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Mullerian adenosarcoma is an uncommon mesenchymal tumor of the gynecologic tract, usually of uterine origin. Tumors are generally low-grade and associated with good prognosis, whereas high-grade adenosarcomas are rare and less well studied. Herein, we sought to characterize the molecular features of 27 adenosarcomas (primary uterine, n = 19, cervical, n = 3, ovarian, n = 4, peritoneal, n = 1), enriched for high-grade tumors (n = 17) subjected to targeted panel sequencing. Recurrent genetic alterations in adenosarcomas included TP53 mutations (n = 4, 15%), restricted to high-grade cases, BAP1 homozygous deletions (n = 4, 15%), DICER1 mutations (n = 4, 15%), ARID1A mutations (n = 3), TERT promoter mutations (n = 2) and amplification (n = 1), ATRX frameshift mutation/homozygous deletions (n = 3), MDM2 (n = 2), CDK4 (n = 2) and CCNE1 (n = 2) amplifications, as well as alterations involving members of the PI3K (PTEN, n = 3; PIK3CA, n = 4; AKT1, n = 2) and MAPK (KRAS, n = 4, BRAF, n = 2) signaling pathways. One tumor harbored an ESR1-NCOA3 fusion and another had an MLH1 homozygous deletion, associated with loss of MLH1 and PMS2 protein expression. The fraction of genome altered was significantly higher in high-grade compared to low-grade adenosarcomas (P = 0.001). Somatic ATRX frameshift mutations were found in two patients with low-grade adenosarcoma with high-grade recurrences and one case of high-grade adenosarcoma with an adjacent low-grade component. Immunohistochemical analysis for BAP1 revealed loss of nuclear expression in 6/24 (25%) cases, including all 4 tumors with BAP1 deletions. Notably, out of 196 mesenchymal neoplasms of gynecologic origin, BAP1 homozygous deletion was only found in adenosarcomas (4/27,15% adenosarcomas vs 0/169, 0% other mesenchymal neoplasms, P = 0.0003). This study demonstrates that high-grade adenosarcomas are heterogeneous at the molecular level and are characterized by genomic instability and TP53 mutations; ATRX loss may be involved in high-grade transformation of low-grade adenosarcoma; and BAP1 inactivation appears to be a specific pathogenic driver in a subset of adenosarcomas.

Uterine perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal neoplasm that occasionally shares morphologic and immunohistochemical overlap with low- and high-grade endometrial stromal sarcoma (LGESS and HGESS). In this study, we sought to characterize the clinical, morphologic, genetic, and epigenetic features of five uterine sarcomas that display histologic features of LGESS, HGESS, and PEComa. All tumors demonstrated epithelioid cells often associated with a low-grade spindled component resembling LGESS, with both regions expressing CD10, ER, PR, variable HMB45, and Melan-A immunoreactivity, and strong cathepsin K and pS6 expression. Targeted massively parallel sequencing analysis revealed the presence of somatic TSC2 mutations in all five cases, of which four harbored concurrent or consecutive JAZF1-SUZ12 gene fusions. Unsupervised hierarchical clustering analysis of methylation profiles of TSC2-mutant uterine sarcomas (n = 4), LGESS (n = 10), and HGESS (n = 12) demonstrated two clusters consisting of (1) all LGESS and TSC2-mutant uterine sarcomas and (2) all HGESS. KEGG pathway analysis detected methylation differences in genes involved in PI3K/AKT, calcium, and Rap1 signaling. TSC2-mutant uterine sarcomas were responsive to hormone suppression, and mTOR inhibition demonstrated clinical benefit in four patients with these neoplasms. Our results suggest that these tumors represent histologically distinctive LGESS with TSC2 mutations. TSC2 mutations and JAZF1-SUZ12 fusion may help diagnose these tumors and possibly direct effective treatment.

High-grade endometrial stromal sarcoma (HGESS) may harbor YWHAE-NUTM2A/B fusion, ZC3H7B-BCOR fusion, and BCOR internal tandem duplication (ITD). NTRK3 upregulation and pan-Trk expression were reported in soft tissue lesions that share similar morphology and genetic abnormalities. To confirm these findings in HGESS, differential expression analysis was performed at gene level comparing 11 HGESS with 48 other uterine sarcomas, including 9 low-grade endometrial stromal sarcomas, 23 undifferentiated uterine sarcomas, and 16 leiomyosarcomas, using targeted RNA sequencing data. Pan-Trk immunohistochemistry was performed on 35 HGESS, including 10 tumors with RNA expression data, with genotypes previously confirmed by targeted RNA sequencing, fluorescence in situ hybridization, and/or genomic PCR. Unsupervised hierarchical clustering of the top 25% of differentially expressed probes identified three molecular groups: (1) high NTRK3, FGFR3, RET, BCOR, GLI1, and PTCH1 and low ESR1 expression; (2) low NTRK3, FGFR3, RET, BCOR, GLI1, and PTCH1 and high ESR1 expression; and (3) low NTRK3, FGFR3, RET, BCOR, GLI1, PTCH1, and ESR1 expression. Among HGESS, 64% of tumors clustered in group 1, while 27% clustered in group 2. Cytoplasmic and/or nuclear pan-Trk staining of variable extent and intensity was seen in 91% of HGESS regardless of cyclin D1 and/or BCOR positivity. ER and PR expression was seen in 44% of HGESS despite ESR1 downregulation. Two patients with ER and PR positive but ESR1 downregulated stage I HGESS were treated with endocrine therapy, and both recurred at 12 and 36 months after primary resection. By RNA expression, HGESS appear homogenous and distinct from other uterine sarcomas by activation of kinases, including NTRK3, and sonic hedgehog pathway genes along with downregulation of ESR1. Most HGESS demonstrate pan-Trk staining which may serve as a diagnostic biomarker. ESR1 downregulation is seen in some HGESS that express ER and PR which raises implications in the utility of endocrine therapy in these patients.

Sinonasal undifferentiated carcinoma (SNUC) is an aggressive malignancy harboring IDH2 R172 mutations in >80% cases. We explored the potential of genome-wide DNA methylation profiling to elucidate tumor biology and improve the diagnosis of sinonasal undifferentiated carcinoma and its histologic mimics. Forty-two cases, including sinonasal undifferentiated, large cell neuroendocrine, small cell neuroendocrine, and SMARCB1-deficient carcinomas and olfactory neuroblastoma, were profiled by Illumina Infinium Methylation EPIC array interrogating >850,000 CpG sites. The data were analyzed using a custom bioinformatics pipeline. IDH2 mutation status was determined by the targeted exome sequencing (MSK- IMPACTTM) in most cases. H3K27 methylation level was assessed by the immunohistochemistry-based H-score. DNA methylation-based semi-supervised hierarchical clustering analysis segregated IDH2 mutants, mostly sinonasal undifferentiated (n = 10) and large cell neuroendocrine carcinomas (n = 4), from other sinonasal tumors, and formed a single cluster irrespective of the histologic type. t-distributed stochastic neighbor embedding dimensionality reduction analysis showed no overlap between IDH2 mutants, SMARCB1-deficient carcinoma and olfactory neuroblastoma. IDH2 mutants demonstrated a global methylation phenotype and an increase in repressive trimethylation of H3K27 in comparison to IDH2 wild-type tumors (p < 0.001). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed no difference in pathway activation between IDH2-mutated sinonasal undifferentiated and large cell neuroendocrine carcinomas. In comparison to SMARCB1-deficient, IDH2-mutated carcinomas were associated with better disease-free survival (p = 0.034) and lower propensity for lung metastasis (p = 0.002). ARID1A mutations were common in small cell neuroendocrine carcinoma but not among IDH2 mutants (3/3 versus 0/18 and p < 0.001). IDH2 mutations in sinonasal carcinomas induce a hypermethylator phenotype and define a molecular subgroup of tumors arising in this location. IDH2- mutated sinonasal undifferentiated carcinoma and large cell neuroendocrine carcinoma likely represent a phenotypic spectrum of the same entity, which is distinct from small cell neuroendocrine and SMARCB1-deficient sinonasal carcinomas. DNA methylation-based analysis of the sinonasal tumors has potential to improve the diagnostic accuracy and classification of tumors arising in this location.

High-grade endometrial stromal sarcoma likely encompasses underrecognized tumors harboring genetic abnormalities besides YWHAE-NUTM2 fusion. Triggered by three initial endometrial stromal sarcomas with ZC3H7B-BCOR fusion characterized by high-grade morphology and aggressive clinical behavior, we herein investigate the clinicopathologic features of this genetic subset by expanding the analysis to 17 such tumors. All of them occurred in adult women with a median age of 54 (range, 28-71) years. They were predominantly based in the endomyometrium and demonstrated tongue-like and/or pushing myometrial invasion. Most were uniformly cellular and displayed haphazard fascicles of spindle cells with mild to moderate nuclear atypia. Myxoid matrix was seen in 14 of 17 (82%) tumors, and collagen plaques were seen in 8 (47%). The mitotic index was ≥10 mitotic figures/10 high-power fields (HPFs) in 14 of 17 (82%) tumors with a median of 14.5 mitotic figures/10 HPFs. No foci of conventional or variant low-grade endometrial stromal sarcoma were seen. All tumors expressed CD10 with only limited or absent desmin, SMA and/or h-caldesmon staining. ER and PR expression in >5% of cells was seen in 4 of 12 (33%) tumors. Diffuse cyclin D1 and BCOR immunoreactivity was present in 7 of 8 (88%) and 7 of 14 (50%) tumors, respectively. Fluorescence in situ hybridization or targeted RNA sequencing confirmed ZC3H7B-BCOR fusion in all tumors, including four and two previously diagnosed as myxoid leiomyosarcoma and undifferentiated uterine sarcoma, respectively. Limited clinical data suggest that patients present at higher stage and have worse prognosis compared with published outcomes in low-grade endometrial stromal sarcoma. Tumors with ZC3H7B-BCOR fusion constitute a distinct group of endometrial stromal sarcomas with high-grade morphology that should be distinguished from other uterine mesenchymal neoplasms that may demonstrate myxoid morphology.

Recognition of high-grade endometrial stromal sarcoma is important because of its aggressive clinical behavior. Morphologic features of YWHAE-NUTM2 high-grade endometrial stromal sarcoma may overlap with other uterine sarcoma types. BCOR immunoexpression was studied in these tumors and their morphologic mimics to assess its diagnostic utility. BCOR immunohistochemical staining was performed on archival tissue from 28 high-grade endometrial stromal sarcomas with classic morphology (20 YWHAE-NUTM2, 5 ZC3H7B-BCOR, 3 BCOR-ZC3H7B), 3 high-grade endometrial stromal sarcomas with unusual morphology and unknown gene rearrangement status, 66 low-grade endometrial stromal sarcomas, 21 endometrial stromal nodules, 38 uterine leiomyosarcomas, and 19 uterine leiomyomas. Intensity of nuclear staining and percentage of positive tumor cells were recorded. Strong diffuse nuclear BCOR staining (defined as >95% of tumor cells) was seen in the round cell component of all 20 (100%) classic YWHAE-NUTM2 high-grade endometrial stromal sarcomas and the 3 unusual high-grade endometrial stromal sarcomas which prompted FISH studies confirming YWHAE rearrangement in 2 tumors. Genomic PCR confirmed the presence of BCOR exon 16 internal tandem duplication in the third case. Diffuse BCOR staining was strong in three and weak in one BCOR-rearranged high-grade endometrial stromal sarcoma while absent in the remaining four BCOR-rearranged tumors. BCOR staining was weakly positive in