Distinct molecular profiles associated with early vs late genome instability in cancer

Abstract The temporal order in which cancer hallmark phenotypes emerge can influence tumor behavior and prognosis. In prior work, we used variant allele frequency (VAF) analysis to infer hallmark acquisition order in over 30,000 tumors, identifying two dominant evolutionary trajectories: one characterized by early genome instability (EGI) and the other by late genome instability (LGI). These trajectories were associated with distinct survival outcomes. Here, we investigate the molecular and microenvironmental features that differentiate these two groups, using data from The Cancer Genome Atlas (TCGA). We show that EGI tumors exhibit higher aneuploidy and are enriched for mutational signatures linked to homologous recombination deficiency, particularly SBS3, while LGI tumors show greater prevalence of environmentally associated signatures, including those linked to UV exposure and haloalkanes. Immune profiling revealed that activated mast cells, dendritic cells, and NK cells are enriched in EGI tumors, whereas resting mast cells are strongly associated with LGI tumors. Stemness scores were also significantly higher in EGI tumors, while epithelial-mesenchymal transition (EMT) metrics did not differ between groups. These findings suggest that the timing of genome instability is associated with distinct mutational processes, immune states, and plasticity features. Understanding these differences may provide insight into tumor evolution and offer opportunities for stratified therapeutic intervention based on hallmark ordering.