Defining a prognostic immune landscape for distant metastases in early-stage melanoma: insights from RNA sequencing and multiplex immunofluorescence in the Dutch Early Stage Melanoma (D-ESMEL) study

Background: The detection of circulating tumor DNA (ctDNA) through liquid biopsy has primarily focused on plasma derived from the blood of cancer patients.However, this method requires trained personnel, specific equipment, and several millilitres of whole blood for plasma collection and subsequent storage at -80C.In this study, we aimed to explore the possibility of detecting ctDNA in archived whole blood samples from cancer patients undergoing treatment for primary or recurrent cutaneous melanoma.Methods: We retrospectively analyzed matched whole blood (n = 10), and plasma samples (n=10) collected more than ten years ago from melanoma patients.We extracted DNA from fixed formalinembedded slides for tumours from nine patients and sequenced at 30X whole exome sequencing (WES) to derive patient-specific mutation lists.We extracted DNA from whole blood ranging from 1mL to 50uL (representing drops of blood) and performed pairedend, shallow 1-2X WGS.We analyzed the data for somatic copy number aberrations (SCNAs) tumour fraction estimation in a tumour-nave analysis, and single nucleotide variants (SNVs) detection in a tumour-informed analysis for increased sensitivity of detection.Results: In DNA extracted from archived whole blood samples, we detected SCNAs that reflected the profile observed in matched plasma DNA and tumour tissue from the same individuals.Using the bioinformatic pipeline ichorCNA to identify SCNAs, we found the tumour fraction ranged from 5% to 40% in whole blood in 6 patients.We observed distinct fragmentation profiles in whole blood with nucleosome fragmentation patterns similar to plasma cfDNA.Analysis of SNVs by Integration of Variant Reads (INVAR2) pipeline, detected ctDNA in nine out of the ten patients with mutant allele fractions ranging from 1% to 30%.Conclusions: Our data proves that ctDNA can be detected directly from thawed whole blood without plasma isolation and suggests the potential for ctDNA analysis in existing archived blood collections.Such archival collections offer the opportunity to evaluate the potential to detect ctDNA with a wide dynamic range of tumour burden, which has implications for liquid biopsy applications.Compared to conventional protocols based on plasma cfDNA, the proposed process is novel, simple and requires minimal blood volume, allowing existing biobanks to maximise discovery research in melanoma trials.