Abstract

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.

MeSH

Cells, Genes, Neoplasms, Therapeutics, Mutation