Malignant Undifferentiated Epithelioid Neoplasms with MAML2 rearrangements: a Clinicopathologic Study of Six Cases Demonstrating a Heterogenous Entity

Abstract

Among mesenchymal tumors, MAML2 gene rearrangements have been described in a subset of composite hemangioendothelioma and myxoinflammatory fibroblastic sarcoma (MIFS). However, we have recently encountered MAML2-related fusions in a group of 6 undifferentiated malignant epithelioid neoplasms that do not correspond to any established pathologic entities. The patients included 5 males and 1 female, aged 41–71 years old (median 61 years). The tumors involved the deep soft tissue of extremities (hip, knee, arm, hand), abdominal wall, and the retroperitoneum. Microscopically, the tumors consisted of solid sheets of highly atypical epithelioid cells with abundant cytoplasm and showed prominent mitotic activity and necrosis. In 3 cases, the cells displayed hyperchromatic nuclei or conspicuous macronucleoli and were admixed with background histiocytoid cells and a lymphoplasmacytic infiltrate. By immunohistochemistry (IHC), the neoplastic cells had a nonspecific phenotype. On targeted RNA sequencing, MAML2 was the 3’ partner and fused to YAP1 (3 cases), ARHGAP42 (2 cases), and ENDOD1 (1 case). One case with YAP1::MAML2 showed strong and diffuse nuclear YAP1 immunostaining and harbored a concurrent RBMS3::RAF1 fusion. In 2 cases with targeted DNA sequencing, mutations in TP53, RB1 and PTEN were detected in 1 case, and PDGFRB mutations, CCNE1 amplifications and CDKN2A/2B deletion were detected in another case, which showed strong and diffuse PDGFRB expression by IHC. Of the 4 cases with detailed clinical history (median follow-up period 8 months), 3 developed distant metastatic disease (one of which died of disease); one case remained free of disease 3 years following surgical excision. In conclusion, we describe a heterogeneous series of MAML2-rearranged undifferentiated malignant epithelioid neoplasms, a small subset of which may overlap with a recently described MIFS variant with YAP1::MAML2 fusions, further expanding the clinicopathologic spectrum of mesenchymal neoplasms with recurrent MAML2 gene rearrangements.

INTRODUCTION

Mastermind-like (MAML) was first identified as a pivotal coactivator of Notch-dependent transcription and belongs to a group of proteins encoded by the eponymous MAML genes: MAML1, MAML2 and MAML3. MAML2 (mastermind-like 2) is a key regulator of various signaling pathways that are essential for developmental processes as well as tumorigenesis, including Notch, nuclear factor-κB (NF-κB), and β-catenin signaling.^1–4

Recurrent gene rearrangements of MAML2 have been described in mucoepidermoid carcinoma,⁵ poromas/porocarcinoma,⁶ metaplastic thymoma,⁷ retiform and composite hemangioendothelioma,^8–10 and a rare “nodular necrotizing” variant of myxoinflammatory fibroblastic sarcoma (MIFS).¹¹ Herein, we report a series of six undifferentiated sarcomas characterized by an epithelioid morphology and the presence of recurrent MAML2 gene rearrangements with various fusion partners.

Materials and Methods

Study Cohort

Archival files from the Memorial Sloan Kettering Cancer Center Department of Pathology, as well as the personal consultation files of the authors (BCD, AJHS, CRA), were reviewed and searched for cases with MAML2 gene rearrangement. 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 necrosis were evaluated. The study was approved by the Institutional Review Board.

Immunohistochemical staining

The relevant antibodies and the dilutions used in this study are as follows: BRG1 (Santa Cruz Technology clone B-7, 1:250), CD163 (Ventana clone MRQ-26, undiluted), CD31 (Ventana clone JC70, undiluted), CD34 (Ventana clone QBEnd10, undiluted), CDK4 (Invitrogen, clone DCS-31, 1:200), desmin (Ventana clone DE-R-11, undiluted), EMA (Ventana clone DE-R-11, undiluted), ERG (Ventana clone E29, undiluted), INI1 (BD Bioscience clone BAF47, 1:200), MDM2 (Millipore clone IF2, 1:50), NUT (Cell Signaling Technology, clone C52B1, 1:100), S100 (Cell Marque clone 4C4.9, 1:600), SMA (Cell Marque clone 1A4, undiluted), SOX10 (Biocare clone BC34, 1:50), and YAP1 (Santa Cruz Technology clone 63.7, 1:1000).

Targeted DNA and RNA Sequencing

For targeted RNA sequencing, an anchored multiplex PCR-based assay (MSK-Fusion) (cases 1, 2, 4, 5, 6) and the TruSight RNA fusion panel (Illumina, San Diego, CA) (cases 3, 5) were used.^13–15 The former is a custom amplicon-based NGS assay using the Archer FusionPlex (Archer, Boulder, CO) standard protocol targeting specific exons in 123 genes, requiring a minimum of 5 unique reads and 3 reads with unique start sites for each fusion call.¹⁴ The latter is a hybrid capture-based assay that targets 507 known fusion-associated genes.^15,16 Detailed descriptions of MSK-IMPACT (performed in cases 1 and 2), a hybridization capture-based targeted DNA NGS assay of 505 cancer genes, were described previously.¹³ Unstained recut slides from archival formalin-fixed, paraffin-embedded (FFPE) blocks were used for sequencing following extraction of DNA and RNA. For mRNA levels, for samples tested on the Illumina panel, the average read counts across the transcript from the bam files were obtained by manual inspection on integrated genome viewer; for samples tested by the Archer panel, the RNA expression levels were provided by the Archer proprietary platform and were arbitrary values normalized to the average expression of Archer’s internal control.

Results

Clinical summary

The cohort consisted of 5 males and 1 female, aged 41 to 71 years old (median 60.5 years). The primary sites of involvement included the extremities (hip, knee, arm, hand) in four patients, the abdominal wall (multifocal lesions) in one patient, and the retroperitoneum in another patient. All tumors occurred in the deep soft tissues, including skeletal muscle, and ranged between 1.3 to 14.8 cm in greatest dimensions (median 5.0 cm) (Figure 1A–B, E–F). For the four cases with detailed clinical data (cases 1, 2, 4, 5): the patients underwent surgical excision and/or neoadjuvant/adjuvant chemotherapy. Over a limited median follow-up period of 8 months (range 2 to 36 months), three of four patients developed distant lung (cases 1, 2, 5) and bone/soft tissue (case 2) metastases (Figure 1C–D), of which two remained alive with disease (cases 1, 5) and one died of disease (case 2). One patient remained free of disease 36 months following surgery (case 4). The detailed clinical presentation and follow-up information is presented in Table 1.

Figure 1. Radiologic presentation.

A, Magnetic resonance imaging (MRI) showing a sagittal view of a large thigh mass involving the left hip and abductor musculature (T2-weighted sequence with fat suppression) (case 1). B, Computed tomography (CT) imaging showing a transverse view of right lower lobe metastases (green arrowhead) from the thigh mass (case 1). C, Positron emission tomography (PET) imaging showing multifocal FDG-avid tumors involving the abdominal wall skeletal muscles and adipose tissue (case 2). D, CT imaging showing extensive bilateral lung metastases (case 2). E-F, MRI showing the axial (T1-weighted, fat suppressed) and coronal (T2-weighted) views of an infiltrative tumor involving subcutaneous tissue and skeletal muscle between the 2^nd and 3^rd digits of the left hand (case 4).

Table 1.

Clinical Summary

Case	Age (years)	Sex	Site	Greatest dimension (cm)	Presentation	Treatment	Status at last follow-up	Follow-up period (months)
1	70	Female	Left hip involving abductor musculature.	11.5	Large thigh mass for 1 year	Wide resection followed by adjuvant chemotherapy for metastatic disease	AWD (metastases to lung)	8
2	71	Male	Multifocal abdominal wall tumors involving skeletal muscles and adipose tissue	Multiple, 2.0 cm in greatest dimension	Multifocal subcutaneous abdominal nodules multiplied and spread to back. Progressive dyspnea	Chemotherapy (decitabine, docetaxel)	DOD (metastases to lung, soft tissue, bone)	2
3	65	Male	Retroperitoneum	14.8	Large retroperitoneal mass on imaging	Neoadjuvant radiation therapy, surgical resection
4	56	Male	Left hand subcutaneous tissue and skeletal muscle	4.5	Pain and mass in left hand for 1 year	2-ray amputation	ANED	36
5	48	Male	Left knee	5.0	Large left knee soft tissue mass	Local radiotherapy and systemic chemotherapy	AWD (metastases to lung)	5
6	41	Male	Left arm	1.3	Painful, enlarging lump for 3–4 months	Biopsy followed by excision	ANED	2

ANED: alive with no evidence of disease; AWD: alive with disease; DOD: dead of disease.

Histopathologic features

Microscopically, all 6 cases consisted of cellular, high-grade, and predominantly epithelioid cells arranged in solid sheets and/or fascicles. Nuclear pleomorphism ranged from mild to moderate. The neoplastic cells displayed abundant eosinophilic to amphophilic cytoplasm and enlarged nuclei with hyperchromatic to vesicular chromatin (Figure 2A–F). In two cases (cases 4 and 5) located in the hand and knee, the neoplastic cells displayed virocyte-like macronucleoli and were scattered among background histiocytoid cells admixed with a prominent mixed inflammatory infiltrate including neutrophils, eosinophils and lymphocytes, resembling MIFS (Figure 3A–E). In one case (case 6) located in the arm, set in an extensively hyalinized/necrotic background, the epithelioid/histiocytoid neoplastic cells displayed hyperchromatic, irregular nuclei and eosinophilic, vacuolated cytoplasm. The hyalinized areas were rimmed by lymphoid tissue, imparting the appearance of lymph node involvement (Figure 3F–G). Mitotic activity was conspicuous and greater than 20 per 10 high power fields in 3 of 6 cases. Tumor necrosis was present in all cases ranging from focal in 3 cases to extensive (> 50%) in 3 cases. Hemosiderotic fibrolipomatous tumor (HFLT)-like areas were not identified in any of the cases. The histopathologic features are summarized in Table 2.

Figure 2. Histologic features (cases 1–3).

A,B, Cellular epithelioid neoplasm showing tumor cells arranged in solid sheets and nests and displaying round to ovoid nuclei, even chromatin, abundant amphophilic cytoplasm and brisk mitotic activity (case 1; A, B: 400X). C,D, Malignant epithelioid neoplasm showing tumor cells arranged in solid sheets with mild to moderate nuclear pleomorphism, prominent nucleoli, frequent mitotic figures, and extensive tumor necrosis (case 2; C: 400X, D: 100X). E,F, Cellular epithelioid and spindled neoplasm showing tumor cells arranged in nests and solid sheets and with ovoid, tapering nuclei, pale eosinophilic cytoplasm, and readily identifiable mitotic figure (case 3; E: 200X, F: 400X). A-F: hematoxylin & eosin.

Figure 3. Histologic features (cases 4–6).

A, B, Pleomorphic epithelioid neoplasm showing tumor cells with prominent macronucleoli and admixed with a neutrophilic and lymphoplasmacytic infiltrate (case 4; A: 200X, B: 400X). C, Scattered punctate necrosis and touton-like giant cells are present (case 4, 200X). D, Epithelioid neoplasm showing tumor cells with prominent macronucleoli and abundant eosinophilic cytoplasm, admixed with eosinophils (case 5, 400X). E, Extensive tumor necrosis is present (case 5, 400X). F, G, Epithelioid/histiocytoid cells set in extensively hyalinized/necrotic tissue rimmed by lymphoid tissue (case 6: F, whole slide image; G, 400X). H, YAP1 immunohistochemistry showing overexpression of YAP1 nuclear immunostaining (case 5, 400X) A-G: hematoxylin & eosin.

Table 2.

Histopathologic Findings

Case	Histology	Cytomorphology	Mitoses (per 10 HPF)	Necrosis
1	Solid sheets; UPS-like	High grade, pleomorphic spindled and epithelioid cells with hyperchromatic chromatin and abundant amphophilic cytoplasm	17	Focal
2	Solid sheets; malignant epithelioid neoplasm	High grade epithelioid cells with abundant amphophilic cytoplasm and prominent nucleoli	> 20	Extensive
3	Malignant epithelioid to spindle cell neoplasm in a fascicular-herringbone pattern; occasional ectatic and branching thin-walled vessels	Ovoid to epithelioid cells with mild pleomorphism, pale and eosinophilic cytoplasm	> 20	Focal
4	MIFS-like: Sheets of plump histiocytoid cells and rare virocyte-like large cells admixed with an eosinophilic and lymphoplasmacytic infiltrate; myxoid to collagenous stroma; diffusely infiltrative growth; occasional touton like giant cells	Plump histiocytoid cells with pale eosinophilic to foamy cytoplasm and round nuclei; occasional large cells with vesicular chromatin and enlarged virocyte-like nucleoli	3	Focal
5	MIFS-like: Sheets of plump histiocytoid cells and rare virocyte-like large cells admixed with a lymphoplasmacytic infiltrate	Predominantly composed of histiocytes and lymphoplasmacytic infiltrate. Admixed rare large cells with prominent nucleoli	> 20	Extensive
6	Malignant neoplasm composed of sheets and clusters of atypical epithelioid/histiocytoid cells against extensively hyalinized/necrotic tissue rimmed by lymphoid tissue	Spindled to epithelioid/histiocytoid cells with atypical, hyperchromatic, irregular nuclei with pink, vacuolated cytoplasm	< 1	Extensive (necrosis/hyalinization)

HPF: high power fields; MIFS: myxoinflammatory fibroblastic sarcoma; UPS: undifferentiated pleomorphic sarcoma.

Immunohistochemical summary

Immunohistochemically, the tumors showed an undifferentiated phenotype with mostly nonspecific staining of patchy to focal SMA/desmin and cytokeratins, but were negative for melanocytic, vascular, hematolymphoid markers and retained INI1 and BRG1 expression. Interestingly, in case 5, where MAML2 was fused to YAP1, the N-terminal YAP1 immunohistochemical antibody was highly expressed diffusely in the tumor nuclei (Figure 3H). In case 2, where there was a concurrent PDGFRB mutation, the neoplastic cells also express strong and diffuse membranous PDGFRB staining (Table 3).

Table 3.

Immunohistochemical (IHC) and Molecular Findings

Case	IHC (+)	IHC (−)	5’ Gene	3’ Gene	Additional Molecular Findings
1	SMA, caldesmon, CK OSCAR (rare)	pan-CK, EMA, CK7, S100, SOX10, HMB45, MDM2, CDK4, Desmin, CD163, CD31, ERG, CD3, CD20, CD45, CD117, DOG1, pan-NTRK, MUC4, ALK, Mel-A, TFE3. INI1, BRG1 and BRM retained.	ARHGAP42 (NM_152432) exon 3	MAML2 (NM_003403.4) exon 3	TP53 p.Y234C, PTEN intragenic deletion, RB1 p.A538Qfs*5, BRCA2 p.A2351P, HNF1A p.A534T, PREX2 p.N64T. TMB 4.1 mt/Mb. mRNA expression: MAML2 and VGLL3 expression levels not upregulated.
2	PDGFRB 4+, vimentin	AE1/AE3, CAM5.2, CK5/6, CK7, CK20, EMA, PSA, Mart-1, S-100, p63, TTF-1, SMA, desmin, myogenin, WT1, MUM1, CD34, ALK (D5F3), NUT, and pan-NTRK. CD45, CD56, CD34, CD20, ERG, CD117, CD21, SF-1, SOX10 and BRAF VE1. INI1, BRG1 and H3K27me3 (retained)	ENDOD1 (NM_015036) exon 1	MAML2 (NM_003403.4) exon 3	19p13.2 amplification (CCNE1, BRD4, NOTCH3), 9p21.3 deletion (CDKN2A/CDKN2B), point mutations including PDGFRB hotspot exon 11 p.1547_1553dup and exon 18 p.R853P. TMB 6.6 mt/Mb. mRNA expression: MAML2 and VGLL3 expression levels not upregulated.
3	Desmin (focal), DOG1 (patchy weak), loss of H3K27me3	AE1/AE3, SMA, myogenin, S100, SOX10, HMB45, CD34 MDM2	ARHGAP42 (NM_152432) exon 2	MAML2 (NM_003403.4) exon 2
4	CD68 (KP1 and PGM1) and CD163 in histiocytes, pan-cytokeratin (rare cells), TFE3	EMA, 34BE12, HMB45, CD15, CD30, cathepsin-K, ALK-D5F3, S100, CD1a, langerin, CD31, CD34, ERG, EMA, desmin BRAF VE1. INI-1 (retained)	YAP1 (NM_001130145) exon 5	MAML2 (NM_003403.4) exon 2	mRNA expression: MAML2 not upregulated.
5	YAP1, AE1/AE3, D2–40, SMA (focal), EMA (focal)	ERG, CD34, desmin, S100, HMB-45, Melan-A, MITF, SOX10, MyoD1. INI-1 (retained)	YAP1 (NM_001130145) exon 1	MAML2 (NM_003403.4) exon 2	RBMS3 (NM_014483.3) exon 11::RAF1 (NM_002880.3) exon 8. mRNA expression: VGLL3 upregulated; MAML2 not upregulated.
6	AE1/AE3, S100	EMA, p63, ERG, SOX10, INI1 (retained)	YAP1 (NM_001130145) exon 5	MAML2 (NM_003403.4) exon 2	mRNA expression: MAML2, VGLL3 and YAP1 not upregulated.

Molecular findings

By RNA sequencing, MAML2 rearrangement was detected in all six cases. The 5’ partners were ARHGAP42 (case 1, 3), ENDOD1 (case 2), and YAP1 (cases 4–6). MAML2 and all three of its 5’ partners are in proximity to each other on chromosome 11q21-q22 (Figure 4A). All fusions were predicted to be in-frame and involved exons 2 and 3 of MAML2 (NM_003403.4). As the 3’ partner, the transcriptional factor IIA (TFIIA) domain of MAML2 is preserved in the predicted chimeric protein (Figure 4B). Interestingly, in case 5 a concurrent fusion RBMS3::RAF1 with adequate read support (339 reads spanning breakpoint, 66 unique start sites by Archer) and its reciprocal fusion RAF1::RBMS3 were also detected. The presence of concurrent YAP1::MAML2 and RBMS3::RAF1 fusions in case 5 was confirmed independently by an amplicon-based, anchored multiplex PCR targeted RNA sequencing assay and a hybrid capture-based Illumina TruSight RNA fusion assay (Dickson 2018).^13,15 VGLL3 RNA expression was upregulated in case 5 but not upregulated in cases 1, 2 or 6 (not available for evaluation for cases 3 and 4). MAML2 expression was not upregulated in cases 1, 2, 4, 5 or 6 (not available for evaluation for case 3).

Figure 4. MAML2 fusions schematic diagrams.

A: Circos plot depicting MAML2 fusions represented by links between cytobands (hg19 genome). Plot generated using R package “circlicize” version 0.4.13.³⁹ B: Schematic of MAML2 fusion transcripts annotated by NCBI RefSeq accession numbers. Numbers and black arrows represent exons and directions of transcript, respectively. Predicted chimeric proteins with protein domains are depicted below the transcripts. Horizontal black arrows designate portions of the protein domains that are present in the chimeric protein. Vertical dotted red line represents fusion breakpoint.

Targeted DNA sequencing data by MSK-IMPACT was available for cases 1 and 2. Case 1 had a tumor mutation burden (TMB) of 4.1 mutations per megabases (mt/Mb), and harbored missense mutations in TP53 (p.Y234C), BRCA2 (p.A2351P), and PREX2 (p.N64T), a truncating frameshift mutation in RB1 p.A538Qfs*5, and an intragenic deletion in PTEN. Case 2 had a TMB of 6.6 mt/Mb, and harbored PDGFRB mutations in exon 11 (p.I547_I553dup) and exon 18 (p.R853P), copy number amplifications on 19p13.2 (contains CCNE1, BRD4, NOTCH3) and deletions on 9p21.3 (contains CDKN2A/CDKN2B). These two cases showed no amplification at chromosome 3p11–12 (where VGLL3 is located) by copy number profiling. Details of the molecular findings are described in Table 3.

DISCUSSION

We report six cases of undifferentiated malignant epithelioid neoplasms with MAML2 gene fusions arising in the deep soft tissues. In our cohort, the 5’ partner of MAML2 were YAP1, ARHGAP42 and ENDOD1, the latter two have not been previously reported as a fusion partner of MAML2. Interestingly, all these genes are located on the same or adjacent bands on chromosome 11: MAML2 and ENDOD1 on 11q21; YAP1 and ARHGAP42 on 11q22. In the predicted chimeric proteins, MAML2 retains the C-terminal TFIIA domain, preserving its function as a transcriptional coactivator.^2–4 Additionally, a recent study identified a PPxY motif in MAML1 and MAML2 that interacts with YAP1 or TAZ (WWTR1) and promotes their nuclear localization, where MAML1 and MAML2 also act as a transcriptional coactivator in the nucleus, thereby promoting oncogenic transformation.¹⁷ As YAP1 retained its N-terminal portion in the chimeric protein, it showed high expression by immunohistochemistry in case 5 using an antibody that recognizes its N-terminal in the case with YAP1::MAML2, where the oncogenic fusion driver is predicted to be overexpressed. Of note that this is the reverse scenario as seen in YAP1::TFE3 epithelioid hemangioendothelioma, where the loss of C-terminal YAP1 immunostaining is detected for the diagnosis.¹⁸

The two cases (cases 4, 5) of this study with YAP1::MAML2 that were located on the hand, and knee and displayed sheets of epithelioid cells with large vesicular nuclei and prominent virocyte-like macronucleoli admixed with a mixed inflammatory infiltrate—histopathologic features that overlap with the recently described “nodular necrotizing” variants of MIFS with recurrent YAP1::MAML2 fusions.¹¹ In an additional case (case 6, arm) the tumor cells were associated with extensive hyalinization and necrosis centrally and surrounded at the periphery by lymphoid aggregates. However, in the study by Perret et al, only 2 of 7 cases were acral location, and they did not comment on whether any of their cases showed HFLT-like areas. In the current study, only one case occurred in acral location (harboring YAP1-MAML2 fusion), and none of our cases showed a nodular configuration or HFLT-like areas, i.e., mixture of mature adipose tissue and bland spindled cells admixed with abundant hemosiderin and background histiocytes away from the main tumor. MIFS, as well as the related HFLT, was first described to harbor rearrangements of TGFBR3 and MGEA5 as a result of translocations between chromosomes 1 and 10 and amplification of VGLL3 gene on 3p11–12.^19–22 Later, recurrent BRAF rearrangements have also been described in MIFS but not HFLT.²³ The genetic heterogeneity of the MIFS spectrum begs the question of whether these MAML2-rearranged tumors should be grouped under the same umbrella of MIFS, based on their limited clinical, morphologic and molecular overlap. Further studies are needed to investigate the pathogenetic relationship between these tumors and “MIFS family” including transcriptional and epigenetic platforms.

On the other hand, in a recent study of MIFS using array comparative genomic hybridization (array CGH), VGLL3 amplification was seen in 8 (40%) of 20 cases of MIFS, regardless of whether these cases harbored t(1;10) or BRAF rearrangements.²² VGLL3 amplification is also strongly correlated with upregulation of VGLL3 RNA expression level.²⁴ In the study by Perret et al, the 4 cases with array CGH showed flat copy number profiles (including 3p11–12 where VGLL3 is located), and the single case with FISH showed no VGLL3 amplification.¹¹ In our study, only one case (case 5, knee) showed evidence of upregulation of VGLL3 RNA expression. All these features cast uncertainty on the diagnosis of MIFS for MAML2-rearranged malignant epithelioid neoplasms. Furthermore, the first three cases in our cohort did not exhibit histologic or clinical features of MIFS and were essentially undifferentiated sarcomas with an epithelioid morphology. Moreover, in contrast to the complete lack of local recurrence or distant metastases in the study by Perret et al,¹¹ the highly aggressive clinical behavior in all except one patient with available follow-up developing distant spread in the current study is not consistent with the biology of MIFS. In fact, the only patient without tumor progression had an acral lesion harboring YAP1-MAML2 (case 4) and displaying a partial morphologic overlap with MIFS. The fact that oncogenic mutations in TP53, BRCA2, RB1, etc., copy number losses in PTEN and CDKN2A/2B, and copy number gains in CCNE1 were also detected in these tumors, coupled with the relatively high TMB of 4 to 6 mt/Mb—a profile akin to other high-grade sarcomas.²⁵

Moreover, it is intriguing that one of the cases from the study by Perret et al describing a “nodular necrotizing” variant of MIFS with YAP1::MAML2 also showed a concurrent BRAF rearrangement, whereas one of the cases with YAP1::MAML2 in the current study (case 5) had a concurrent RAF1 rearrangement. The concurrent YAP1::MAML2 and RBMS3::RAF1 fusions in case 5 were confirmed independently by two different targeted RNA sequencing assays (amplicon-based and hybrid capture based). In almost all tumors, one dominant driver fusion is sufficient to induce oncogenic transformation. Thus, it is highly unusual for sarcomas to harbor two concurrent driver fusions. The biological relevance of this rare phenomenon remains to be investigated.

Additionally, recurrent MAML2 fusions have been described in rare cases of retiform and composite hemangioendothelioma (CHE) with neuroendocrine differentiation.^8–10 Although this type of CHE may show severe nuclear atypia, they also display areas with clear vascular differentiation and consistently express vascular markers such as ERG, CD31 and CD34. Additionally, MAML2 rearrangements are known to be recurrent in certain carcinomas including mucoepidermoid carcinoma (CRTC1::MAML2),⁵ metaplastic thymomas (YAP1::MAML2),⁷ and poromas/porocarcinomas (YAP1::MAML2),⁹ which have specific clinicopathologic features that do not overlap with the MAML2-rearranged undifferentiated malignant neoplasm in this study.

The differential diagnosis of MAML2-rearranged undifferentiated malignant neoplasm is broad and includes any high-grade sarcomas with epithelioid morphology as well as undifferentiated malignant neoplasms. Proximal-type epithelioid sarcoma also consists of solid sheets of large epithelioid cells with abundant glassy eosinophilic cytoplasm and enlarged nuclei that is eccentrically placed, imparting a rhabdoid cytomorphology. Immunophenotypically, epithelioid sarcoma expresses cytokeratin consistently, CD34 in approximately half of the cases, and loses INI1 nuclear staining in all cases as a result of SMARCB1 biallelic inactivation.²⁶ SMARCB1 deficiency is mutually exclusive from inactivation of SMARCA4, which encodes for BRG1 (SMARCA4), another catalytic subunit of the SWI/SNF complex. SMARCA4-deficient malignant neoplasms, initially reported in the thoracic cavity,²⁷ have now been now recognized at many locations in the body,^28,29 and can also display rhabdoid features. The loss of BRG1 immunoreactivity represents a useful diagnostic marker. Another emerging class of malignant neoplasms that may be included in the differential diagnosis is the family of tumors with NUTM1 gene rearrangements, formerly described mainly as NUT-midline carcinoma, but recently being found at various body sites including visceral organs and deep soft tissues and lacking convincing epithelial differentiation.^15,30,31 The latter cases of NUTM1-rearranged undifferentiated soft tissue and visceral tumors often display a round-epithelioid-rhabdoid phenotype arranged in solid sheets, nests and cords,¹⁵ with strong and diffuse NUT nuclear immunopositivity, which is helpful in its diagnostic workup.³⁰ Additionally, the epithelioid-spindled morphology one of the cases (case 3), as well as its retroperitoneal location and loss of H3K27me3 immunostaining, raise the possibility of malignant peripheral nerve sheath tumor (MPNST).³² However, MPNST has never been shown to harbor oncogenic fusions, and loss of H3K27me3, albeit a highly sensitive marker of MPNST, has been shown to lack specificity and could be seen in a wide range of mimics.³³

Furthermore, a rare type of malignant epithelioid neoplasm characterized by the presence of EWSR1/FUS::CREM fusions was recently described having predilection for mesothelial-lined cavities, which can be an additional diagnostic consideration.³⁴ No specific immunoprofile is available to diagnose such tumors. Therefore, the use of molecular testing using a panel that covers common recurrent gene fusions may be useful for the workup of undifferentiated/poorly differentiated sarcomas. Finally, other poorly differentiated or dedifferentiated tumors should also be considered. Dedifferentiated liposarcoma is the top diagnostic consideration of any malignant mesenchymal tumors in the retroperitoneum but will show MDM2/CDK4 amplification by genetic analysis.³⁵ Dedifferentiated melanoma—a melanoma that loses melanocytic markers expression including SOX10 and S100 and may resemble any other high-grade tumors—is a highly challenging diagnosis, and may require demonstration of an ultraviolet mutational signature or the presence of certain driver mutations, such as BRAF V600 and NRAS mutations in combination with a clinical history of melanoma to establish the diagnosis.^36–38

In conclusion, we describe a series of MAML2-rearranged undifferentiated sarcomas with an epithelioid morphology and aggressive clinical behavior. Based on these findings, we recommend maintaining a high index of suspicion and suggest testing for potential oncogenic fusions when encountering an undifferentiated sarcoma with epithelioid morphology and monomorphic cytology, and inclusion of MAML2 on targeted next-generation fusion panels. Although a subset of our cases showed partial overlap with the recently described ‘nodular necrotizing’ variant of MIFS harboring YAP1::MAML2 fusions, their pathogenetic relationship with the well-established family of MIFS remains unclear, given the low frequency of acral presentation and VGLL3 upregulation/amplification, as well as the high rate of distant metastases. Further larger clinicopathologic studies with molecular correlates are needed to establish if MAML2 gene rearrangements define a heterogeneous group of tumors or merely a wide morphologic spectrum of a single pathologic entity of soft tissue sarcomas with epithelioid phenotype.

Data Availability Statement

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.