EWSR1::YY1 fusion positive peritoneal epithelioid mesothelioma harbors mesothelioma epigenetic signature: report of 3 cases in support of an emerging entity

Abstract

Mesothelioma is a rare, aggressive malignant neoplasm of mesothelial origin. A small subset of peritoneal mesothelioma is driven by recurrent gene fusions, mostly EWSR1/FUS::ATF1 fusions, with predilection for young adults. To date, only two cases of mesothelioma harboring EWSR1::YY1 fusions have been described. We present three additional cases of EWSR1::YY1-fused peritoneal mesotheliomas, two localized and one diffuse, all occurring in the peritoneum of middle-aged adults (2 females and 1 male), and discovered incidentally by imaging or during surgery performed for unrelated reasons. None presented with symptoms or had a known history of asbestos exposure. All three cases were cellular epithelioid neoplasms with heterogeneous architectural patterns comprising mostly solid nests and sheets with variably papillary and trabecular areas against collagenous stroma. Cytologically, the cells were monomorphic, polygonal, epithelioid cells with dense eosinophilic cytoplasm and centrally located nuclei. Overt mitotic activity or tumor necrosis was absent. All cases showed strong diffuse immunoreactivity for pancytokeratin, CK7, and nuclear WT1, patchy to negative calretinin, retained BAP1 expression, and were negative for Ber-EP4 and MOC31. RNA-sequencing confirmed in-frame gene fusion transcripts involving EWSR1 exon 7/8 and YY1 exon 2/3. By unsupervised clustering analysis, the methylation profiles of EWSR1::YY1-fused mesotheliomas clustered similarly with EWSR1/FUS::ATF1-fused mesotheliomas and conventional mesotheliomas, suggesting a mesothelioma epigenetic signature. All three patients underwent surgical resection or cytoreductive surgery of the masses. On follow-up imaging, no recurrence or progression of disease was identified. Our findings suggest that EWSR1::YY1-fusion defines a small subset of peritoneal epithelioid mesothelioma in middle-aged adults without history of asbestos exposure.

Introduction

Mesothelioma is a rare and aggressive tumor arising from the mesothelium.^1–3 Most tumors develop from the pleura and have a strong association with asbestos exposure. Inactivation of BRCA1 associated protein 1 (BAP1) is the most common pathogenetic alteration in malignant mesotheliomas.^4–8 Additionally, genetic alterations in Hippo pathway genes [neurofibromatosis type 2/merlin (NF2), LATS1/2, RASSF1, SAV1], as well as SETD2, SETDB1, DDX3X, DDX51, TP53, CDKN2A, were also reported in a subset of malignant mesotheliomas.^4–6,9,10

Rare cases of peritoneal mesotheliomas, typically in children or young adults, harboring recurrent gene fusions in ALK^11–14 and EWSR1/FUS::ATF1^15–17 have been reported without associated asbestos exposure. Moreover, an EWSR1::YY1 fusion was reported in a single case study of 2 patients with peritoneal mesothelioma.¹⁸ We report herein 3 additional cases of peritoneal mesothelioma with EWSR1::YY1 fusions and provide detailed clinical, pathologic and molecular characterization.

Materials and Methods

Study Cohort

Clinical data, including age, sex, and anatomic site were retrieved from pathology reports. Hematoxylin and eosin–stained slides from resection specimens were rereviewed. The study was approved by the Institutional Review Board.

Immunohistochemistry

The relevant antibodies and the dilutions used in this study are as follows: BAP1 (Santa Cruz #SC-28383 clone C-4, 1:25); Ber-EP4 (Ventana #760–4383, predilute); Calretinin (Ventana #790–4467 clone SP65, predilute); CK5/6 (Ventana #790–4554 clone D5/16B4, predilute); CK7 (Dako #M7018 clone OV-TL 12/30, 1:800); D2–40 (podoplanin) (Ventana #760–4395, predilute); pan-CK (AE1/AE3) (Dako #M3515, 1:1600); WT1 (N-terminal) (Leica #PA0562 clone WT49, predilute).

Targeted RNA Sequencing

Detailed descriptions of MSK-Fusion, an amplicon-based targeted RNA NGS assay using the Archer^™ FusionPlex^™ standard protocol, were described previously.¹⁹ Briefly, RNA is extracted from tumor formalin-fixed paraffin-embedded material followed by cDNA synthesis. cDNA libraries were made using the Archer^™ FusionPlex^™ standard protocol. Fusion unidirectional GSPs have been designed to target specific exons in 123 genes known to be involved in chromosomal rearrangements based on current literature. The final targeted amplicons are ready for 2×150bp sequencing on an Illumina MiSeq sequencer. FASTQ files are automatically generated using the MiSeq reporter software (Version 2.6.2.3) and analyzed using the Archer^™ analysis software (Version 5.0.4). Each fusion call should be supported with a minimum of 5 unique reads and a minimum of 3 reads with unique start sites.

Methylation Profiling and Clustering Analysis

For methylation analysis, we included a total of 23 in-house samples: 2 EWSR1::YY1-fused tumors 2 EWSR1::ATF1 and 1 FUS::ATF1-fused mesotheliomas,¹⁵ 7 angiomatoid fibrous histiocytomas (AFH), 4 clear cell sarcomas (CCS), and 7 gastrointestinal neuroectodermal tumor (also known as clear cell sarcoma-like tumor of the gastrointestinal tract) (GICCS). All samples were obtained from the pathology archives of Memorial Sloan Kettering Cancer Center (MSKCC). Details on methylation profiling were published previously.²⁰ Briefly, genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections for each of the 23 samples. Next, 250 ng of genomic DNA was subjected to bisulfite conversion and processed on the Illumina (San Diego, CA) methylation EPIC/850k platform according to manufacturer’s instructions. We used several external, publicly available data sets to enhance the analysis. First, we obtained raw IDAT files for 83 samples from the Heidelberg sarcoma methylation classifier reference cohort²¹ [Gene Expression Omnibus (GEO) study accession number GSE140668]: 8 AFH, 7 CCS, 18 desmoplastic small round cell tumor (DSRCT), 11 Ewing sarcoma (ES), 35 gastrointestinal stromal tumor (GIST), and 4 sclerosing epithelioid fibrosarcoma (SEF). Second, we obtained 79 mesothelioma samples from the Heidelberg mesothelioma study (GSE164269).²² Finally, we obtained processed 450k beta values for 173 samples from The Cancer Genome Atlas (TCGA) cohort: 50 colorectal adenocarcinoma (COAD), 50 lung adenocarcinoma (LUAD), and 73 mesotheliomas.²³

IDAT processing and data analysis on all 358 samples was performed using R version 4.1.0 and the “minfi” package version 1.38.0.²⁴ Normalization was performed using the preprocess Illumina function and probes with a detection P value > 0.01 were filtered as were single nucleotide polymorphisms (SNP)-related probes, and probes on sex chromosomes. Methylation levels were measured using beta values for all cases.²⁵ Batch effects for all mesothelioma samples were removed using the ComBat function from the R “sva” package version 3.40.0 with parametric adjustment.²⁶

For unsupervised clustering, dimensionality reduction was performed by the T-distributed stochastic neighborhood embedding (t-SNE) method.²⁷ After normalizing the input data matrix (centering the mean of each column to zero), the top 10,000 most variable CpGs by variance were analyzed using the “Rtsne” package version 0.15 with the following non-default parameters: perplexity =  10, max_iter = 5000, and theta = 0. Unsupervised hierarchical clustering and heatmap generation were performed using the “pheatmap” R package version 1.0.12 with Ward’s linkage as the clustering method and Euclidean distance for clustering of rows and columns.

Results

Clinical summary

Case 1: A 56-year-old female presented with a left lower quadrant mass in proximity to the bowel that appeared partially calcified on computed tomograpy (CT) imaging performed after a fall. Patient underwent resection of the mass along with segmental small bowel resection. The mass was located in the mesenteric adipose tissue, attached to the small bowel.

Case 2: A 49-year-old male was found to have omental thickening in the pelvis and right lower quadrant, reminiscent of peritoneal “carcinomatosis”, incidentally found during a hernia repair. Patient had a remote history of osteosarcoma more than 3 decades ago that was treated with chemotherapy with complete response. After the diagnosis of (malignant) mesothelioma via biopsy, patient underwent cytoreductive surgery that included total pelvic peritonectomy to resect all gross lesions and removal of hernia mesh, followed by 4 cycles of adjuvant intraperitoneal chemotherapy (cisplatin/mitomycin).

Case 3: A 67-year-old female presented with a mass at the gastrohepatic ligament incidentally identified by surveillance imaging. Patient has a history of hemicolectomy for an early-stage colonic adenocarcinoma 5 years ago. The mass was resected surgically.

None of the 3 patients had a known history of asbestos exposure, nor were any of them symptomatic from disease at the time of presentation. On follow-up imaging, no recurrence or progression of disease was identified. The clinical presentation of the three cases is summarized in Table 1.

Table 1.

Patients Demographics, Pathologic Findings and Clinical Follow-up

Case	Age/Sex	Site	Size	Presentation	Treatment	Status at last follow-up	Follow-up period (months)
1	56/F	Peritoneum (mesentery): unifocal	7.0 cm	Incidentally identified left lower quadrant partially calcified mass by imaging	Surgical resection	Alive with no evidence of disease	1
2	49/M	Peritoneum (bladder, side wall, mesoappendix, bowel, perirectal): multifocal	Vague nodularity, largest nodule 0.8 cm	Incidentally identified during hernia repair	Surgical cytoreduction followed by intraperitoneal chemotherapy (cisplatin, mitomycin)	Alive with no evidence of disease	24
3	67/F	Gastrohepatic omentum: unifocal	3.5 cm	Incidentally identified by imaging	Surgical resection	Alive with no evidence of disease	24
Panagopoulos Case 1	61/M	Peritoneum (diaphragm, ligamentum falciforme, appendix, spleen, omentum)	Thickened omentum	Peritoneal carcinomatosis (clinical + radiological)	Surgical resection	N/A	4
Panagopoulos Case 2	68/M	Peritoneum (inguinal, omentum)	N/A	Incidentally identified during hernia repair, massive ascites	Surgical resection	N/A	N/A

Macroscopic features

On gross pathologic examination, all three cases showed solitary or multinodular masses (Figure 1A–B), displaying mostly cellular and solid epithelioid phenotypes with heterogenous architectural patterns. Case 1 and Case 3 were both solitary, circumscribed masses measuring 7 cm and 5 cm, respectively, in greatest dimensions. For Case 2, grossly there were no discrete masses, with only a vague nodularity discerned within a diffusely thickened omentum.

Figure 1. Histologic architecture.

A-B. Low power view of the multinodular implants within peritoneal adipose tissue (A, case 3; B, case 2). C-E, Epithelioid cells arranged in cords and trabeculae set against abundant background stromal collagen or lightly myxoid background (C, case 2: 200X; D, case 3: 200X; E, case 1: 200X). F, Tumor cells in case 1 also showed papillary areas (200X). A-F, Hematoxylin and eosin stain.

Histopathologic features

On low power, the minute tumor nodules identified microscopically scattered in the omental adipose tissue were associated with prominent lymphoid aggregates. One dominant pattern common to all three cases was that of epithelioid cells arranged in cords and trabeculae set against abundant stromal collagen or lightly myxoid stroma (Figure 1C–E). Case 1 also displayed areas where cells were arranged in a (pseudo)papillary pattern. (Figure 1F). There were also extensive areas where cells were arranged in solid nests (Figure 2A–B) and solid sheets, with occasional multinucleated giant cells (Figure 2C–D). Case 1 also contained areas where cells with higher nuclear-to-cytoplasmic (N:C) ratio and lower N:C ratio were juxtaposed against each other (Figure 2D). Cytologically, the cells were large, polygonal, monomorphic epithelioid cells with abundant eosinophilic to clear cytoplasm, well-demarcated cell borders, and enlarged, centrally located nuclei with fine chromatin and inconspicuous nucleoli (Figure 2E–F). Overt mitotic activity or tumor necrosis was not appreciated. The histopathologic features are summarized in Table 2.

Figure 2. Architectural and cytomorphologic findings.

A-D, Epithelioid cells arranged in a nested pattern (A, case 1: 400X; B, case 3: 400X) and in solid sheets with occasional multinucleated giant cells (C-D, case 3: 400X). D, Biphasic area where cells with higher nuclear-to-cytoplasmic (N:C) and low N:C ratio are juxtaposed against each other. E-F, Cytologically, the epithelioid cells are monomorphic and show abundant pale eosinophilic to clear cytoplasm, centrally located nuclei with even chromatin and inconspicuous nucleoli (E, case 2: 400X; F, case 1: 400X). A-F, Hematoxylin and eosin stain.

Table 2.

Microscopic, Immunohistochemical and Molecular Findings

Case	Histology	Cytomorphology	Immunohistochemistry	EWSR1 (NM_005243.3)	YY1 (NM_003403.4)	Chromosomal breakpoint (hg19)
1	Trabeculae, cords, papillae, solid nests, set against abundant stromal collagen or lightly myxoid stroma	Epithelioid cells with ample pale eosinophilic to clear cytoplasm, well-demarcated cell borders, and large central nuclei with prominent nucleoli	(+) CK OSCAR, AE1/AE3, CK7, WT1 (−) CK20, CDX2 MOC31, calretinin, CD34, DOG1, SOX10, S100, HMB45, ERG, STAT6, desmin, synaptophysin, chromogranin, ER, ALK-D5F3, BAP1 (retained)	Exon 8	Exon 3	chr22:29,684,775(+)::chr14:100,741,035(+)
2			(+) EMA (patchy), CK5 (patchy), calretinin (focal), WT1 (−) BerEP4, MOC31, CEA, claudin-4, BAP1 (retained), MTAP (retained)	Exon 7	Exon 2	chr22:29,683,123(+)::chr14:100,728,641(+)
3			(+) Pan-CK, calretinin (patchy), WT1, ALK (focal) (−) CK5/6, D2–40, GATA3, ER, PR, PAX8, synaptophysin, chromogranin, melan A, SMA, desmin, caldesmon, S100, SOX10, CD34, ERG, CD31	Exon 8	Exon 3	chr22:29,684,775(+)::chr14:100,741,035(+)
Panago-poulos18 Case 1	Papillary, acinar, tubular, trabecular	Epithelioid, cuboid cells with densely eosinophilic cytoplasm	(+) AE1/AE3, CK7, calretinin (focal), CD5/6 (focal) (−) BerEP4, B72.3, CEA, CK20, CDX2, CD31, WT1	Exon 7	Exon 2	chr22:29,683,123(+)::chr14:100,706,260(+)
Panago-poulos18 Case 2			(+) CK5/6, CK7, CK19, WT1, calretinin, thrombomodulin (−) BerEP4, CEA, CK20, CDX2, PSA, TTF1	Exon 7	Exon 2	chr22:29,683,123(+)::chr14:100,706,260(+)

HPF: high power fields

Immunohistochemical summary

All three cases showed strong and diffuse staining for pan-cytokeratin, CK7, and nuclear WT1, patchy to negative expression of calretinin, retained BAP1 expression, and were negative for other markers, such as MOC31, CEA, BerEP4 (Table 2) (Figure 3).

Figure 3. Immunohistochemical findings.

In all three cases, the cells are diffusely and strongly positive for pancytokeratin (A, case 1, CK OSCAR; B, case 3, AE1/AE3) and CK7 (C, case 1). In case 2 and case 3, the cells show patchy calretinin expression (D, case 3). All three cases exhibit diffuse and strong WT1 nuclear reactivity (E, case 2; F, case 3; G, case 1). Neoplastic cells retain BAP1 nuclear staining (H, case 1). A-F, 200X.

EWSR1::YY1 fusion transcript

A targeted RNA sequencing fusion panel (Archer) or RNA-sequencing confirmed in-frame gene fusion transcripts of exons 7 or 8 of EWSR1 [NM_005243.2] and either exon 2 or 3 of YY1 [NM_003403.4] (Table 2). In all cases, in the predicted chimeric protein product, while the serine-tyrosine-glycine-glutamine (SVGQ)-rich transactivation domain of EWSR1 was preserved, its Zinc finger RNA/DNA binding domain was replaced by the Zinc finger DNA binding domain of YY1 (Figure 4).

Figure 4. Schematic of predicted fusion proteins encoded by gene fusions.

The fusions involve exons 7 or 8 of EWSR1 [NM_005243.2] and either exon 2 or 3 of YY1 [NM_003403.4]. The EWSR1::YY1 chimeric protein products are predicted to be in-frame. In all three cases, while the transactivation domain of EWSR1 is preserved, its Zinc finger RNA/DNA binding domain is replaced by the Zinc finger DNA binding domain of YY1. Vertical dotted lines represent exon boundaries. Abbreviations: aa, amino acids.

Methylation profiling

Methylation profiling on the Illumina EPIC 850k methylation array and unsupervised learning were performed on a large cohort of mesotheliomas, soft tissue tumors, and carcinomas. Both t-SNE and hierarchical unsupervised clustering showed that the EWSR1::YY1-fused cases (cases 1 and 2) and the three cases of EWSR1/FUS::ATF1-fused mesotheliomas cluster with conventional mesotheliomas (Figure 5A, B). In both unsupervised clustering methods, all three (sub)types of mesotheliomas were distinct from lung adenocarcinomas (LUAD) and colorectal adenocarcinomas (COAD). The mesotheliomas were also distinct from CCS, GICCS, DSRCT, SEF and ES, which are soft tissue tumors that harbor EWSR1 rearrangements, as well as GISTs, which could potentially be included in the differential diagnosis. Interestingly, AFH appeared to be related to mesothelioma from the clustering pattern. Overall, these findings suggests that EWSR1::YY1 fused neoplasms express a mesothelioma epigenetic signature.

Figure 5. Unsupervised clustering.

A. t-SNE clustering. B. Hierarchical clustering. Both methods showed all three mesotheliomas subtypes: conventional, EWSR1::YY1 fused, and EWSR1/FUS::ATF1 fused overlap to form a loose cluster that is distinct from colorectal (COAD) and lung (LUAD) adenocarcinomas, clear cell sarcomas (CCS), clear cell sarcoma-like tumor of the gastrointestinal tract (GICCS), Ewing sarcoma (ES), desmoplastic small round cell tumor (DSRCT), gastrointestinal stromal tumor (GIST), and sclerosing epithelioid fibrosarcoma (SEF). The EWSR1-rearranged mesotheliomas were also closely related to angiomatoid fibrous histiocytomas (AFH).

On the other hand, mesotheliomas with EWSR1::YY1 fusions did not match any methylation class in the previously published sarcoma methylation classifier by Koelsche et al.²¹

Discussion

Mesothelioma is a rare malignant tumor arising from the mesothelial lining of pleura, peritoneum, or tunica vaginalis, pericardium, with an annual incidence of approximately 3300 cases per year in the United States.^1,3 Peritoneal mesotheliomas constitute approximately 15–20% of all mesotheliomas and carry extremely poor prognosis, likely due to a period of asymptomatic but extensive peritoneal dissemination and delayed diagnosis owing to nonspecific clinical presentation. Unlike pleural mesotheliomas, which commonly occur in men greater than 60 years old at diagnosis, peritoneal mesotheliomas affect men and women equally, with a median age of 66 years, and only 8–14% of patients reported occupational exposure.^28,29 Similar to pleural mesotheliomas, the majority of peritoneal mesotheliomas are epithelioid type (approximately 80–90%), with the remaining being biphasic and sarcomatoid type. They can also be classified as localized or diffuse type based on clinical growth distribution.³⁰ Cytoreductive surgery combined with intraperitoneal and/or systemic chemotherapy is the mainstay of treatment modalities for peritoneal mesothelioma.

Mesotheliomas are characterized immunohistochemically by the expression of epithelial markers (CK5/6, CK7), mesothelial markers (D2–40, WT1, calretinin), and lack of markers such as B72.3, BerEP4, MOC31, Claudin-4,^31,32 as well as loss of BAP1.^33–37 For the three cases in this study, the cytomorphology of large epithelioid cells with dense eosinophilic cytoplasm, well demarcated cell borders, presence of papillary/trabecular architecture, strong and diffuse expression of cytokeratins and mesothelial markers, coupled with the lack of certain adenocarcinoma markers were consistent with the diagnosis of peritoneal mesothelioma, epithelioid type. Additionally, the striking monotonous cytomorphology in our cases hinted at a fusion-driven entity. However, we do not think that this feature is specific enough to distinguish this subtype from conventional epithelioid mesotheliomas, which also tend to be cytologically uniform.³⁰ As two of our three cases presented as a solitary mass, the presence of an intraabdominal, cytologically monomorphic epithelioid neoplasms also raises the differential diagnosis of epithelioid gastrointestinal stromal tumors and in females, sex cord stromal tumors. However, the lack of expression of markers such as DOG1 and SF1 argue against these diagnostic considerations. Immunohistochemically, our cases consistently express cytokeratins (pan-CK), WT1 and variably expresses calretinin. However, conventional mesotheliomas could also show inconsistent expression of mesothelial markers and often require a combination of multiple markers for diagnosis.^31–37 Additionally, none of our cases showed loss of BAP1, which may be one of the features that distinguishes EWSR1::YY1 fused mesotheliomas with conventional epithelioid mesotheliomas. However, this will require studies of larger sample sizes for confirmation, as BAP1 was not performed in the 2 previously published cases of this entity.

The molecular pathogenesis of malignant mesothelioma is complex and heterogeneous. Inactivation of BAP1 via loss-of-function mutations and/or copy number loss is a frequent event in both pleural and peritoneal mesotheliomas (70–85%).^4–8,12 A significant proportion of these cases are due to germline BAP1 mutations in BAP1 tumor predisposition syndrome.^37–40 In peritoneal mesotheliomas, recurrent gene mutations in the epigenetic regulatory genes SETD2, and DDX3 have also been reported.⁶ Another study of peritoneal mesotheliomas also identified mutations in TP53, TRAF7, SUZ12 and CHEK2 in BAP1-wildtype tumors, and frequent copy number loss of ARID1B, PRDM1, PBRM1, SETD2, NF2, and CDKN2A in BAP1-altered cases.³⁸ Copy number loss of CDKN2A and NF2,⁴¹ as well as TP53 mutations,⁴ have been associated with poor clinical outcome in peritoneal mesothelioma. Further, a recent study showed that alterations in BAP1, NF2, TP53, SETD2, and LATS2 were common in peritoneal mesothelioma, and BAP1 alterations were associated with shorter survival.⁴²

On the other hand, in children and young adults, rare cases of peritoneal mesotheliomas unrelated to asbestos exposure harboring recurrent gene fusions in ALK,^11–14 NTRK¹⁴ and EWSR1/FUS::ATF1^15–17 have been reported. EWSR1::YY1 fusion was reported only in 2 cases of peritoneal mesothelioma, both in males in their sixties.¹⁸ In these cases, loss of BAP1 by immunohistochemistry (IHC)^33–36 was not a useful feature since BAP1 genetic alterations are not the underlying molecular driver, as evidenced by the fact that BAP1 is retained by IHC in the reported literature among these cases. We report an additional 3 cases of peritoneal mesothelioma with EWSR1::YY1 fusions in three middle-aged adults (median 56), occurring in two females and one male, in support of this fusion being a recurrent genetic alteration in peritoneal mesothelioma with an epithelioid phenotype and lack of asbestos exposure. Consistent with previously described morphologic spectrum of peritoneal mesothelioma, our cases displayed tubulopapillary and solid growth patterns.²⁸ Based upon clinical and gross disease growth, cases 1 and 3 would be classified as localized peritoneal mesothelioma, and case 2 as diffuse peritoneal mesothelioma. Of interest, prior intra-abdominal surgery was documented in two of these patients, a finding which is often documented in patients with peritoneal mesothelioma.²⁸ The surgical history of the first two cases of EWSR1::YY1-fused peritoneal mesothelial was not available in the initial report.¹⁸

Ewing Sarcoma breakpoint region 1/EWS RNA binding protein (EWSR1) is the most commonly rearranged gene in diverse bone and soft tissue tumors. EWSR1 is ubiquitously expressed in most cell types and belongs to the FET family of RNA-binding proteins, which also include Fused in Sarcoma (FUS) and TATA-box binding protein Associated Factor 15 (TAF15). EWSR1 plays biological roles in epigenetic regulation of gene expression, RNA processing, signal transduction, and microRNA regulation.^43–45 Yin Yang 1 (YY1) is a ubiquitous transcription factor that plays a fundamental role in normal biological processes including embryogenesis, cellular differentiation, and proliferation. When YY1 is overexpressed or activated, it is associated with unmitigated cellular proliferation, apoptotic resistance, tumorigenesis and metastasis.^46–48 Similar to the two previously reported cases, the three cases of peritoneal mesotheliomas with EWSR1::YY1 fusions in our study preserve the transactivation domain of EWSR1, but its Zinc finger RNA/DNA binding domain is replaced by the Zinc finger DNA binding domain of YY1. As such, we postulate that the RNA/DNA binding functionality of EWSR1 and thereby its epigenetic/gene regulatory functions are preserved in tumor cells harboring the EWSR1::YY1 chimeric protein. In the 2 published cases, EWSR1 (exon 7)::YY1 (exon 2) were identified. In our study, 2 cases harbored EWSR1 (exon 8)::YY1 (exon 3) and one case harbored EWSR1 (exon 7)::YY1 (exon 2).

DNA methylation profiling has recently been shown to discriminate between malignant pleural mesothelioma and reactive/neoplastic mimics in the chest.²² In our study, by two different unsupervised clustering of the methylation profiles of multiple mesothelioma subtypes in comparison to a variety of EWSR1/FUS::CREB translocation family of tumors (AFH, CCS, GICCS), lung and colorectal carcinomas, and other soft tissue tumors (ES, DSRCT, GIST), peritoneal mesotheliomas with EWSR1::YY1 fusions appear to be closely related to other malignant mesotheliomas driven by gene fusions EWSR1/FUS::ATF1 and conventional malignant mesotheliomas. Since DNA methylation is a form of epigenetic memory of cellular lineage inherited through cell divisions and cancer progression,⁴⁹ these findings supports the mesothelioma-like differentiation of EWSR1::YY1 tumors, and illustrates the utility of methylation profiling in elucidation of tumor classification. Moreover, the “diffuse” nature of the mesothelioma clusters indicates the possibility of subclusters. With bigger sample sizes, we may be able to identify such subclusters in future studies. On the other hand, to confirm a mesothelial epigenetic signature, comparison to normal mesothelium, as well as non-neoplastic/reactive mesothelial lesions and benign mesothelial neoplasms, such as multicystic peritoneal mesothelioma or adenomatoid tumors, would be necessary. Regrettably, we were unable to retrieve methylation profile data of such samples for the current study.

Further, it is intriguing that AFH clusters closely with the fusion-associated mesothelioma cases in our study. This raises the question of whether AFH could at least in part display mesothelioma-like tissue differentiation, an interesting angle for further exploration as the precise histogenesis of AFH is yet to be clarified. Not surprisingly, mesotheliomas with EWSR1::YY1 fusions did not classify to any methylation classes in the previously published sarcoma methylation classifier, most likely because mesotheliomas are not included in the reference and training cohort originally used to develop this classifier.²¹

In conclusion, we report 3 new cases of peritoneal mesothelioma harboring EWSR1::YY1 fusions, lending support that EWSR1::YY1 represents a recurrent genetic alteration in peritoneal mesothelioma, epithelioid type. Whether or not peritoneal mesotheliomas with EWSR1::YY1 fusions represent a distinct entity in itself or are related to other malignant mesotheliomas driven by gene fusions, specifically those by ALK and EWSR1/FUS::ATF1, warrants further investigations in future studies that could involve transcriptomic and additional epigenetic profiling. Furthermore, long-term studies with larger sample sizes are warranted to investigate the clinical prognosis of this group of mesotheliomas, specifically in comparison with mesotheliomas characterized by alterations in BAP1 and Hippo pathway genes.