{"paper_id":"0ce88323-6ab7-4faf-b339-037b2c07acf1","body_text":"Endometrial stromal tumors (EST) represent rare uterine neoplasms of mesenchymal origin [ 1 ]. The 2014 WHO classification of tumors of the female reproductive tract distinguishes four categories of EST: endometrial stromal nodule (ESN), low‐grade endometrial stromal sarcoma (LGESS), high‐grade endometrial stromal sarcoma (HGESS), and undifferentiated uterine sarcoma (UUS) [ 2 ]. While ESN and LGESS histologically resemble stromal cells of the proliferating endometrium and harbor recurrent chromosomal translocations most frequently associated with a  JAZF1‐SUZ12  gene fusion, UUS comprises endometrial and myometrial sarcomas which lack specific mesenchymal differentiation and are molecularly heterogenous [ 3 ,  4 ]. As defined by the 2014 WHO classification, HGESS harbor a t(10;17)(q22;p13) chromosomal translocation resulting in a  YWHAE‐NUTM2  fusion [ 2 ,  5 ]. Such tumors represent a clinically more aggressive entity with patients diagnosed at higher stages and more likely to die of disease when compared to LGESS [ 6 ].\nRecently, a rare subtype of ESS with high‐grade features and  BCOR  alterations, caused by either a gene fusion between  BCOR  and  ZC3H7B  or a mutually exclusive somatic internal tandem duplication (ITD) of exon 15 of  BCOR , has been described (HGESS‐BCOR) [ 7 ,  8 ]. Although such  BCOR  alterations may well be the molecular driver in these tumors, little is known about their biology, or about potential cooperative and co‐occurring genetic events.\nWe recently identified a case of HGESS‐BCOR that carried an amplification of the 12q15 region involving the  MDM2  locus. This observation prompted us to compile a multicenter cohort to investigate  MDM2  amplification in HGESS‐BCOR.\n\nA study cohort including HGESS‐BCOR, LGESS, HGESS, uterine tumors resembling ovarian sex cord tumors (UTROSCT), uterine leiomyomas (ULMO), and uterine leiomyosarcomas (ULMS) was collected from the referral center archives of two of the authors (DS and FK), the Department of Pathology, University of Heidelberg, and the KK Women's and Children's Hospital, Singapore. All cases were subject to expert pathology review including molecular pathology [ 9 ]. Fusion status of HGESS‐BCOR has previously been reported in part [ 10 ,  11 ]. This study was performed in accordance with the ethical standards of the institutional research committee and the Declaration of Helsinki.\nDNA of all tumors was extracted from formalin‐fixed paraffin‐embedded (FFPE) tissue samples. Extracted DNA was quantified using the QuantiFast SYBR Green PCR Kit (Qiagen, Duesseldorf, NW, Germany).\nA total of >100 ng DNA was available for array‐based DNA methylation analysis in all cases. Samples were analyzed using the Illumina Infinium HumanMethylation450 (450k) or EPIC (850k) BeadChip (Illumina, San Diego, IL, USA), according to the manufacturer's instructions at the Genomics and Proteomics Core Facility of the German Cancer Research Center (DKFZ), Heidelberg. DNA methylation data were normalized by performing background correction and dye bias correction as previously described [ 11 ]. Probes targeting sex chromosomes, probes containing multiple single nucleotide polymorphisms, and those which could not be uniquely mapped, were removed.\nMDM2 ‐specific fluorescence  in situ  hybridization (FISH) analysis was performed on whole tissue sections using the ZytoLight® SPEC MDM2/CEN 12 Dual Color Probe (ZytoVision GmbH, Bremerhaven, Germany) as previously described [ 12 ]. Amplification of  MDM2  was defined as an  MDM2 /centromere 12 (CEN12) ratio ≥2.0 or an average number of  MDM2  signals per tumor cell nucleus ≥6 or large clusters of MDM2 signals in ≥10% of tumor cells.\nFour micrometer sections were cut and mounted on StarFrost Advanced Adhesive slides (Engelbrecht, Kassel, Germany) followed by heat induced antigen retrieval in high pH buffer. MDM2 immunohistochemistry was performed using a monoclonal mouse antibody (dilution 1:100, clone IF2, Invitrogen by Thermo Fisher Scientific Inc., Waltham, MA, USA) as previously described [ 12 ]. Specimens were examined according to their nuclear staining for MDM2.\n\nFive HGESS‐BCOR were available for analysis. All tumors showed a hypercellular appearance with haphazard fascicular architecture. Tumor cells were spindled with irregular nuclear contours and an even chromatin pattern (Figure  1A,B ). More notable atypia was only seen in one tumor (case 2; Figure  1C ). Island‐like myxoid stromal change was present in three tumors (cases 1, 3, and 4; Figure  1D ). RNA‐seq analysis identified a  ZC3H7B‐BCOR  gene fusion in four cases (cases 1, 2, 4, and 5). In case 3, the fusion detection algorithm identified a rearrangement between  BCOR  and the  LPP  gene, including RNA reads spanning parts of both genes. This event, however, appears to result in overexpression of a C‐terminally truncated BCOR protein, without generation of a fusion protein (no coding part of LPP is included). Thus, while RNA‐seq data support the presence of a  BCOR  alteration, the precise functional consequences of this aberration remain unclear. Clinicopathological data of all HGESS‐BCOR cases are summarized in Table  1 .\nHistologic features of HGESS‐BCOR: sarcomatous proliferation of atypical and spindled neoplastic tumor cells with fascicular architecture (A and B), notable atypia (C) and prominent myxoid stroma (D). H&E stain; bar equals 50 μm.\nClinicopathological data of HGESS‐BCOR cases\nCopy number analysis of HGESS‐BCOR showed an amplification of the 12q15 region involving the  MDM2  locus in all five cases (Figure  2A ). For subsequent MDM2 validation analyses sufficient material was only available for three tumors (cases 1–3). FISH and IHC confirmed the amplification of  MDM2  and overexpression (Figure  2B ) of MDM2 in all three tumors.  MDM2  amplification by FISH was present diffusely across the tumors analyzed. In addition to an  MDM2  amplification, copy number analyses also revealed amplification of  CDK4  in three tumors (cases 1–3) and a deletion of  CDKN2A  in one tumor (case 5).\nMDM2  amplification in HGESS‐BCOR: (A) copy number profiles (cases 1–5). (B)  MDM2  FISH (MDM2: green, CEN12: red) and immunohistochemical staining for MDM2 (cases 1–3). Bar equals 50 μm.\nNext, we analyzed copy number profiles of potential differential diagnoses of HGESS‐BCOR including 17 LGESS, 6 HGESS with  YWHAE‐NUTM2  gene fusion, 16 UTROSCT, 7 ULMO, and 8 ULMS. None of these cases carried an amplification of the 12q15 region involving the  MDM2  locus (Table  2 ).\n12q15 ( MDM2 ) amplification status of mesenchymal uterine neoplasms including LGESS, HGESS‐BCOR, HGESS with  YWHAE ‐rearrangement, UTROSCT, ULM, and ULMS\n\nHGESS‐BCOR represents a new subtype in the spectrum of EST, defined by genetic alterations involving  BCOR . ESS with  BCOR  alteration show a greater degree of atypia when compared to LGESS, and available clinical data suggest an aggressive clinical course similar to that of classical HGESS [ 13 ]. Thus, it has been proposed to place such tumors in the HGESS category. We have recently shown  BCOR ‐ and  YWHAE ‐rearranged HGESS to share similar DNA methylation profiles, distinct from LGESS and other high‐grade uterine sarcomas such as ULMS, further supporting the proposed classification [ 11 ]. In the current study, we show HGESS‐BCOR to harbor  MDM2  amplifications, while all HGESS with  YWHAE‐NUTM2  gene fusion in our series were  MDM2 ‐balanced.\nMDM2 is the primary negative regulator of p53 and is overexpressed in cancers,  e.g . certain subtypes of sarcomas [ 14 ]. Mechanistically, MDM2 overexpression functions as a powerful oncogene by negatively regulating  TP53  transcriptional activity and therefore has been suggested as a druggable target for small molecule inhibitors [ 15 ]. Given the above, p53 immunohistochemistry in a subset of HGESS‐BCOR of our cohort did not exhibit strong or diffuse p53 expression ( n  = 3). In line with this staining pattern, additional DNA sequencing did not identify pathogenic  TP53  mutations in any HGESS‐BCOR of our series ( n  = 5; data not shown). Thus, detecting  MDM2  amplification in HGESS‐BCOR might have clinical implications in affected patients. To date, therapeutic strategies in cases of high‐grade uterine sarcoma are usually limited to surgery and radio‐/chemotherapy, and prognosis is generally dismal [ 16 ]. Targeting MDM2 might therefore be a promising way to extend the spectrum of therapeutic options for this aggressive neoplasm.\nMDM2 has previously been investigated in EST ( n  = 43) including ESN, LGESS, HGESS with  YWHAE ‐rearrangement, and UUS [ 17 ]. Schoolmeester  et al  reported  MDM2  amplification in two of 43 (5%) tumors, both of which showed an adverse clinical course. One of the latter tumors showed morphological features consistent with LGESS, and it was reported to harbor a  JAZF1‐ rearrangement. The second tumor harboring a  MDM2  amplification had high‐grade morphology, polysomy of  JAZF1 ,  PHF1 , and  YWHAE  but no rearrangements were reported, leading to a diagnosis of UUS. While the latter data imply that  MDM2  amplification is also present in a subset of LGESS and UUS, we did not identify copy number gains of  MDM2  in any other mesenchymal uterine tumor of our series including 17 LGESS. Interestingly, a recent study suggests that UUS often represents under‐recognized HGESS including tumors with  BCOR  alteration [ 18 ]. Moreover, ESS may rarely present as a primary intrabdominal soft tissue tumor or arise at distant sites in extragenital endometriosis. This is important to know when encountering undifferentiated mesenteric or retroperitoneal soft tissue tumors. While  MDM2  amplification in such tumors usually supports a diagnosis of dedifferentiated liposarcoma, HGESS‐BCOR should also be considered in the differential diagnoses in such cases [ 19 ]. Notably, MDM2 positivity by immunohistochemistry has been reported in cases of uterine leiomyosarcoma and primary liposarcoma of the uterus [ 20 ,  21 ]. However, to date no systematic analysis of  MDM2  amplification has been performed in such cases.\nAs a limitation of our study, it is important to mention that our study cohort did not include cases of Müllerian adenosarcoma, a lesion known to harbor  MDM2  amplifications in up to 28% of cases [ 22 ]. Further, our study did not investigate tumors with  BCOR‐ ITD, which are exceedingly rare with only few cases reported in the literature to date [ 8 ]. Thus, future studies of larger cohorts of mesenchymal uterine neoplasms are needed to confirm, and to expand on, our results.\nIn conclusion, our data suggest that detection of  MDM2  amplicons could be a useful tool in uterine sarcoma pathology and implicate MDM2 as a potential therapeutic target in HGESS‐BCOR.\n\nFKFK and CK conceptualized the project and wrote the original draft. FKFK and CK coordinated data generation. FS and AvD supervised array‐based analysis. DSt, DTWJ, and AB analyzed array data. WH supervised the FISH and IHC analysis. FKFK, KTC, CEH, SF, AS, GM, HPS, DS, and FK provided tumor samples and corresponding metadata. All authors contributed to and approved of the final manuscript.","source_license":"CC-BY-4.0","license_restricted":false}