Endometrial polyps are non-neoplastic but harbor epithelial mutations in endometrial cancer drivers at low allelic frequencies.

Endometrial polyps (EMPs) are common exophytic masses associated with abnormal uterine bleeding and infertility. Unlike normal endometrium, which is cyclically shed, EMPs persist over ovulatory cycles and after the menopause. Despite their usual classification as benign entities, EMPs are paradoxically associated with endometrial carcinomas of diverse histologic subtypes, which frequently arise within EMPs. The etiology and potential origins of EMPs as clonally-derived neoplasms are uncertain, but previous investigations suggested that EMPs are neoplasms of stromal origin driven by recurring chromosomal rearrangements. To better define benign EMPs at the molecular genetic level, we analyzed individual EMPs from 31 women who underwent hysterectomy for benign indications. The 31 EMPs were subjected to comprehensive genomic profiling by exome sequencing of a large panel of tumor-related genes including oncogenes, tumor suppressors, and chromosomal translocation partners. There were no recurring chromosomal rearrangements, and copy-number analyses did not reveal evidence of significant chromosome-level events. Surprisingly, there was a high incidence of single nucleotide variants corresponding to classic oncogenic drivers (i.e., definitive cancer drivers). The spectrum of known oncogenic driver events matched that of endometrial cancers more closely than any other common cancer. Further analyses including laser-capture microdissection showed that these mutations were present in the epithelial compartment at low allelic frequencies. These results establish a link between EMPs and the acquisition of endometrial cancer driver mutations. Based on these findings, we propose a model where the association between EMPs and endometrial cancer is explained by the age-related accumulation of endometrial cancer drivers in a protected environment that-unlike normal endometrium-is not subject to cyclical shedding.

Serial genomic analysis of endometrium supports the existence of histologically indistinct endometrial cancer precursors.

The endometrium is unique as an accessible anatomic location that can be repeatedly biopsied and where diagnostic biopsies do not extirpate neoplastic lesions. We exploited these features to retrospectively characterize serial genomic alterations along the precancer/cancer continuum in individual women. Cases were selected based on (1) endometrial cancer diagnosis/hysterectomy and (2) preceding serial endometrial biopsies including for some patients an early biopsy before a precancer histologic diagnosis. A comprehensive panel was designed for endometrial cancer genes. Formalin-fixed, paraffin-embedded specimens for each cancer, preceding biopsies, and matched germline samples were subjected to barcoded high-throughput sequencing to identify mutations and track their origin and allelic frequency progression. In total, 92 samples from 21 patients were analyzed, providing an opportunity for new insights into early endometrial cancer progression. Definitive invasive endometrial cancers exhibited expected mutational spectra, and canonical driver mutations were detectable in preceding biopsies. Notably, ≥1 cancer mutations were detected prior to the histopathologic diagnosis of an endometrial precancer in the majority of patients. In 18/21 cases, ≥1 mutations were confirmed by abnormal protein levels or subcellular localization by immunohistochemistry, confirming genomic data and providing unique views of histologic correlates. In 19 control endometria, mutation counts were lower, with a lack of canonical endometrial cancer hotspot mutations. Our study documents the existence of endometrial lesions that are histologically indistinct but are bona fide endometrial cancer precursors. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Fbxw7 is a driver of uterine carcinosarcoma by promoting epithelial-mesenchymal transition.

Uterine carcinosarcoma is an aggressive variant of endometrial carcinoma characterized by unusual histologic features including discrete malignant epithelial and mesenchymal components (carcinoma and sarcoma). Recent studies have confirmed a monoclonal origin, and comprehensive genomic characterizations have identified mutations such as Tp53 and Pten However, the biological origins and specific combination of driver events underpinning uterine carcinosarcoma have remained mysterious. Here, we explored the role of the tumor suppressor Fbxw7 in endometrial cancer through defined genetic model systems. Inactivation of Fbxw7 and Pten resulted in the formation of precancerous lesions (endometrioid intraepithelial neoplasia) and well-differentiated endometrioid adenocarcinomas. Surprisingly, all adenocarcinomas eventually developed into definitive uterine carcinosarcomas with carcinomatous and sarcomatous elements including heterologous differentiation, yielding a faithful genetically engineered model of this cancer type. Genomic analysis showed that most tumors spontaneously acquired Trp53 mutations, pointing to a triad of pathways (p53, PI3K, and Fbxw7) as the critical combination underpinning uterine carcinosarcoma, and to Fbxw7 as a key driver of this enigmatic endometrial cancer type. Lineage tracing provided formal genetic proof that the uterine carcinosarcoma cell of origin is an endometrial epithelial cell that subsequently undergoes a prominent epithelial-mesenchymal transition underlying the attainment of a highly invasive phenotype specifically driven by Fbxw7.

FOXA2 suppresses endometrial carcinogenesis and epithelial-mesenchymal transition by regulating enhancer activity.

FOXA2 encodes a transcription factor mutated in 10% of endometrial cancers (ECs), with a higher mutation rate in aggressive variants. FOXA2 has essential roles in embryonic and uterine development. However, FOXA2's role in EC is incompletely understood. Functional investigations using human and mouse EC cell lines revealed that FOXA2 controls endometrial epithelial gene expression programs regulating cell proliferation, adhesion, and endometrial-epithelial transition. In live animals, conditional inactivation of Foxa2 or Pten alone in endometrial epithelium did not result in ECs, but simultaneous inactivation of both genes resulted in lethal ECs with complete penetrance, establishing potent synergism between Foxa2 and PI3K signaling. Studies in tumor-derived cell lines and organoids highlighted additional invasion and cell growth phenotypes associated with malignant transformation and identified key mediators, including Myc and Cdh1. Transcriptome and cistrome analyses revealed that FOXA2 broadly controls gene expression programs through modification of enhancer activity in addition to regulating specific target genes, rationalizing its tumor suppressor functions. By integrating results from our cell lines, organoids, animal models, and patient data, our findings demonstrated that FOXA2 is an endometrial tumor suppressor associated with aggressive disease and with shared commonalities among its roles in endometrial function and carcinogenesis.

Reliable Identification of Endometrial Precancers Through Combined Pax2, ß-Catenin, and Pten Immunohistochemistry.

The diagnosis of endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (AH/EIN) remains challenging and subjective in some cases, with variable histologic criteria and differences of opinion among gynecologic pathologists, potentially leading to under/overtreatment. There has been growing interest in the use of specific immunohistochemical markers as adjuncts in AH/EIN diagnosis. For example, the World Health Organization 2020 Classification specifies that loss of Pten, Pax2, or mismatch repair proteins are desirable diagnostic criteria. Other markers, most notably ß-catenin and Arid1a, are also aberrantly expressed in some AH/EIN. However, the performance of some markers individually-and more importantly as a group-has not been rigorously explored, raising questions as to which marker(s) or combination(s) is the most effective in practice. Formalin-fixed paraffin-embedded tissue sections from AH/EIN cases (n=111) were analyzed by immunohistochemistry for 6 markers: Pax2, Pten, Mlh1, ß-catenin, Arid1a, and p53. Aberrant expression was tabulated for each case and marker. An additional set of normal endometria (n=79) was also analyzed to define optimal diagnostic criteria for marker aberrance. The performance characteristics of each marker, the entire panel, and subsets thereof were quantitatively and statistically analyzed. In order of number of cases detected, the most frequently aberrant markers in AH/EIN were Pax2 (81.1% of cases), Pten (50.5%), ß-catenin (47.7%), Arid1a (7.2%), Mlh1 (4.5%), and p53 (2.7%). The majority of cases showed aberrant expression of ≥2 markers. All 6 markers together identified 92.8% of cases. Arid1a, Mlh1, and p53 were robust and readily scored markers, but all cases showing aberrant expression of these 3 markers were also detected by Pax2, Pten, or ß-catenin. A focused panel of only 3 markers (Pax2, Pten, and ß-catenin) showed optimal performance characteristics as a diagnostic adjunct in the histopathologic diagnosis of AH/EIN. Use of this panel is practicable and robust, with at least 1 of the 3 markers being aberrant in 92.8% of AH/EIN.

A PoleP286R mouse model of endometrial cancer recapitulates high mutational burden and immunotherapy response.

Cancer is instigated by mutator phenotypes, including deficient mismatch repair and p53-associated chromosomal instability. More recently, a distinct class of cancers was identified with unusually high mutational loads due to heterozygous amino acid substitutions (most commonly P286R) in the proofreading domain of DNA polymerase ε, the leading strand replicase encoded by POLE. Immunotherapy has revolutionized cancer treatment, but new model systems are needed to recapitulate high mutational burdens characterizing human cancers and permit study of mechanisms underlying clinical responses. Here, we show that activation of a conditional LSL-PoleP286R allele in endometrium is sufficient to elicit in all animals endometrial cancers closely resembling their human counterparts, including very high mutational burden. Diverse investigations uncovered potentially novel aspects of Pole-driven tumorigenesis, including secondary p53 mutations associated with tetraploidy, and cooperation with defective mismatch repair through inactivation of Msh2. Most significantly, there were robust antitumor immune responses with increased T cell infiltrates, accelerated tumor growth following T cell depletion, and unfailing clinical regression following immune checkpoint therapy. This model predicts that human POLE-driven cancers will prove consistently responsive to immune checkpoint blockade. Furthermore, this is a robust and efficient approach to recapitulate in mice the high mutational burdens and immune responses characterizing human cancers.

Restoring transcription factor HoxA5 expression inhibits the growth of experimental hemangiomas in the brain.

Hemangiomas are angiogenesis-dependent benign vascular tumors that can rupture and cause intracranial hemorrhages. We previously showed that the transcription factor homeobox A5 (HoxA5), which is absent in activated angiogenic endothelial cells can block angiogenesis. Here, we investigated whether restoring expression of HoxA5 blocks hemangioma growth by transplanting mouse hemangioendothelioma endothelial cells (EOMA) or HoxA5-expressing EOMA cells into the brains of mice. The EOMA cells induced brain hemangiomas characterized by large cystlike spaces lined by thin walls of endothelial cells surrounded by scant smooth muscle cells. When HoxA5-expressing EOMA cells were injected, lesion volumes were reduced between 5- and 20-fold compared with the EOMA control group (p < 0.05). Restoration of HoxA5 was associated with increased thrombospondin-2, which inhibits angiogenesis and reduced hypoxia-inducible factor 1alpha expression. These data suggest that restoring HoxA5 can attenuate experimental brain hemangioma development.

Meningioma transcript profiles reveal deregulated Notch signaling pathway.

Meningiomas constitute the second most common central nervous system tumor, and yet relatively little is known about the molecular events that are important for the pathogenesis and malignant progression of these tumors. We have used serial analysis of gene expression to compare the transcriptomes of nonneoplastic meninges and meningiomas of all malignancy grades. A novel finding from this screen is the induction of three components of the Notch signaling pathway: the transcription factor, hairy and enhancer of Split1 (HES1) and two members of the Groucho/transducin-like enhancer of Split family of corepressors, TLE2 and TLE3. TLE corepressors interact and modulate the activity of a wide range of transcriptional regulatory systems, one of which is HES1. We have shown that the transcript and protein levels of HES1, the Notch2 and Notch1 receptors and the Jagged1 ligand are induced in meningiomas of all grades, whereas induction of TLE2 and TLE3 occurs specifically in higher-grade meningiomas. Meningioma cell lines express components of the Notch signaling pathway and an inhibitor of this pathway suppresses meningioma cell survival. These results suggest that deregulated expression of the Notch pathway is a critical event in meningioma pathogenesis and that modulation of this and potentially other signaling pathways by TLE corepressors leads to a more malignant phenotype.

Insights into a CRM1-mediated RNA-nuclear export pathway in Trypanosoma cruzi.

Nuclear export and import of proteins and RNAs is a regulated process that permits the control of protein expression during cell development and differentiation. In all eukaryotic organisms transport of proteins to specific cellular compartments requires specific signaling sequences. Proteins that shuttle between nucleus and cytoplasm bear nuclear localization signals (NLS) and/or nuclear export signals (NES) and some of them can carry mRNAs, as part of shuttling ribonucleoprotein complexes. In this work we describe in the protozoan parasite Trypanosoma cruzi, a CRM1/exportin1 nuclear export factor named TcCRM1. This protein contains the conserved central region (CCR) that interacts with NES sequences present within cargo molecules, and the Cys residue involved in covalent binding to the Streptomyces metabolite leptomycin B (LMB). By subcellular fractionation we show that TcCRM1, a protein of about 117 kDa, has nuclear localization. We also demonstrate that LMB inhibits the replication of T. cruzi in a dose-dependent manner. In situ hybridization experiments performed with a Texas red-coupled oligo(dT) probe revealed that LMB produced a partial short-term accumulation of a poly(A)+RNA subset in the nucleus. Some mRNAs such as HSP70, TcUBP2/1 and TcPABP1 are reduced or disappeared from the cytoplasm of LMB treated cells. In sharp contrast with metazoans, no effect was observed on two U snRNAs subcellular localization, implying that a different export route might exist for these RNAs in trypanosomes.

Characterization of farnesylated protein tyrosine phosphatase TcPRL-1 from Trypanosoma cruzi.

Protein tyrosine kinases and phosphatases play important roles in the regulation of cell growth, development, and differentiation. We report here the identification in Trypanosoma cruzi of a gene (TcPRL-1) encoding a protein tyrosine phosphatase. The predicted protein (TcPRL-1) shares ca. 35% identity with the mammalian protein tyrosine phosphatase known as phosphatase of regenerating liver 1 (PRL-1). Four copies of this protein tyrosine phosphatase are present in the T. cruzi genome, and Northern blot assays showed a transcript of approximately 750 bases. TcPRL-1 was detected by Western blot analysis only in amastigote extracts as a 21-kDa protein. TcPRL-1 was expressed in Escherichia coli, and its phosphatase activity was determined by using p-nitrophenylphosphate and a phosphorylated protein as substrates. In contrast to other PRLs, TcPRL-1 activity was not affected by pentamidine, and it was inhibited by very low concentrations of o-vanadate. TcPRL-1 has a C-terminal CAAX motif (CAVM) and is farnesylated in vitro by T. cruzi epimastigote extracts and in vivo according to the transfection results. After transfection of T. cruzi with a vector that expresses TcPRL-1 as a C-terminal fusion to green fluorescent protein, GFP-TcPRL-1 was detected in the endocytic pathway of epimastigotes, amastigotes, and trypomastigotes by colocalization with cruzipain and concanavalin A. Interestingly, a mutant form without the CAAX motif localized to the cytoplasm, in contrast to its mammalian counterparts that localize to the nucleus. The results of these studies on TcPRL-1 reveal that, even though the animal and parasite PRLs share similar kinetic properties, their susceptibilities to inhibitors, as well as their localization, are distinct, implying that they may be involved in different cellular processes.

gp63 homologues in Trypanosoma cruzi: surface antigens with metalloprotease activity and a possible role in host cell infection.

gp63 is a highly abundant glycosylphosphatidylinositol (GPI)-anchored membrane protein expressed predominantly in the promastigote but also in the amastigote stage of Leishmania species. In Leishmania spp., gp63 has been implicated in a number of steps in establishment of infection. Here we demonstrate that Trypanosoma cruzi, the etiological agent of Chagas' disease, has a family of gp63 genes composed of multiple groups. Two of these groups, Tcgp63-I and -II, are present as high-copy-number genes. The genomic organization and mRNA expression pattern were specific for each group. Tcgp63-I was widely expressed, while the Tcgp63-II group was scarcely detected in Northern blots, even though it is well represented in the T. cruzi genome. Western blots using sera directed against a synthetic peptide indicated that the Tcgp63-I group produced proteins of approximately 78 kDa, differentially expressed during the life cycle. Immunofluorescence staining and phosphatidylinositol-specific phospholipase C digestion confirmed that Tcgp63-I group members are surface proteins bound to the membrane by a GPI anchor. We also demonstrate the presence of metalloprotease activity which is attributable, at least in part, to Tcgp63-I group. Since antibodies against Tcgp63-I partially blocked infection of Vero cells by trypomastigotes, a possible role for this group in infection is suggested.