Tumour-informed liquid biopsies to monitor advanced melanoma patients under immune checkpoint inhibition

Abstract

Immune checkpoint inhibitors (ICI) have significantly improved overall survival in melanoma patients. However, 60% experience severe adverse events and early response markers are lacking. Circulating tumour DNA (ctDNA) is a promising biomarker for treatment-response and recurrence detection. The prospective PET/LIT study included 104 patients with palliative combined or adjuvant ICI. Tumour-informed sequencing panels to monitor 30 patient-specific variants were designed and 321 liquid biopsies of 87 patients sequenced. Mean sequencing depth after deduplication using UMIs was 6000x and the error rate of UMI-corrected reads was 2.47×10^-4. Variant allele fractions correlated with PET/CT MTV (rho=0.69), S100 (rho=0.72), and LDH (rho=0.54). A decrease of allele fractions between T1 and T2 was associated with improved PFS and OS in the palliative cohort (p = 0.008 and p < 0.001). ctDNA was detected in 76.9% of adjuvant patients with relapse (n = 10/13), while all patients without progression (n = 9) remained ctDNA negative. Tumour-informed liquid biopsies are a reliable tool for monitoring treatment response and early relapse in melanoma patients with ICI.

Fig. 1. Study design for patients with combined ICI.

PET/CT and sequencing of tumour normal pairs for patients under palliative ICI were done before treatment start. Blood samples were taken at T0, T1 (3 weeks) and T2 (second PET/CT, 12 weeks). Additional blood samples were collected during therapy and follow-up to increase the sensitivity for changes in allele fractions during and after therapy completion.

Fig. 2. Examples of ctDNA sequencing results of advanced melanoma patients.

A shows ctDNA sequencing results (allele fractions of all monitored variants over time) of two patients under combined ICI with progressive disease. An early decrease in variant AF is followed by a subsequent increase. Due to the minimal invasive nature of liquid biopsies, closer monitoring over time can be obtained compared to PET/CT. The different allele fractions of the somatic variants reflect the clonal structure of the primary tumour which remained stable in most cases. B illustrates the course of ctDNA AFs of two patients responding to combined ICI. For patient 23, the variant AFs quickly decrease and remain below the detection limit. C shows two patients with adjuvant ICI and a relapse during follow-up. The treatment of patient 74 was changed upon progression and the clinical response to the new treatment is reflected in decreasing AFs below the detection limit. For patient 85, we detected progression under adjuvant treatment at day 43 (64 days before PET/CT). All panels: Each line represents a single somatic variant before, during or after treatment. Source data are provided as a Source Data file.

Fig. 3. Correlation of mean AFs and known melanoma biomarkers.

A, B and C show the mean allele fraction of patient-specific variants in liquid biopsy samples from patients under palliative combined ICI and S100 (µg/l), LDH (U/l) and MTV (ml). The Spearman’s rank correlation coefficients were calculated to determine the correlation between AF and S100 (rho = 0.72, t = 8.17, df = 62, p = 2.01 × 10⁻⁹), LDH (rho = 0.54, t = 5.06, df = 62, p = 4.12 × 10⁻⁴) and MTV (rho = 0.69, t = 7.26, df = 58, p = 1.1 × 10⁻⁶). Axes were log transformed and the minimum of all non-zero values was divided by two and added to zero values. S100 - µg/l, LDH - U/l, MTV - ml. Source data are provided as a Source Data file.

Fig. 4. Survival analysis in advanced melanoma patients and relapse detection in adjuvant patients.

Kaplan-Meier curves and differences in survival for patients with combined ICI grouped by ctDNA AF changes between T0 and T1 for PFS (A), OS (B) and between T1 and T2 for PFS (C) and OS (p = 3.712 × 10⁻⁴) (D). AF asc—ascending or stable allele fractions over time, AF desc—descending allele fractions. E A swimmer plot with patients with adjuvant ICI and follow-up. Relapse-free patients (blue bars) had no ctDNA-positive liquid biopsy indicating a high specificity of the approach. ctDNA-negative: samples with less than three significant variants, ctDNA-positive: samples with at least three significant variants. Source data are provided as a Source Data file.