Edaise M. da Silva

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.

Genomic profiling of primary and recurrent adult granulosa cell tumors of the ovary

Adult-type granulosa cell tumor (aGCT) is a rare malignant ovarian sex cord-stromal tumor, harboring recurrent FOXL2 c.C402G/p.C134W hotspot mutations in 97% of cases. These tumors are considered to have a favorable prognosis, however aGCTs have a tendency for local spread and late recurrences, which are associated with poor survival rates. We sought to determine the genetic alterations associated with aGCT disease progression. We subjected primary non-recurrent aGCTs (n = 7), primary aGCTs that subsequently recurred (n = 9) and their matched recurrences (n = 9), and aGCT recurrences without matched primary tumors (n = 10) to targeted massively parallel sequencing of ≥410 cancer-related genes. In addition, three primary non-recurrent aGCTs and nine aGCT recurrences were subjected to FOXL2 and TERT promoter Sanger sequencing analysis. All aGCTs harbored the FOXL2 C134W hotspot mutation. TERT promoter mutations were found to be significantly more frequent in recurrent (18/28, 64%) than primary aGCTs (5/19, 26%, p = 0.017). In addition, mutations affecting TP53, MED12, and TET2 were restricted to aGCT recurrences. Pathway annotation of altered genes demonstrated that aGCT recurrences displayed an enrichment for genetic alterations affecting cell cycle pathway-related genes. Analysis of paired primary and recurrent aGCTs revealed that TERT promoter mutations were either present in both primary tumors and matched recurrences or were restricted to the recurrence and absent in the respective primary aGCT. Clonal composition analysis of these paired samples further revealed that aGCTs display intra-tumor genetic heterogeneity and harbor multiple clones at diagnosis and relapse. We observed that in a subset of cases, recurrences acquired additional genetic alterations not present in primary aGCTs, including TERT, MED12, and TP53 mutations and CDKN2A/B homozygous deletions. Albeit harboring relatively simple genomes, our data provide evidence to suggest that aGCTs are genetically heterogeneous tumors and that TERT promoter mutations and/or genetic alterations affecting other cell cycle-related genes may be associated with disease progression and recurrences.

Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

AbstractSclerosing stromal tumor (SST) of the ovary is a rare type of sex cord-stromal tumor (SCST), whose genetic underpinning is currently unknown. Here, using whole-exome, targeted capture and RNA-sequencing, we report recurrent FHL2-GLI2 fusion genes in 65% (17/26) of SSTs and other GLI2 rearrangements in additional 15% (4/26) SSTs, none of which are detected in other types of SCSTs (n = 48) or common cancer types (n = 9,950). The FHL2-GLI2 fusions result in transcriptomic activation of the Sonic Hedgehog (SHH) pathway in SSTs. Expression of the FHL2-GLI2 fusion in vitro leads to the acquisition of phenotypic characteristics of SSTs, increased proliferation, migration and colony formation, and SHH pathway activation. Targeted inhibition of the SHH pathway results in reversal of these oncogenic properties, indicating its role in the pathogenesis of SSTs. Our results demonstrate that the FHL2-GLI2 fusion is likely the oncogenic driver of SSTs, defining a genotypic–phenotypic correlation in ovarian neoplasms.

Genomic characterization of small cell carcinomas of the uterine cervix

Small cell carcinoma (SCC) of the uterine cervix is a rare and aggressive form of neuroendocrine carcinoma, which resembles small cell lung cancer (SCLC) in its histology and poor survival rate. Here, we sought to define the genetic underpinning of SCCs of the uterine cervix and compare their mutational profiles with those of human papillomavirus (HPV)‐positive head and neck squamous cell carcinomas, HPV‐positive cervical carcinomas, and SCLCs using publicly available data. Using a combination of whole‐exome and targeted massively parallel sequencing, we found that the nine uterine cervix SCCs, which were HPV18‐positive (n = 8) or HPV16‐positive (n = 1), harbored a low mutation burden, few copy number alterations, and other than TP53 in two cases no recurrently mutated genes. The majority of mutations were likely passenger missense mutations, and only few affected previously described cancer‐related genes. Using RNA‐sequencing, we identified putative viral integration sites on 18q12.3 and on 8p22 in two SCCs of the uterine cervix. The overall nonsilent mutation rate of uterine cervix SCCs was significantly lower than that of SCLCs, HPV‐driven cervical adeno‐ and squamous cell carcinomas, or HPV‐positive head and neck squamous cell carcinomas. Unlike SCLCs, which are reported to harbor almost universal TP53 and RB1 mutations and a dominant tobacco smoke‐related signature 4, uterine cervix SCCs rarely harbored mutations affecting these genes (2/9, 22% TP53; 0% RB1) and displayed a dominant aging (67%) or APOBEC mutational signature (17%), akin to HPV‐driven cancers, including cervical adeno‐ and squamous cell carcinomas and head and neck squamous cell carcinomas. Taken together, in contrast to SCLCs, which are characterized by highly recurrent TP53 and RB1 alterations, uterine cervix SCCs were positive for HPV leading to inactivation of the suppressors p53 and RB, suggesting that these SCCs are convergent phenotypes.