Fresia Pareja

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

Abstract Purpose: 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. Experimental Design: 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. 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% 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. Conclusions: 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.

Clonal relationship and directionality of progression of synchronous endometrial and ovarian carcinomas in patients with DNA mismatch repair-deficiency associated syndromes

Sporadic synchronous endometrial (ECs) and ovarian cancers (OCs), although clinically considered to be independent primaries, have been shown to be clonally related and likely constitute metastases from each other. We sought to define whether synchronous ECs/OCs in patients with DNA mismatch repair (MMR)-deficiency syndromes would be clonally related. We subjected synchronous ECs/OCs from four patients (LS3-LS6) with clinically confirmed Lynch syndrome (LS) and one patient with constitutional mismatch repair-deficiency syndrome (CMMRD) to massively parallel sequencing targeting 468 cancer-related genes. Somatic mutations, copy number alterations (CNAs), clonal relatedness and clonal decomposition analyses were performed using previously described bioinformatics methods. All synchronous ECs/OCs analyzed were considered independent primaries based on clinicopathologic criteria. Sequencing analysis revealed that the ECs/OCs of three cases (LS2-CMMRD, L3, L4) harbored similar repertoires of somatic mutations and CNAs and were clonally related. In these three cases, a subset of subclonal mutations in the EC became clonal in the OC, suggesting that the EC was likely the substrate from which the OC developed. LS5's EC/OC had distinct mutational profiles but shared specific CNAs. In contrast, LS6's EC/OC harbored distinct somatic mutations and lacked CNAs, consistent with each tumor constituting an independent primary lesion. In LS5 and LS6, PTEN mutations and PTEN loss of protein expression were found to be restricted to the EC. Finally, re-analysis of sequencing data of sporadic synchronous ECs/OCs supported the observations made in the current study that the directionality of progression is likely from the endometrium to the ovary. In conclusion, contrary to sporadic synchronous ECs/OCs, which are almost invariably clonally related, ECs/OCs simultaneously involving the uterus and ovary in LS patients may represent distinct primary tumors. A subset of MMR-deficiency syndrome-related synchronous ECs/OCs, however, may originate from a single primary tumor at variance with their clinical diagnosis, with the endometrium being the likeliest site of origin.

Ovarian cancer mutational processes drive site-specific immune evasion

High-grade serous ovarian cancer is a genomically complex malignancy. Here the authors integrate whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence across 160 tumour sites from 42 patients, revealing that mutational processes shape site-specific immune evasion.