV-negative) treated with curative intent. Plasma was collected pre-treatment, at 4–6 weeks (FU1) and 8-12 weeks (FU2) post-treatment. Circulating tumor DNA (ctDNA) was analyzed using a tumor-informed (RaDaR®) and a tumor-naïve (CAPP-seq) assay. HPV DNA was measured using HPV-sequencing (HPV-seq) and digital PCR (dPCR). A total of 86 plasma samples from 32 patients were analyzed; all patients with at least 1 follow-up sample. Most patients were stage III HPV-positive (50%) and received chemoradiation (78%). No patients had radiological residual disease at FU2. With a median follow-up of 25 months, there were 7 clinical relapses. ctDNA at baseline was detected in 15/17 (88%) by RaDaR and was not associated with recurrence free survival (RFS). Two patients relapsed within a year after definitive therapy and showed MRD at FU2 using RaDaR; detection of ctDNA during follow-up was associated with shorter RFS (p < 0.001). ctDNA detection by CAPP-seq pre-treatment and during follow-up was not associated with RFS (p = 0.09). HPV DNA using HPV-seq or dPCR during follow-up was associated with shorter RFS (p < 0.001). Sensitivity and specificity for MRD at FU2 using RaDaR was 40% and 100% versus 20 and 90.5% using CAPP-seq. Sensitivity and specificity for MRD during follow-up using HPV-seq was 100% and 91.7% versus 50% and 100% using dPCR. In conclusion, HPV DNA and ctDNA can be detected in LA-HNSCC before definitive therapy. The RaDaR assay but not CAPP-seq may detect MRD in patients who relapse within 1 year. HPV-seq may be more sensitive than dPCR for MRD detection.

高达 30% 的局部晚期头颈鳞状细胞癌 (LA-HNSCC) 患者会复发。尚未报道明确治疗后使用多种检测进行分子残留病（MRD）检测。在这项研究中，我们纳入了接受治疗的 LA-HNSCC（III 期人乳头瘤病毒 (HPV) 阳性、III-IVB HPV 阴性）患者。在治疗前、治疗后 4-6 周 (FU1) 和 8-12 周 (FU2) 收集血浆。使用肿瘤知情 (RaDaR®) 和肿瘤初始 (CAPP-seq) 测定法对循环肿瘤 DNA (ctDNA) 进行分析。使用 HPV 测序 (HPV-seq) 和数字 PCR (dPCR) 测量 HPV DNA。总共分析了 32 名患者的 86 份血浆样本；所有患者至少有 1 个随访样本。大多数患者为 III 期 HPV 阳性 (50%)，并接受了放化疗 (78%)。FU2 时没有患者出现放射学残留病。中位随访 25 个月，有 7 例临床复发。RaDaR 在 15/17 (88%) 中检测到基线 ctDNA，并且与无复发生存 (RFS) 无关。两名患者在确定性治疗后一年内复发，并使用 RaDaR 显示 FU2 级 MRD；随访期间 ctDNA 检测与较短的 RFS 相关 ( p  < 0.001)。CAPP-seq 预处理和随访期间的 ctDNA 检测与 RFS 无关（p  = 0.09）。随访期间使用 HPV-seq 或 dPCR 检测 HPV DNA 与较短的 RFS 相关 ( p  < 0.001)。使用 RaDaR 对 FU2 的 MRD 的敏感性和特异性分别为 40% 和 100%，而使用 CAPP-seq 的敏感性和特异性分别为 20% 和 90.5%。使用 HPV-seq 进行随访期间 MRD 的敏感性和特异性分别为 100% 和 91.7%，而使用 dPCR 的敏感性和特异性分别为 50% 和 100%。总之，在最终治疗前可以在 LA-HNSCC 中检测到 HPV DNA 和 ctDNA。RaDaR 检测（而非 CAPP-seq）可以检测 1 年内复发的患者的 MRD。对于 MRD 检测，HPV-seq 可能比 dPCR 更敏感。