Elucidating the impact of the immune microenvironment on the evolutionary dynamics of early-stage ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is an aggressive subtype of epithelial ovarian cancer, characterized by high recurrence rates and poorer prognosis compared to other ovarian cancers. It has an elevated mortality worldwide, particularly in Taiwan. Strikingly, OCCC cases with an immune-hot microenvironment exhibited worse survival outcomes, challenging the conventional understanding that 'hot' tumors confer a better prognosis. This paradox prompts our investigation into how the immune-hot microenvironment influences OCCC progression and leads to these counterintuitive survival outcomes In this study, we utilized insertion-deletion (indel) variants within hypermutable polyguanine (polyG) sequences (mutation rate: ~10 -6 -10 -4 ) to reconstruct the phylogeny of a cohort of 20 early-stage OCCC patients. These patients were evenly stratified into immune-hot and immune-cold phenotypes based on their predefined immune status. From a subset of 11 cases (7 immune-hot and 4 immune-cold), we collected a total of 201 samples. Using the published mutation rate for polyG loci (9.43*10 -5 ), we converted the polyG phylogenies into cell-division trees to objectively quantify the lineages. We further examined phylogenetic patterns and clonality through whole-exome sequencing (WES). By comparing evolutionary processes across phylogenies, we identified a distinct subgroup through total cell divisions and MRCA division within the immune-hot tumors that may explain unexpected survival outcomes. We compared three groups: immune-hot Subgroup 1, immune-hot Subgroup 2, and immune-cold tumors. Phylogenetic analysis revealed that Subgroup 1 underwent significantly more cell divisions from the zygote to the MRCA compared to Subgroup 2 and immune-cold tumors (1744 vs. 742 vs. 936, p = 0.012), despite maintaining similar clonal complexity (CV: 26.04% vs. 29.34% vs. 34.18%, p = 0.52). However, Subgroup 1 exhibited a significantly lower pairwise genetic distance compared to the other groups (0.08 vs. 0.10 vs. 0.10, p = 2.73 × 10 -9 ), implying a recent clonal sweep. This finding was further supported by the sample-wise coalescence ratio (CoaR), a metric that quantifies the shared evolutionary history between samples. Subgroup 1 exhibited a significantly higher ratio than Subgroup 2 and immune-cold tumors (0.96 vs. 0.80 vs. 0.80, p = 1.52 × 10 - 46 ), indicating high phylogenetic similarity. Conversely, when quantifying clonal diversity from WES data (using CONIPHER and the Shannon diversity index), Subgroup 1 displayed higher diversity than Subgroup 2 and immune-cold tumors (1.32 vs. 0.70 vs. 0.87, p = 0.012). This discrepancy, high genomic diversity yet low phylogenetic heterogeneity, suggests intense selection pressure in Subgroup 1 that eliminates neo-clones. Finally, we observed that while Subgroup 2 shared a similar clonal structure to immune-cold tumors, it was associated with the worst survival outcomes (p = 0.0019). This suggests that Subgroup 2 may represent a distinct evolutionary trajectory or intermediate state, possessing adaptive features that facilitate aggressive progression. These findings highlight the heterogeneity within immune-hot tumors and offer a potential explanation for why high immune infiltration does not always correlate with favorable survival in OCCC."