Integrated analysis of oral rinse-derived and plasma circulating tumour DNA for mutation profiling and outcome prediction with oral squamous cell carcinoma

Abstract

This study evaluates the potential of oral rinse-derived and plasma circulating tumour DNA (ctDNA) in HPV-negative oral squamous cell carcinoma (OSCC), where early recurrence occurs in a significant proportion of patients, contributing to poor prognosis. Analysis of paired tissue, oral rinse, and plasma samples from 123 patients revealed ctDNA detection rates of 94.3% in oral rinse and 80.5% in plasma samples. Combined testing improved mutation detection sensitivity to 48.6%. A machine learning model integrating seven mutated genes (TP53, TERT, IKZF1, EP300, MYC, EGFR, PIK3CA) and clinical factors demonstrated robust prediction of recurrence (validation AUC: 0.854) and survival outcomes. Integration of pretreatment plasma ctDNA status further enhanced predictive performance. In longitudinal analysis, ctDNA detected recurrence approximately four months before clinical manifestation. These findings suggest that integrated ctDNA analysis offers improved mutation profiling and outcome prediction, potentially enabling earlier interventions in OSCC.

Fig. 1. The study design, sample collections, and patients’ responses.

Detectable mutation refers to patients with any identifiable somatic mutation using our NGS panel.

Fig. 2. Comprehensive mutational profiling of cell-free DNA from oral rinse and plasma samples.

Heatmap illustrating the mutational profile of 116 oral rinse-derived cfDNA samples (A) and 99 plasma cfDNA samples (B). Each column represents a tumour sample, and each row represents a gene. Only genes mutated in more than two cases are shown. The upper bar indicates the number of mutations per patient, and the right bars show gene mutation frequencies across the cohort. Samples are clustered by pathological subtype. cfDNA cell-free DNA.

Fig. 3. Concordance between oral rinse-derived cfDNA, plasma cfDNA, and tumour gDNA genotyping.

A–C Venn diagrams showing the number of mutations in oral rinse-derived cfDNA and tumour gDNA (A), plasma cfDNA and tumour gDNA (B), and those shared between tissue and oral rinse-derived or plasma cfDNA (C). D Bar graph showing the allele frequency of variants in tumour gDNA. Blue bars indicate mutations detected in both tumour gDNA and cfDNA (oral rinse-derived or plasma), while red bars show mutations detected only in tumour gDNA. E Correlation between tumour DNA content in oral rinse and plasma samples. cfDNA cell-free DNA, gDNA genomic DNA.

Fig. 4. Overview of clinical and mutational characteristics.

A The most frequently mutated genes. B Significantly different SNVs included in the 1-year recurrence model. C Significantly different CNVs included in the 1-year recurrence model. D Differences in clinical characteristics based on 1-year recurrence status. p-values were calculated using Fisher’s exact test. The size of the white dots in (C) represents sample size. SNV single-nucleotide variant, CNV copy number variation.

Fig. 5. Predicting 1-year recurrence and PFS/OS by combining mutation and clinical characteristics.

A ROC curve for the 1-year recurrence predictive model in the training and validation cohorts. B A nomogram based on stepwise logistic regression to predict 1-year recurrence. C Predicting PFS and OS by combining key mutation and clinical characteristics. D, E Kaplan–Meier curves comparing high- and low-risk groups for PFS (D) and OS (E). PFS progression-free survival, OS overall survival.

Fig. 6. Mutation landscape of pre-treatment oral rinse-derived ctDNA.

A Overview of oral rinse-derived ctDNA status, clinical characteristics, and 1-year recurrence at baseline. B, C Kaplan–Meier estimates of PFS (B) and OS (C) according to pre-treatment oral rinse-derived ctDNA status. ctDNA circulating tumour DNA, PFS progression-free survival, OS overall survival.

Fig. 7. Mutation landscape of pre-treatment plasma ctDNA.

A Overview of plasma ctDNA status, clinical characteristics, and 1-year recurrence at baseline. B, C Kaplan–Meier estimates of PFS (B) and OS (C) according to pre-treatment plasma ctDNA status. ctDNA circulating tumour DNA, PFS progression-free survival, OS overall survival.

Fig. 8. Prediction of 1-year recurrence and prognosis using a combined model with ctDNA status.

A 1-year recurrence prediction based on the combined model incorporating DNA mutations, clinical factors, and ctDNA status from oral rinse and plasma samples. B 1-year recurrence prediction using the combined model with ctDNA concentration from oral rinse and plasma samples. C, D Kaplan–Meier curves comparing high- and low-risk groups for PFS (C) and OS (D). ctDNA circulating tumour DNA, PFS progression-free survival, OS overall survival.

Fig. 9. Longitudinal monitoring of oral rinse-derived and plasma ctDNA.

A–D Changes in ctDNA status and concentration from T0 to T2 in the oral rinse recurrence cohort (A), oral rinse non-recurrence cohort (B), plasma recurrence cohort (C), and plasma non-recurrence cohort (D). E, F Monitoring of serial oral rinse-derived (E) and plasma (F) samples from 19 patients, showing ctDNA detection (red circle) and subsequent relapse (yellow triangle). ctDNA was detected at all pre-operative time points (purple square). The dashed line indicates the total follow-up duration. ctDNA circulating tumour DNA.T0, pre-treatment. T1, post-operation. T2 post-operation detectable ctDNA.