Expanding the molecular spectrum of gene fusions in endometrial stromal sarcoma: Novel subunits of the chromatin remodeling complexes PRC2 and NuA4/TIP60 as alternative fusion partners.

The ESS harboring an ING3::PHF1 fusion (case 1) presented as a large dominant 11.3 cm extrauterine mass in a 58-year-old female infiltrating the mesocolon and muscularis propria, likely arising from endometriosis, plus a separate 1 cm lesion at the right ovary. The uterus, left adnexa, and all 17 lymph nodes were free of tumor. Due to extensive involvement of the abdominopelvic organs, the patient underwent pelvic exenteration including total abdominal hysterectomy and bilateral salpingo-oophorectomy (TAH-BSO) and left colectomy. She also received adjuvant letrozole treatment. The patient remained alive with disease at 13 months following surgery ( Table 1 ). The three cases of uterine ESS with EPC1::EED fusion were diagnosed in female patients aged 27–62 years (median 59 years). On clinical follow-up, one patient (case 2) experienced local recurrence of a 4 cm pelvic mass after a long interval of 12 years following total abdominal hysterectomy and chemotherapy (unknown original tumor size). Another patient (case 4) developed local pelvic recurrence, as well as distant metastasis to the peritoneum and kidney 20 months after TAH (25 cm primary uterine mass), and received multiple lines of chemotherapy. Both patients remained alive with disease on last follow up. One patient (case 3) had no evidence of disease 14 months following TAH (unknown original tumor size) ( Table 1 ). The EPC1::EZH2- fusion positive ESS (case 5) arose in a 52-year-old female as a 13 cm mass that was confined to the uterus. The patient underwent TAH-BSO and thus far remains free of disease on limited follow-up of 3 months after surgery ( Table 1 ). Case 1 ( ING3::PHF1 ) and case 2 ( EPC1::EED ) consisted of uniformly large, ovoid to epithelioid cells with moderate to abundant eosinophilic cytoplasm, open chromatin, and variably prominent nucleoli. On low power, the tumor (case 1) showed tongue-like invasion of the muscularis propria of the colon. The cells were arranged in solid sheets and showed vascular invasion. Mitotic activity was up to 9 per 10 high power fields (HPF). Tumor cell necrosis was multifocal and partially confluent with hemorrhage, occupying about 10% of tumor volume. Case 2 was characterized by well-demarcated cellular membranes and a nested appearance, imparting a perivascular epithelioid cell tumor (PEComa)-like appearance ( Figure 1A – B , E – F ). Mitotic activity was inconspicuous (1 per 50 HPF). There was approximately 10% tumor necrosis. Histologic assessment was performed on the recurrent tumor only as the primary tumor was no longer available for evaluation. Cases 3 and 4 ( EPC1::EED ) were primarily spindled ESS consisting of monomorphic fusiform cells arranged in fascicles or solid sheets with scattered delicate branching vasculature. The cells displayed open to fine chromatin and scant pale eosinophilic cytoplasm ( Figure 2A – D ). Areas with severe cytologic atypia were not identified. Both cases showed elevated mitotic figures (8–9 per 10 HPF), focal tumor cell necrosis (5–10%) and lymphovascular invasion. Of note, for case 4, histologic assessment was performed for both the primary and recurrent/metastatic tumors, which showed similar morphology. Case 5 ( EPC1::EZH2 ) consisted of uniform short spindled cells arranged in intersecting fascicles with vague whorls around arteriolar-like vessels. The tumor showed irregular borders that invaded into the underlying myometrium. The cells were set in a variably collagenous stroma with distinct areas of collagen plaques. The cells contained scant lightly eosinophilic cytoplasm without significant cytologic atypia ( Figure 2E – F ). Focal mitotic activity up to 6 per 10 HPF was present. Tumor cell necrosis and lymphovascular invasion were absent. The four cases of ESS with EPC1::EED fusion and EPC1::EZH2 were diffusely and strongly positive for CD10. Estrogen receptor (ER) and progesterone receptor (PR) were diffusely and strongly positive in three cases (cases 2, 3 and 5). Cyclin D1 was focally positive (5–20%) in two cases (cases 3 and 4). The ESS with ING3::PHF1 fusion was diffusely positive for ER, PR, CD10, focally positive for cyclin D1 and desmin, and negative for SMA and h-caldesmon ( Figure 1C – D , G – H ; Figure 2H ; Table 2 ). A fusion involving exons 6 (2 cases) or 10 (1 case) of EPC1 ( NM_019071 ) on 10p11 and exon 2 of EED ( NM_003797 ) on 1q14 was identified in cases 2–4. In the resulting chimeric protein, which is predicted to be in frame, the enhancer of polycomb-like 1 (EPC1) domain of EPC1 is preserved and fused to the WD40 domain of EED. A fusion involving exon 5 of EPC1 ( NM_019071 ) and exon 2 of EZH2 ( NM_004456 ) was identified in case 5 ( Figure 3A , Table 2 ). In the resulting chimeric protein, which is also predicted to be in frame, the EPC1 domain is preserved and fused to the entire coding region of EZH2. Case 3 was also negative for gene rearrangements in JAZF1 , PHF1 , and YWHAE by FISH. 25 In case 1, a fusion involving exon 9 of ING3 ( NM_019071 ) on 7q31 and exon 2 of PHF1 ( NM_024165 ) on 6p21 was detected. In the resulting chimeric protein, which is predicted to be in frame, the inhibitor of growth (ING) domain of ING3 and part of the chromatin remodeling (TNG2) domain are preserved and fused to the entire coding region of PHF1 ( Figure 3B ). Of note, the patient with ING3::PHF1 ESS also developed a secondary mutation in ESR1 ( NM_001122740 ) p.Tyr537Ser as an acquired resistance mechanism to letrozole treatment ( Table 2 ). 26

Archival files from the Memorial Sloan Kettering Cancer Center Department of Pathology, as well as the personal consultation files of the authors (NKD, GM, AA, BCD, CRA), were reviewed and searched for ESS with previously unreported novel gene fusions. Clinical data, including age, sex, and anatomic site were retrieved from pathology reports. Hematoxylin and eosin–stained slides from resection specimens were rereviewed. Histopathologic parameters, including architectural patterns, cytomorphology, degree of cytologic atypia, mitotic activity, and tumor cell necrosis were evaluated. The study was approved by the Institutional Review Board. The relevant antibodies and the dilutions used in this study are as follows: CD10 (Ventana clone SP67, ready- to-use, Washington, DC, USA), cyclin D1 (Thermo Fisher clone SP4, 1:200, Waltham, MA, USA), ER (Leica Biosystems clone 6F11, ready-to-use, Buffalo Grove, IL, USA), PR (Leica Biosystems, clone 16, ready-to-use), pan-cytokeratin (AE1/AE3) (Dako #M3515, 1:1600, Santa Clara, CA, USA), SMA (Cell Marque clone 4C4.9, ready-to-use, Burlington, MA, USA), desmin (Ventana clone DE-r-11, ready-to-use), H-caldesmon (Cell Marque clone E89, ready-to-use), SOX10 (Biocare clone BC34, 1:50, New York, NY, USA), and S100 (Cell Marque clone 4C4.9, 1:600). For targeted RNA sequencing, the TruSight RNA fusion panel (Illumina, San Diego, CA) (cases 2, 3, 5) and an anchored multiplex PCR-based assay (cases 1 and 4) were used. The former is a hybrid capture-based assay that targets 507 known fusion-associated genes. 22 The latter is a custom amplicon-based NGS assay using the Archer FusionPlex (Archer, Boulder, CO) standard protocol. 23 Case 1 (recurrent tumor) was also sequenced with a multiplex PCR-based Ion AmpliSeq cancer panel. 24 Unstained recut slides from archival formalin-fixed, paraffin-embedded (FFPE) blocks were used for sequencing following extraction of RNA.

Despite their molecular heterogeneity, with the exception of YWHAE:NUTM2A/B in HGESS, the fusion genes driving the pathogenesis of most LGESS and a small subset of HGESS, such as SUZ12, PHF1, EPC1 and BCOR rearrangements, encode protein products associated with chromatin remodeling complexes, including NuA4 acetyltransferase complex and Polycomb group of Protein, mainly the Polycomb Repressive Complex 2 (PRC2) subunits. 3 , 27 Herein, we report three additional novel gene fusions involving EED and EZH2 subunits of PRC2 and ING3 of the NuA4 acetyltransferase complex. Among the 5 ESS cases included in our cohort, three cases harbored an EPC1::EED fusion, and one case each an EPC1::EZH2 and ING3-PHF1. Endometrial stromal tumors are clinically and histologically heterogeneous and are classified as endometrial stromal nodule (ESN), LGESS and HGESS in the latest World Health Organization (WHO) Classification of Female Genital Tumors (5th ed.). 4 , 13 Compared to uterine smooth muscle tumors, endometrial stromal tumors are relatively rare. The classic histology of tumors of endometrial stromal origin, which is typified in ESN and LGESS, is that of monotonous small cells with oval to fusiform nuclei and scant cytoplasm that are usually described as resembling proliferative phase endometrial stroma. Tumor cells display minimal or no cytologic atypia. Mitotic activity is usually low but can be brisk. Unlike ESNs, which are well-circumscribed and lack myometrial and lymphovascular invasion, LGESS shows a “tongue-like” pattern of myometrial infiltration and/or lymphovascular invasion. Collagen-rich hyaline plaques, such as those seen in case 5, as well as cystic changes and foamy macrophages, can also be seen. Additionally, a characteristic network of small arterioles surrounded by whorls of tumor cells is a helpful feature. Smooth muscle-like differentiation may be present and occasionally present challenges in the distinction from conventional leiomyoma. 28 The JAZF1::SUZ12 fusion is by far the most common gene fusion in endometrial stromal tumors. It is present in at least 50% of ESNs, a significant subset (20–35%) of LGESS, 29 and in a minority of undifferentiated uterine sarcomas (originally termed “undifferentiated endometrial sarcomas with nuclear uniformity”). 30 Some of these cases might also represent HGESS, which was not reflected in the WHO classification used at that time. In rarer subsets of ESS (~1%), JAZF1::PHF1 and EPC1::PHF1 fusions are detected. 13 These fusions have also been seen in ESS morphologic variants displaying smooth muscle, fibromyxoid, epithelioid, and sex cord-like differentiation, 31 – 35 as well as primary extrauterine ESS. 29 , 31 , 33 , 34 There appears to be no clear-cut correlation between histologic variants or uterine versus extrauterine locations and specific gene fusions. 28 On the other hand, ESS with YWHAE::NUTM2A/B fusions are characterized by sheets or nests of relatively uniform but highly atypical round-to-epithelioid cells with scant to moderate eosinophilic cytoplasm and enlarged nuclei with irregular nuclear contours, vesicular chromatin, and variably distinct nucleoli. Brisk mitotic activity and extensive areas of necrosis and hemorrhage are common. 15 These high-grade areas may be associated with a fibromyxoid or conventional LGESS component. 13 As a result, YWHAE::NUTM2A/B- fused ESS currently represents a well-defined group of HGESS. 15 Because of the high-grade morphology, the differential diagnosis of YWHAE::NUT2MA/B- fused ESS with other more common uterine sarcomas, including epithelioid leiomyosarcomas and sarcomatous overgrowth component in a müllerian adenosarcoma can be challenging, particularly in the absence of a LGESS component. More recently, another distinctive subset of ESS characterized by the presence of BCOR rearrangements has been described: most commonly ZC3H7B::BCOR fusion, variant BCOR and BCORL1 fusions with rare novel partners, and BCOR-ITD . These tumors display haphazardly arranged fascicles of uniform spindle cells with variable degrees of cytologic atypia admixed with epithelioid and round cell areas. Myxoid matrix is commonly seen. Mitotic activity is typically brisk. Importantly, these tumors are associated with worse prognosis compared to LGESS and are thus classified under the category of HGESS. 16 – 21 Recent studies revealed a common methylation signature among ESS with YWHAE::NUTM2A/B and BCOR rearrangements, distinct from low-grade ESS. 36 , 37 Since both LGESS and HGESS may show elevated mitotic activity and tumor cell necrosis, based solely on these morphologic descriptions, case 1 ( ING3::PHF1 ) and case 2 ( EPC1::EED ) may be classified as HGESS while cases 3–5 ( EPC1::EED and EPC1::EZH2 ) display mostly LGESS features. Nevertheless, it is also possible that cases 1 and 2 were a result of high-grade transformation from a LGESS in the primary tumor. 38 Of note, an ESR1 Y537S mutation was detected in the pelvic recurrence for case 1 after hormonal therapy. Incidentally, a recent study demonstrated the same ESR1 Y537S hotspot mutations in LGESS that underwent high-grade transformation and endocrine treatment. 26 On the other hand, the two cases with the most aggressive clinical courses, cases 1 and 4, showed distinct morphologies (high-grade round/epithelioid cells versus uniform low- grade fusiform/spindled cells) and fusions ( ING3::PHF1 and EPC1::EED ). However, both cases displayed brisk mitotic activity, evidence of lymphovascular invasion, and importantly, were very large tumors (> 10 cm). Large- scale clinical studies of uterine sarcomas have shown that disease stage is the most important prognostic factor regardless of tumor type. 39 , 40 For example, for LGESS, a 5-year survival rate of > 90% is associated with stage I/II versus 50% for stage III/IV. 40 For tumors that are confined to the uterus, tumor size and mitotic index were shown to be significant prognostic factors. Taken together, these observations raise the question of whether a strict dichotomy between LGESS and HGESS can be established solely based on the type of gene fusions and cytomorphologic features. In terms of IHC expression, all 5 cases expressed strong and diffuse CD10, and all but one cases expressed strong and diffuse ER and PR. Cyclin D1 was only focal in three cases. Desmin/h-caldesmon, markers of smooth muscle differentiation, were only weakly and focally positive in two cases. This pattern of strong, diffuse CD10 and focal desmin and h-caldesmon expression favors endometrial stromal over smooth muscle differentiation, 41 albeit not entirely specific, since CD10 expression in uterine smooth muscle tumors and desmin positivity in ESS with smooth muscle differentiation are well documented. 42 The percentage of ER/PR expression in our cohort (80%) also matches the general frequency of 70–95% in LGESS. 43 Cyclin D1, on the other hand, is typically diffusely positive in HGESS, including those with YWHAE::NUTM2A/B and BCOR rearrangements, 14 , 17 , 18 but negative to focal only in LGESS. Taken together, the IHC expression pattern of the EPC1/EZH2::EED and ING3::PHF1- rearranged ESS in our cohort was more in keeping with LGESS, most of which harbor the genetically related JAZF1::SUZ12 and JAZF1/EPC1/MEAF6::PHF1 fusions (see below). In a large portion of ESS, formation of oncogenic fusion proteins that disrupt PRC function represent a common mechanism for tumorigenesis. 3 , 26 The notable exception is the chimeric protein YWHAE:NUTM2A/B in HGESS. Neither YWHAE, which encodes for a member of the 14-3-3 family, 44 or NUTM2A/2B, which belongs to the NUT (nuclear protein in testis) family, are members of protein complexes involved in epigenetic modification. PRC, including PRC1.1 and PRC2, create transcriptionally repressed domains by depositing trimethylation of histone H3 K27 (H3K27me3). The core components of PRC2 include enhancer of zeste homologue 2 (EZH2, the catalytic subunit), suppressor of zeste 12 (SUZ12), PHD finger protein 1 (PHF1), and embryonic ectoderm development (EED). 45 , 46 Mutually exclusive loss of the PRC2 components, SUZ12 or EED, leads to suppressed PRC2 activity and decreased H3K27me3 in the majority of sporadic, NF-1 associated (syndromic) and radiation- associated malignant peripheral nerve sheath tumor (MPNST). 47 Therefore, loss of H3K27me3 by IHC is a useful diagnostic marker for MPNST. 48 Since SUZ12 and NF1 are both located on 17q11.2, they are often co-deleted in MPNST. In many LGESS, SUZ12 or PHF1 are fused to JAZF1, a zinc finger protein, or EPC1. 6 , 7 , 10 The JAZF1::SUZ12 fusion protein reduces PRC2 activity and thus disrupts transcriptional repression. 48 Other related fusions in LGESS include EPC2::PHF1 , MEAF6::PHF1 , and BRD8-PHF1 . 8 , 9 , 11 The NuA4/TIP60 histone acetyltransferase (HAT) multisubunit complex contains EPC1, inhibitor of growth family member 3 (ING3), MBT domain-containing protein 1 (MBTD1) and TIP60, the HAT component of the NuA4 complex. 49 NuA4/TIP60 is a key regulator of gene expression and stability; it is involved in transcriptional activation by regulating acetylation of nucleosomal histone proteins in yeast and mammalian cells, and also plays a direct role in DNA double-stranded breaks repair. 50 – 52 In a subset of ESS cases, EZHIP (also known as CATACOMB or CXORF67), a PRC2 subunit which inhibits the catalytic activity of PRC2, is fused to MBTD1, a member of the NuA4/TIP60 HAT complex. 12 , 52 In the current study, we identified novel fusions of ESS with EPC1::EED , EPC1::EZH2 , and ING3:PHF1 fusions, all involving genes that encode for subunits of the PRC2 and/or NuA4/TIP60 complexes, consistent with the overarching theme that the various ESS fusions commonly involve components of histone posttranslational modification ‘writer’ complexes. The major limitation of this study is the small sample size. The actual incidence of these fusions among ESS and the clinical behavior of ESS with these novel fusions are yet to be defined. Additionally, since most of the cases in this study were personal consults, we were limited by our ability to adequately assess the quality of the initial surgical resections for recurrent cases. These limitations prevent us from drawing definitive conclusions of the natural disease course and clinical behavior of these tumors. In conclusion, we report for the first time, 3 cases of ESS with EPC1::EED , one case with EPC1::EZH2 , and one case with ING3::PHF1 , further expanding the molecular spectrum of ESS.

Endometrial stromal sarcoma (ESS) represents a morphologically and molecularly heterogenous group of malignant stromal tumors that mostly arise from the uterine corpus, but may occasionally originate from extra- uterine sites associated with endometriosis. 1 – 3 Low-grade ESS (LGESS) is composed of cytologically bland and monomorphic small ovoid cells resembling proliferative-type endometrial stroma, usually with minimal to low mitotic activity and a background delicate arteriolar network. 4 By immunohistochemistry (IHC), most LGESS express estrogen receptor (ER), progesterone receptor (PR) and CD10. 4 , 5 About two-thirds of LGESS harbor gene fusions involving the polycomb repressive complexes (PRC), most commonly JAZF1::SUZ12 , followed by JAZF1 :: PHF1 , EPC1 :: PHF1 , and MEAF6 :: PHF1 . 3 , 6 – 8 Rare cases with BRD8 :: PHF1 , EPC2 :: PHF1 , EPC1 :: SUZ12 , and MBTD1 :: EZHIP ( CXorf67 ) fusions have also been reported. 9 – 12 High-grade ESS (HGESS) contains monomorphic cells with increased cytologic atypia and mitotic activity. Cytologically, the neoplastic cells have high-grade round cells and/or spindle cell morphology cells with enlarged, irregular nuclei. 13 By IHC, HGESS are typically diffusely positive for cyclin D1 (Lee 2012) with variable ER and PR expression. 14 YWHAE::NUTM2A/B (formerly named YWHAE::FAM22 ) was the first gene fusion identified in HGESS. 15 More recently, gene fusions ( ZC3H7B::BCOR ) or internal tandem duplications (ITD) involving BCOR , a PRC1.1 component, and the related BCORL1 , have been identified in a subset of HGESS. 16 – 21 Herein, we report three novel gene fusions in 5 ESS patients, including EPC1::EED , EPC1::EZH2, and ING3::PHF1 , and describe their clinicopathologic and molecular characteristics.