At the ESMO Congress 2026 in Madrid, Spain, Jean-Pascal Machiels presented a retrospective circulating tumor DNA (ctDNA) analysis from the phase III KEYNOTE-412 trial (NCT03040999), examining the association between ctDNA dynamics and clinical outcomes in patients with locally advanced head and neck squamous cell carcinoma (LA-HNSCC).
Presented as Abstract 1536RO, the analysis evaluated baseline ctDNA levels and early ctDNA clearance during treatment with pembrolizumab or placebo in combination with concurrent chemoradiotherapy (CRT).
The findings demonstrated important differences according to human papillomavirus (HPV) status. Baseline ctDNA levels were associated with survival outcomes in HPV-negative patients but not in HPV-positive patients. Furthermore, early ctDNA clearance was associated with improved clinical outcomes, although the strength and significance of these associations differed between treatment arms and HPV subgroups.
KEYNOTE-412: Investigating the Prognostic Role of ctDNA
Circulating tumor DNA has emerged as a potential biomarker for monitoring treatment response and evaluating prognosis across several solid tumor types. However, its clinical significance in locally advanced head and neck squamous cell carcinoma remains uncertain.
The phase III KEYNOTE-412 trial evaluated pembrolizumab plus concurrent chemoradiotherapy compared with placebo plus chemoradiotherapy in patients with newly diagnosed, unresected LA-HNSCC.
In this retrospective analysis, investigators examined whether ctDNA measurements obtained before treatment and during chemoradiotherapy were associated with event-free survival (EFS) and overall survival (OS).
Two time points were evaluated:
- Baseline (BL): ctDNA levels measured before treatment initiation.
- Cycle 3, Day 1 (C3D1): Early on-treatment ctDNA measurements obtained in patients who had completed five of the planned six to seven weeks of chemoradiotherapy.
The analysis used the tumor-informed Natera Signatera 16-plex assay to evaluate ctDNA levels.
Associations between baseline ctDNA, early ctDNA clearance and survival outcomes were assessed separately by treatment arm and HPV status, determined through p16 testing, using Cox proportional hazards regression.
Baseline ctDNA: Prognostic Significance Differs by HPV Status
Of the 804 patients enrolled in KEYNOTE-412, 473 had evaluable ctDNA samples at both baseline and C3D1, including 233 patients in the pembrolizumab arm and 240 in the placebo arm.
Baseline ctDNA detection rates were high in both HPV subgroups:
- HPV-positive patients: 95.6%.
- HPV-negative patients: 95.3%.
Baseline ctDNA levels were positively correlated with target lesion size in both HPV-negative and HPV-positive patients, with Spearman correlation coefficients of 0.49 and 0.34, respectively.
However, the association between baseline ctDNA levels and survival differed according to HPV status.
Among HPV-negative patients, higher baseline ctDNA levels were associated with worse overall survival in the pembrolizumab arm and with worse event-free and overall survival in the placebo arm (P < 0.05).
In contrast, no statistically significant associations between baseline ctDNA levels and survival outcomes were observed in HPV-positive patients in either treatment arm (P > 0.05).
The investigators also reported that the prognostic association of baseline ctDNA in HPV-negative patients was independent of ECOG performance status but dependent on tumor size and stage.
These findings suggest that the prognostic significance of pretreatment ctDNA in LA-HNSCC may vary according to HPV status and established clinical disease characteristics.
Early ctDNA Clearance: Higher Rates With Pembrolizumab
The analysis also examined ctDNA clearance at C3D1, following approximately five weeks of concurrent chemoradiotherapy.
Early ctDNA clearance rates were consistently higher among patients receiving pembrolizumab than among those receiving placebo.
| HPV status | Pembrolizumab + CRT | Placebo + CRT |
|---|---|---|
| HPV-negative | 72.6% | 63.6% |
| HPV-positive | 76.8% | 62.7% |
In HPV-negative patients, ctDNA clearance was observed in 72.6% of those receiving pembrolizumab compared with 63.6% in the placebo group.
Among HPV-positive patients, the corresponding clearance rates were 76.8% and 62.7%, respectively.
Although the pembrolizumab arm demonstrated numerically higher clearance rates in both HPV subgroups, the analysis primarily examined the association between ctDNA clearance and subsequent clinical outcomes.
HPV-Negative Disease: ctDNA Clearance Associated With Improved Survival
Among patients with HPV-negative LA-HNSCC, early ctDNA clearance was associated with improved event-free survival in both treatment groups.
The association was statistically significant in patients receiving pembrolizumab (P = 0.001) and those receiving placebo (P = 0.011).
However, the relationship with overall survival differed between the two arms.
In the pembrolizumab group, ctDNA clearance at C3D1 was significantly associated with improved overall survival (P < 0.001).
In the placebo group, the corresponding association did not reach statistical significance (P = 0.075).
These results indicate a stronger association between early ctDNA clearance and long-term survival among HPV-negative patients receiving pembrolizumab with chemoradiotherapy.
Importantly, these findings describe prognostic associations within treatment groups and do not establish that pembrolizumab provides a survival benefit based on ctDNA clearance.
HPV-Positive Disease: A Different Pattern of Prognostic Associations
The results were different among patients with HPV-positive LA-HNSCC.
In this subgroup, early ctDNA clearance was significantly associated with improved event-free and overall survival only in the placebo arm.
The reported associations were:
- Event-free survival: P = 0.028.
- Overall survival: P = 0.013.
No statistically significant associations between C3D1 ctDNA clearance and these survival endpoints were reported in the pembrolizumab arm.
These findings further demonstrate that the prognostic relevance of ctDNA clearance may differ between HPV-positive and HPV-negative head and neck cancers.
The differences observed across treatment arms also suggest that the relationship between early molecular response and long-term outcomes may depend on treatment modality.
Clinical Implications: Toward More Individualized Molecular Monitoring
The KEYNOTE-412 ctDNA analysis provides additional evidence regarding the potential role of molecular biomarkers in locally advanced head and neck cancer.
The high baseline ctDNA positivity rates in both HPV-positive and HPV-negative patients demonstrate that tumor-informed ctDNA testing was able to detect circulating tumor DNA in most evaluable patients before treatment.
However, the prognostic significance of these measurements was not uniform.
In HPV-negative disease, higher pretreatment ctDNA levels were associated with poorer survival outcomes, while early ctDNA clearance was associated with improved event-free survival across both treatment arms.
The particularly strong association between ctDNA clearance and overall survival in the pembrolizumab arm suggests that early molecular response may provide prognostic information in this population.
In HPV-positive disease, the absence of a significant association between baseline ctDNA and survival, together with the different pattern of clearance-related outcomes, highlights the importance of considering HPV status when interpreting ctDNA measurements.
Nevertheless, the retrospective nature of the analysis limits its clinical interpretation. The results establish associations between ctDNA measurements and outcomes but do not demonstrate that ctDNA-guided treatment modifications improve survival.
Prospective validation will be necessary before these findings can be incorporated into routine treatment decision-making.
Conclusion: HPV Status May Influence the Prognostic Value of ctDNA
The ctDNA analysis of the phase III KEYNOTE-412 trial, presented at ESMO Congress 2026, demonstrated that baseline ctDNA levels and early treatment-related ctDNA clearance have distinct prognostic associations in locally advanced head and neck squamous cell carcinoma according to HPV status.
In HPV-negative patients, higher baseline ctDNA levels were associated with poorer outcomes, while early ctDNA clearance was associated with improved event-free survival in both treatment arms and improved overall survival in the pembrolizumab arm.
In HPV-positive patients, baseline ctDNA levels were not significantly associated with survival, and early ctDNA clearance was associated with improved outcomes only in the placebo arm.
The findings suggest that the prognostic interpretation of ctDNA in LA-HNSCC should account for HPV status and treatment modality. Further prospective investigation is needed to determine whether ctDNA monitoring can support individualized treatment strategies and improve clinical decision-making.
Study Information
- Abstract: 1536RO – ctDNA analysis of the phase 3 KEYNOTE-412 trial: Pembrolizumab or placebo plus concurrent chemoradiotherapy for locally advanced head and neck squamous cell carcinoma.
- Presenter: Jean-Pascal Machiels (Woluwe-Saint-Lambert, Belgium).
- Clinical trial registration: NCT03040999.
- Study sponsor: Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc.
- Funding: Merck Sharp & Dohme LLC.
- ctDNA assay: Natera Signatera 16-plex.
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