Gene expression profiling beyond Breslow thickness and ulceration for prediction of distant metastases in early-stage melanoma: the population-based Dutch Early-Stage Melanoma (D-ESMEL) study

Purpose Despite their central role in the current staging system, Breslow thickness and ulceration do not fully identify early-stage melanoma patients who will develop distant metastases. We assessed whether gene expression profiling (GEP) improves prediction of distant metastases beyond standard staging factors in early-stage melanoma.

Data were derived from the population-based Dutch Early-Stage Melanoma (D-ESMEL) study, including a matched discovery set of 442 stage I/II melanomas (221 case-control pairs) and a validation cohort of 308 melanomas nested within 5,815 patients. The discovery set was used to identify genes associated with distant metastases, independent of age, sex, Breslow thickness, and ulceration. The validation cohort was partitioned into model development and independent validation subsets. Candidate genes from the discovery set were used to develop and validate a GEP model, evaluated by weighted area under the curve (AUC) and concordance index (C-index).

RNA sequencing succeeded for 356 melanomas in the discovery set, 200 in the model development subset, and 94 melanomas in the independent validation subset. Differential gene expression analyses and modeling identified 558 candidate genes. In the independent validation subset, the GEP model achieved a weighted AUC of 0.77 (95% CI, 0.66-0.86) and weighted C-index of 0.79 (95% CI, 0.69-0.88), comparable to the clinical model based on Breslow thickness and ulceration (weighted AUC 0.82 (95% CI, 0.73-0.90), weighted C-index 0.84 (95% CI, 0.76-0.91)). Integration of GEP with the clinical model did not improve accuracy. Gene set enrichment analyses showed enrichment of proliferative and stress-related pathways.

While GEP captured biologically relevant signals, its predictive accuracy for distant metastases was comparable to that of Breslow thickness and ulceration in a population-based early-stage melanoma cohort. Competing Interest Statement Y.T.C, E.T.V., L.P. and D.M.S.H. are employees and option holders of SkylineDx BV. All other authors declare no competing interests. Funding Statement The project was co-funded by the PPP Allowance made available by Health~Holland (Grant Number: EMCLSH19008), Top Sector Life Sciences & Health. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Erasmus MC Ethics Committee (MEC-2018-1738), the scientific committee Dutch Nationwide Pathology Databank and the Netherlands Cancer Registry. The code of conduct for responsible use of human residual tissue by the Federation of Dutch Medical Scientific Societies was followed. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes Funding sources: The project was co-funded by the PPP Allowance made available by Health∼Holland (Grant Number: EMCLSH19008), Top Sector Life Sciences & Health. Previous presentations: This study was presented in part at the European Association of Dermato-Oncology (EADO) Congress 2025 and the Society for Melanoma Research (SMR) Congress 2025. Preprint availability statement: A preprint of this manuscript is available on medRxiv (doi: 10.1101/2025.11.10.25339906). Authors’ Disclosures of Potential Conflicts of Interest: Y.T.C, E.T.V., L.P. and D.M.S.H. are employees and option holders of SkylineDx BV. All other authors declare no competing interests. Textual changes, number of words cut down. Data Availability The clinical data used in this study were obtained from the Netherlands Cancer Registry (NCR) and are available through the Netherlands Comprehensive Cancer Organization. These data are not publicly available, and restrictions apply to their use. Access may be granted upon reasonable request and with permission from the Netherlands Comprehensive Cancer Organization (request numbers K19.037 and K21.277). The list of 558 candidate genes is provided in Supplementary Table 1. Gene expression data (counts per gene), including sample ID, pair ID, and case-control status will be made available via a public repository (pending).