Liquid BIOpsy for MiNimal RESidual DiSease Detection in Head and Neck Squamous Cell Carcinoma (LIONESS)-a personalised circulating tumour DNA analysis in head and neck squamous cell carcinoma

Abstract

Background: Head and neck squamous cell carcinoma (HNSCC) remain a substantial burden to global health. Cell-free circulating tumour DNA (ctDNA) is an emerging biomarker but has not been studied sufficiently in HNSCC.

Methods: We conducted a single-centre prospective cohort study to investigate ctDNA in patients with p16-negative HNSCC who received curative-intent primary surgical treatment. Whole-exome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) tumour tissue. We utilised RaDaR^TM, a highly sensitive personalised assay using deep sequencing for tumour-specific variants, to analyse serial pre- and post-operative plasma samples for evidence of minimal residual disease and recurrence.

Results: In 17 patients analysed, personalised panels were designed to detect 34 to 52 somatic variants. Data show ctDNA detection in baseline samples taken prior to surgery in 17 of 17 patients. In post-surgery samples, ctDNA could be detected at levels as low as 0.0006% variant allele frequency. In all cases with clinical recurrence to date, ctDNA was detected prior to progression, with lead times ranging from 108 to 253 days.

Conclusions: This study illustrates the potential of ctDNA as a biomarker for detecting minimal residual disease and recurrence in HNSCC and demonstrates the feasibility of personalised ctDNA assays for the detection of disease prior to clinical recurrence.

Fig. 1. RaDaR^TM workflow.

Tumour tissue from surgical resection was macrodissected and used for whole-exome sequencing to identify somatic mutations. A personalised ctDNA assay was developed for each patient. Tumour and buffy coat DNA were analysed using personalised assays to confirm somatic mutations and exclude clonal haematopoiesis of indeterminate potential (CHIP). Plasma samples were analysed using RaDaR^TM panels and high-depth sequencing and ctDNA detection reported per patient.

Fig. 2. Box plots of estimated median variant allele frequency percentage (% VAF).

a ctDNA levels in baseline samples taken prior to surgery ranged from 0.001% to 2.737% estimated variant allele frequency (%VAF). b In post-surgery samples, ctDNA could be detected at levels as low as 0.0006% VAF, with levels below 0.01% VAF in 20% of ctDNA-positive samples.

Fig. 3. Longitudinal monitoring of serial plasma samples.

Longitudinal monitoring of serial plasma samples from 17 patients, indicating when ctDNA was detected (red circle) or not detected (black circle) and whether the patient subsequently relapsed (inverted yellow triangle). ctDNA was detected in all pre-operative time points (purple square). The black dashed line extends across the x axis to the last clinical visit to date to indicate the total duration of follow-up for each patient. Patients were followed up for a median duration of 371 days (292–532 days) in the overall cohort, excluding one patient lost to follow-up (patient 12) and four deceased patients (2, 9, 13 and 14). Clinical or CT-morphological evidence of disease recurrence was not observed at the time of last follow-up visit in 12/17 cases profiled. The solid blue line indicates the lead time which is the interval between the first ctDNA-positive post-surgery sample and clinical confirmation of disease recurrence.

Fig. 4. Examples of longitudinal monitoring of ctDNA in five patients.

ctDNA detection is indicated with red circles, not detected (ND) with black circles, clinical progression with an inverted yellow triangle and lead time from ctDNA detection to clinical progression with black lines. Periods of adjuvant treatment are shaded in blue. Baseline (time point 0 on the x axis) is the pre-surgical plasma collection time point. a Patient 14—ctDNA was not detected in the pT1 tumour at any time point for this patient (top panel) but was detected at all time points pre- and post-operatively in the pT4a tumour (bottom panel), with a lead time of 108 days from ctDNA detection to clinical progression. b Patient 13—ctDNA detected at all time points, 110 days prior to progression. c Patient 9—ctDNA detected before surgery, but not 3 or 13 days post surgery. ctDNA detected at 21 days post surgery, which decreased after completion of adjuvant treatment only to rise again by day 128, prior to clinical progression. ctDNA levels were undetectable following a second surgical intervention at 161 days. d Patient 2—ctDNA detected before surgery but not at day 7 after surgery. ctDNA levels rose to detectable levels by day 17 and continued to rise >253 days prior to progression. e Patient 6—ctDNA detected before surgery but not at 6, 21 and 90 days post surgery, following adjuvant therapy. Rising ctDNA levels were detected from 132 days, 160 days ahead of clinical progression. For all patients, heatmap on the right of the figure shows the signal from different variants. Each column represents a different variant and each row a different sample type. Variants absent in the tumour DNA or present in the buffy coat DNA were excluded from the analysis.