Next Generation Sequencing based ctDNA Profiling as a Predictor of Immunotherapy Outcomes in Advanced Melanoma

Introduction

Advanced melanoma remains a highly aggressive malignancy in which immune checkpoint inhibitors (ICIs) have transformed patient outcomes, yet resistance and heterogeneous treatment responses persist. Many clinical assays rely either on tumour informed strategies requiring tissue samples or tumour agnostic tests with limited genomic breadth, restricting their utility in routine practice.  Sensitive biomarkers capable of predicting therapeutic benefit and monitoring disease evolution are urgently needed. Circulating tumour DNA (ctDNA) offers a minimally invasive avenue to assess tumour burden, genomic alterations, and early treatment response in real time. A recent study by Heidrich et al. J. Exp Clin Cancer Res. 44:202 (2025) evaluated a broad, tumour agnostic Next Generation Sequencing (NGS) panel covering 1,114 COSMIC mutations across five melanoma relevant genes (BRAF, NRAS, KRAS, EGFR, and PIK3CA) to determine its clinical value in patients with unresectable stage III/IV melanoma undergoing ICI therapy. Through longitudinal ctDNA analysis of 241 plasma samples from 39 patients, the study examined concordance with tumour tissue, mutation dynamics, associations with clinical response, and prognostic value.

Main Points

• Longitudinal ctDNA profiles were assessed in advanced melanoma patients receiving ICIs by applying a high sensitivity NGS panel capable of detecting a mutant allele frequency of just 0.07%. At baseline, ctDNA was detectable in 64.5% of patients, with BRAFV^600E and NRAS^G12D emerging as the most frequent alterations. BRAF mutations dominated tumour tissue analyses, but plasma profiling revealed additional variants, including KRAS, EGFR, and PIK3CA mutations (absent from archival tumour samples), highlighting the complementary value of real time blood-based testing. Tissue–plasma concordance was moderate (51.6%) and strongly influenced by the time gap between biopsy and blood collection, consistent with tumour evolution under therapeutic pressure.
• Persistent ctDNA detection during therapy correlated with significantly shorter progression free survival (PFS). Patients who remained ctDNA negative throughout treatment had not reached median PFS, indicating durable disease control, whereas those with persistent ctDNA positivity or conversion from negative to positive experienced markedly shorter survival. Notably, even very low level ctDNA persistence (5–10 mutant molecules/mL) was associated with adverse outcomes, underscoring the need for highly sensitive assays to detect minimal residual disease. NRAS and KRAS ctDNA levels assessed during the early phase of treatment were strongly associated with disease progression and outperformed baseline ctDNA measurements in predicting lack of response to immunotherapy.
• Both canonical hotspot and rarer non-hotspot mutations were linked with unfavourable outcomes, indicating that broader genomic coverage is essential for capturing clinically meaningful variants. Mutation patterns also exhibited organ specific associations: KRAS^G12D was enriched in lung metastases, EGFR^G719A in lymph nodes, and BRAF^G469A in liver lesions. These observations suggest possible metastatic tropism, although they remain exploratory.
• Overall, the findings demonstrate that dynamic ctDNA monitoring provides substantially greater prognostic insight than single time point assessment. The tumour agnostic NGS panel successfully identified clinically relevant mutations without requiring tumour tissue, supporting its feasibility and utility in real world melanoma care. Additionally, ctDNA outperformed conventional biomarkers such as LDH and S100 in capturing early treatment resistance, though elevated S100 levels were associated with ctDNA positivity.

Conclusion

This study showed that broad, tumour agnostic ctDNA profiling offers powerful real time insight into molecular tumour dynamics and treatment response in advanced melanoma patients receiving ICIs. Persistent or rising ctDNA during treatment, regardless of mutation type, was strongly associated with shorter progression free survival, whilst NRAS and KRAS levels evaluated during the early phase of treatment served as effective predictors of non-response to immunotherapy. By enabling sensitive, tissue independent monitoring, ctDNA analysis has the potential to guide clinical decision making and should be further explored in larger, prospective interventional trials.

EPISTEM SERVICES

What we do

Epistem is a GCLP-accredited laboratory specialising in biomarker discovery, target identification and personalised medicine to inform drug development and clinical studies. We routinely deliver multi-omic analysis across a broad range of tissues and cells. As part of our integrated GCLP accredited platform, Epistem provides gene expression, whole exome and epigenetic analysis across all species using our microarray, qPCR and Next Generation Sequencing (NGS) platforms.

NGS

We offer a comprehensive ctDNA analysis service using next generation sequencing of targeted panels. Using UMI-based error correction and deep sequencing key cancer associated mutations can be detected in plasma, including single nucleotide variants, small insertions/deletions and some copy number changes.

All sequencing is performed in a GCLP compliant laboratory, supporting clinical trials evaluating solid tumours such as melanoma, lung, colorectal, breast, prostate and other metastatic cancers, that all shed ctDNA into the bloodstream. The service is particularly valuable for tracking treatment response, identifying resistance mutations and monitoring relapse when a tumour is known or suspected.

Oncology

Epistem’s pre-clinical portfolio includes several human cancer models (including breast cancer) enabling robust evaluation of cancer therapies as single agents or in combination with chemo- and/or local radiotherapy. To support advanced analysis of treatment response and immune mechanisms in vitro and ex vivo, we utilise a Cytation 5 live-cell imaging system and a 15-colour Novocyte flow cytometer.