Supplementary Figure S4 from Evolution and Co-occurrence of PI3K Pathway Gene Mutations in Endometrial Carcinoma Molecular Subtypes at the Single-Cell Level

Da Cruz Paula, Arnaud; Zhu, Yingjie; Brown, David N.; Issa Bhaloo, Shirin; Pareja, Fresia; Hoang, Timothy J.; Green, Hunter; Basili, Thais; Dopeso, Higinio; Selenica, Pier; da Silva, Edaise M.; Riaz, Nadeem; Chandarlapaty, Sarat; Abu-Rustum, Nadeem R.; Reis-Filho, Jorge S.; Weigelt, Britta

doi:10.1158/1078-0432.30617839

Supplementary Figure S4 from Evolution and Co-occurrence of PI3K Pathway Gene Mutations in Endometrial Carcinoma Molecular Subtypes at the Single-Cell Level

https://doi.org/10.1158/1078-0432.30617839

journal contribution

posted on 2025-11-14, 08:21 authored by Arnaud Da Cruz Paula, Yingjie Zhu, David N. Brown, Shirin Issa Bhaloo, Fresia Pareja, Timothy J. Hoang, Hunter Green, Thais Basili, Higinio Dopeso, Pier Selenica, Edaise M. da Silva, Nadeem Riaz, Sarat Chandarlapaty, Nadeem R. Abu-Rustum, Jorge S. Reis-Filho, Britta Weigelt

<p>Supplementary Figure S4. Single-nucleus genotyping and clonal architecture analysis of endometrioid endometrial carcinomas of different molecular subtypes. A, Representative micrograph of hematoxylin-and-eosin (H&E)-stained endometrioid endometrial cancer EEC132 of CN-low/NSMP molecular subtype and single-nucleus sequencing data information, including allele dropout (ADO) rate and number of tumor cells found (left). Single-nucleus genotyping analysis of each variant detected in our single-nucleus data (middle left). Cells are shown in columns and variants in rows. Clonal evolution phylogenetic three depicting the emergence of PI3K-derived clones in tumor evolution (middle right). Alignment of sequencing reads showing the two PTEN hotspot variants detected in bulk and matched single-nucleus sequencing data (right). B, Representative H&E micrograph of MMRd/MSI-high EEC87 and single-nucleus sequencing data information, including ADO rate and number of tumor cells found (left). Single-nucleus genotyping analysis of each variant detected in our single-nucleus data, (middle). Cells are shown in columns and variants in rows. Fish plot showing the emergence of the subclones in tumor evolution (right). The dotted lines depict the type of variants that constitutes each of the subclones. Clonal evolution phylogenetic three depicting the emergence of PI3K-derived clones in tumor evolution (bottom right). C, Representative H&E micrograph of <i>POLE</i>-mutant EEC136 and single-nucleus sequencing data information, including ADO rate and number of cells found (left). Single-nucleus sequencing analysis of each variant detected in our single-nucleus data (middle). Cells are shown in columns and variants in rows. Fish plot showing the emergence of the subclones in tumor evolution (right). The dotted lines depict the type of variants that constitutes each of the subclones. Clonal evolution phylogenetic three depicting the emergence of PI3K-derived clones in tumor evolution (bottom right). The mutation status is color-coded according to the legend. ADO, allele dropout; CN, copy number; EEC, endometrioid endometrial carcinoma; Hist., histologic; MMRd, mismatch repair-deficient; Mol., molecular; MSI, microsatellite instability; NSMP, no specific molecular profile.</p>

Funding

National Institutes of Health (NIH)

Cycle for Survival

Breast Cancer Research Foundation (BCRF)

History

Related Materials

1.
DOI - Is supplement to Evolution and Co-occurrence of PI3K Pathway Gene Mutations in Endometrial Carcinoma Molecular Subtypes at the Single-Cell Level

ARTICLE ABSTRACT

The PI3K pathway is altered in >85% of endometrioid endometrial carcinomas (EEC), 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. 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) subtypes 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 nonmalignant samples were used to define error rates and filter false-positive calls. 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% to 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% to 27% of the tumor cells sequenced. Our exploratory, hypothesis-generating analysis suggests that PI3K pathway alterations evolve distinctly in MMRd/POLE compared with 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.