Evolution and co-occurrence of PI3K pathway gene mutations in endometrial carcinoma molecular subtypes at the single-cell level.

Purpose: The PI3K pathway is altered in >85% of endometrioid endometrial carcinomas (EECs), with multiple mutations commonly co-occurring. Yet, the therapeutic effects of single-agent PI3K pathway inhibitors have been limited. We used single-cell sequencing to determine whether co-occurring PTEN, PIK3CA, and/or PIK3R1 somatic mutations in EECs stratified by molecular subtype originated through convergent or linear evolution.

Methods: Banked frozen EECs with co-occurring PI3K pathway mutations of no specific molecular profile (NSMP; n=5), mismatch repair-deficient (MMRd;n=3), and POLE (n=3) subtype were selected for single-nucleus DNA sequencing targeting hotspot variants of 64 cancer-related genes and the PTEN, PIK3R1 and PIK3CA coding sequences. EEC cell lines and non-malignant samples were used to define error rates and filter false-positive calls.

Results: Single-nucleus analyses (n=50,009 cells) revealed that in NSMP EECs, the co-occurring PIK3CA, PIK3R1, and/or PTEN mutations affected nearly all cells through linear evolution. MMRd EECs displayed higher levels of genetic heterogeneity, harboring PI3K pathway gene mutations in subsets of cells ranging from 3.9%-96%. POLE EECs had the highest level of clonal diversity and harbored multiple minor subclonal structures in all cases, through convergent evolution. We found a clear distinction between nearly clonal PI3K pathway gene alterations (>95%) and multiple minor, mutually-exclusive subclones only affecting 1.4%-27% of the tumor cells sequenced.

Conclusions: Our exploratory, hypothesis-generating analysis suggest that PI3K pathway alterations evolve distinctly in MMRd/POLE compared to NSMP EECs, which may have therapeutic consequences. Further studies on the signaling output and PI3K pathway inhibitor response in EECs with subclonal PI3K pathway alterations are warranted.