Post-Operative Lymphatic ctDNA Data Shows Superior Sensitivity for Detecting Residual Disease in HPV-Independent HNSCC

Circulating tumor DNA (ctDNA) captured from post-operative lymphatic fluid may offer a more sensitive signal of residual disease than traditional pathology in patients with human papillomavirus (HPV)–independent head and neck squamous cell carcinoma (HNSCC), according to findings from a study published in Clinical Cancer Research.^1,2

Investigators reported that lymph collected from surgical drains contained markedly higher levels of tumor-informed ctDNA compared with that collected from matched plasma samples, and that the presence of ctDNA in this lymphatic exudate at 24 hours post-surgery was strongly associated with subsequent disease recurrence.

Notably, lymph-derived ctDNA outperformed standard high-risk pathological features in identifying patients at risk for relapse. Among 44 evaluable patients with intermediate-risk disease, detection of ctDNA from post-operative lymph achieved 88% sensitivity and 67% specificity for predicting recurrence (HR, 8.4; 95% CI, 1.9-37.1; P = .0008). These results highlight the emerging potential of lymph-based sampling as an early, sensitive marker of residual disease that may help refine adjuvant treatment strategies in HPV-independent HNSCC.

“Our results highlight the incredible potential of LymphDetect to improve survival through precision adjuvant therapy, especially in patients with intermediate-risk cancer where oncologic outcomes are balanced with toxicity of treatment,” Jose Zevallos, MD, MPH, FACS, senior author on the study, stated in a news release.¹ Zevallos is also the deputy director of Hillman Cancer Center, Eugene N. Meyers, MD Professor and chair of the Department of Otolaryngology-Head & Neck Surgery at the University of Pittsburgh Medical Center (UPMC) School of Medicine in Pennsylvania, as well as the co-founder of Droplet Biosciences.

What were the design and methodology of this study?

This prospective observational study enrolled patients with HPV-independent head and neck squamous cell carcinoma (HNSCC) undergoing surgical resection with concomitant neck dissection.² Recruitment occurred at Washington University between August 2019 and November 2021 (n = 62) and at 2 UPMC sites between April 2022 and August 2023 (n = 14).

Eligibility criteria required patients to have HPV-independent HNSCC, be at least 18 years of age, and have undergone primary tumor resection with planned neck dissection and postoperative surgical drain placement. Patients were excluded if they had HPV-positive HNSCC, presence of distant metastatic disease at the time of surgery, synchronous primary tumors, or a history of another primary malignancy within the preceding 5 years. All adjuvant treatment decisions were made independently of assay results. Clinical data were extracted from electronic health records.

Baseline clinical and pathological features were documented at enrollment. Prospective biospecimen collection included lymph (n = 76), plasma (n = 42), peripheral blood (n = 76), and resected tumor tissue (n = 76), obtained approximately 24 hours after surgery. Longitudinal follow-up occurred every 4 months and included documentation of adjuvant therapy, treatment outcomes, disease progression, death, and surveillance imaging.

Tumor samples were snap-frozen in liquid nitrogen, embedded in optimal cutting temperature compound, and stored at –80 °C. Lymph was collected from Jackson-Pratt surgical drains in 50-mL tubes at 24 hours postoperatively, transported on ice, filtered, stabilized with K2EDTA, centrifuged to isolate cell-free DNA–containing supernatant, aliquoted, and stored at –80 °C. For patients with bilateral dissections, lymph was obtained from the side containing biopsy-proven or suspicious nodal disease.

What were the baseline clinical characteristics of the patients enrolled?

The study initially enrolled 76 patients with HPV-independent HNSC. Three patients were excluded, 1 due to a blood sample mismatch and 2 because tumor specimens failed sequencing, resulting in a final study population of 73 evaluable patients. Of these, 36 patients comprised the initial cohort, and 37 patients were allocated to the replication cohort.

Baseline comparisons demonstrated modest differences between cohorts in extranodal extension, immunotherapy exposure, and cumulative smoking history; no other clinical or demographic characteristics differed significantly between groups. The median age of patients in the study population was 63 years, and most patients were male (67.1%). Adjuvant therapy was administered to 71.2% of patients, including chemotherapy (1.4%), radiotherapy (46.6%), chemoradiation (20.5%), and immunotherapy (2.7%).

The median follow-up duration was 37.4 months. Disease recurrence occurred in 34 of 73 patients, evenly distributed across the initial (n = 17) and replication (n = 17) cohorts. Thirty-nine patients exhibited no evidence of disease; this incidence was similarly balanced between cohorts (initial, n = 19; replication, n = 20). Matched plasma samples were available for 42 patients, including 29 in the initial cohort and 13 in the replication cohort.

What are the potential predictive applications of lymph ctDNA in intermediate-risk HPV-independent HNSCC?

Under current NCCN guidelines, patients with HPV-independent HNSCC with high-risk features such as extranodal extension or positive surgical margins are recommended to receive adjuvant chemoradiation, whereas those with intermediate-risk pathology typically receive radiotherapy with consideration of systemic therapy.

Given the observed synergy between pathological features and lymph-derived ctDNA, investigators evaluated whether postoperative lymph ctDNA could further stratify recurrence risk within this intermediate-risk subgroup.

Notably, all patients who received radiotherapy alone and later relapsed (n = 9) were ctDNA-positive in lymph at 24 hours. Locoregional predictive performance was even stronger, with 92% sensitivity and 67% specificity (HR, 14.7; 95% CI, 1.9-113.4; P = .0007). Lymph ctDNA demonstrated higher early predictive accuracy than any available pathological feature, supporting its use as a complementary molecular biomarker for identifying patients who may benefit from treatment intensification.

Case examples underscored this utility. Two patients with intermediate-risk disease had positive lymph ctDNA at 24 hours despite limited pathological risk features, and both experienced recurrence, 1 locoregional and 1 distant, within months.

“We’re thrilled to share our findings in Clinical Cancer Research and to collaborate with such outstanding partners,” Wendy Winckler, PhD, chief scientific officer at Droplet Biosciences, shared in the news release.¹ “Our results validate the hypothesis that lymph—collected directly from surgical drain fluid—is a powerful new biofluid for advancing precision oncology. We also look forward to presenting the first results from our second indication, muscle-invasive bladder cancer, in the Top Abstracts session at the Society of Urologic Oncology meeting [in] December [2025].”

References

1. Droplet Biosciences study reveals post-surgical lymph fluid as a powerful source of ctDNA for early recurrence detection in head and neck cancer. BioSpace. News Release. November 14, 2025. Accessed November 14, 2025. https://www.biospace.com/press-releases/droplet-biosciences-study-reveals-post-surgical-lymph-fluid-as-a-powerful-source-of-ctdna-for-early-recurrence-detection-in-head-neck-cancer
2. Lazare S, Gu Z, Earland, N, et al. Postoperative lymph is a proximal source of ctDNA for detection of recurrence in HPV-independent head and neck cancer. Clinical Cancer Research. Published online November 2, 2025. doi:10.1158/1078-0432.CCR-25-1221