A novel WWTR1::AFF2 fusion in an intra‐abdominal soft tissue sarcoma with associated endometriosis

Dashti NK, Nooshin K. Dashti, Josephine K. Dermawan, Schoolmeester JK, J. Kenneth Schoolmeester, Kevin C. Halling, Halling KC, Cristina R. Antonescu
Genes, chromosomes & cancer · 2022 · vol. 61(8) , pp. 497–502 · doi:10.1002/gcc.23045 · PMID:35429182 · PMC9233893 · W4223892980
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⚙ AI-generated summary by claude@2026-06, 2026-06-10 ⓘ

Targeted RNA sequencing identified a novel WWTR1::AFF2 fusion in a challenging intra-abdominal spindle cell sarcoma with associated endometriosis, raising possibilities for its histogenesis and pathogenesis.

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⚙ AI-generated deep summary by claude@2026-06, 2026-06-13 · read from full text ⓘ

This brief communication reports a first description of an intra-abdominal spindle cell soft tissue sarcoma harboring a novel WWTR1::AFF2 fusion, identified in a 37-year-old woman with abdominal pain. Using RNA-based sequencing with fusion-detection bioinformatics, supported by FISH break-apart testing for WWTR1, the authors found a transcript fusion joining WWTR1 exon 2 to AFF2 exon 10; histology showed a highly cellular fascicular tumor adjacent to areas of endometriosis, with diffuse desmin, WT-1, ER, PR, and AR expression but negative SMA and caldesmon. A major caveat is that this is a single case report, leaving the tumor’s histogenesis and clinical behavior uncertain and requiring additional studies to determine recurrence risk and classification. Relevance to endometriosis: endometriosis was identified at the tumor periphery and further confirmed on subsequent hysterectomy, though the study’s primary focus is discovery and characterization of a WWTR1::AFF2 fusion sarcoma.

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Abstract

Application of molecular testing in clinical practice has led to significant advances in the classification of soft tissue sarcomas. Despite remarkable progress, there are still challenging cases that remain unclassified. In this study, we present an unusual spindle cell sarcoma arising in the abdominal cavity of a 37-year-old female. An extensive panel of immunostains was nonspecific for a line of differentiation and the tumor was subjected to targeted RNA sequencing for further classification. The findings showed a novel WWTR1::AFF2 fusion, which was further confirmed by break-apart FISH analysis for WWTR1 gene rearrangement. The tumor was attached to the wall of sigmoid colon and showed a highly cellular proliferation of plump spindle to epithelioid cells arranged in intersecting fascicles. Areas of extensive endometriosis were identified adjacent to the tumor. The immunoprofile was significant for reactivity with desmin, calponin, WT-1, ER, and PR, while negative for CD10, SMA, caldesmon, pan-keratin, ALK, CD117, and S100. The patient is alive and well after 11 months of follow-up. The exact histogenesis of this sarcoma remains unclear, however, the presence of adjacent endometriosis and coexpression of WT1/ER/PR raises the possibility of an unusual endometrioid stromal sarcoma, occurring outside the GYN tract. Additional cases are needed to establish the recurrent potential of this fusion event and to better define its pathogenesis and clinical behavior.
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Results

The patient was a 37-year-old female who presented with abdominal pain. Imaging and exploratory laparotomy identified a 4.5 × 4.5 × 2.6 cm abdominal mass attached to the wall of sigmoid colon. With the surgical impression of a gastrointestinal stromal tumor and marked pelvic adhesions, the patient underwent sigmoid colectomy with side-to-side anastomosis and partial omentectomy. Grossly, the mass appeared well-circumscribed, solid and lobulated. Microscopically, the neoplasm was highly cellular and composed of rather uniform plump spindle to epithelioid cells arranged in long fascicular architecture. The tumor showed alternating hypercellular and more myxoid hypocellular areas with scattered lymphocytic infiltrate throughout the tumor ( Figure 1 ). There were areas of perivascular edema. The cells demonstrated plump vesicular nuclei, prominent nucleoli and dark amphophilic cytoplasm with ill-defined cell borders. Mitotic figures were inconspicuous and tumor necrosis was absent. Endometriosis was identified at the periphery of the tumor ( Figure 1 ). By immunohistochemistry, the neoplastic cells showed diffuse expression of desmin, WT-1, ER, PR and AR ( Figure 2 ). There was patchy calponin staining, while the remaining stains, including myogenin, MyoD1, CD10, SMA, CD34 and caldesmon were negative. Surgical complications, including anastomosis leakage and sepsis with abscess formation, prompted subsequent total hysterectomy and bilateral salpingo-oophorectomy and abdominal debulking, which showed abscess and endometriosis, with no residual tumor. Moreover, microscopic examination of uterus, ovaries or fallopian tubes was unremarkable. The patient is alive and well with no evidence of disease after 11 months of follow-up. Targeted RNA sequencing identified a novel WWTR1::AFF2 fusion. The specific gene transcript based on GRCh37 (hg19) was composed of exon 2 of WWTR1 ( NM_015472 ) at genomic position Chr3:g.149374663 and exon 10 of AFF2 ( NM_002025 ) at genomic position ChrX:g.148035110 ( Figure 3 ). The presence of WWTR1 gene rearrangements was demonstrated by FISH break-apart analysis ( Supplem Figure 1 ).

Materials

Immunostains for desmin, caldesmon, SMA, ALK-1, CD99, cytokeratin AE1/AE3, ERG, myogenin, MyoD1, CD10, calponin, ER, PR, AR, WT-1, CD117, DOG1, S100 and CD34 were performed according to standard protocols. RNA-seq was performed as described previously 1 . mRNA isolation, cDNA synthesis and library preparation were performed using an Illumina TruSeq Library Preparation Kit version 2 following the manufacturer’s protocol, and sequencing was performed on an Illumina HiSeq 2500 instrument. A customized bioinformatics pipeline for RNA-Seq analysis known as MAP-RSeq was used to assess specimens for fusions and gene expression 2 . Fluorescence in situ hybridization (FISH) for WWTR1 was performed using standard methods as previously reported 3 . Briefly, custom bacterial artificial chromosome (BAC) clone probes were designed to flank the WWTR1 gene based on the UCSC genome browser ( http://genome.ucsc.edu ),and obtained from BACPAC sources of Children’s Hospital of Oakland Research Institute (Oakland, CA; http://bacpac.chori.org ) 4 . DNA from each BAC was isolated and then labeled with fluorochromes by nick translation. The slides were deparaffinized, pretreated, and then hybridized with the denatured probes. Following an overnight incubation, the slides were rinsed, stained with 4’,6-diamidino-2-phenylindole, mounted, and examined using a Zeiss fluorescence microscope (Zeiss Axioplan, Oberkochen, Germany), controlled by Isis 5 software (Metasystems). A positive score was interpreted when at least 20% of the nuclei showed a break apart signal. Nuclei with incomplete sets of signals were omitted from the score.

Discussion

In this brief communication, we report an intra-abdominal spindle cell sarcoma which, we believe, defies current tumor classification categories. Mesenchymal neoplasms of the abdominal cavity represent a heterogenous group of tumors, with many entities being specific to abdominal visceral sites, while others overlap with sarcomas of other locations. Non-organ specific tumors like the current case, which was attached to the wall of sigmoid colon, may pose major classification challenges. Targeted RNA-based next generation sequencing was performed and a novel WWTR1::AFF2 fusion was detected. WWTR1 gene (WW domain containing transcription regulator 1) encodes for TAZ, a transcriptional coactivator with PDZ-binding motif, which is a downstream regulator in Hippo pathway and plays a critical role by restricting proliferation and promoting apoptosis 5 , 6 . WWTR1 fusions have been reported in a number of vascular neoplasms, including epithelioid hemangioendothelioma partnering with CAMTA1 3 , 7 , MAML2 and ACTL6A 4 , pseudomyogenic hemangioendothelioma 8 and epithelioid hemangioma partnering with FOSB 9 as well as other tumors such as ossifying fibromyxoid tumor partnering with KDM2A 10 , biphenotypic sinonasal carcinoma with PAX3 11 , and poroma with NUTM1 12 . AFF2 gene (AF4/FMR2 family member 2) is located at Xq28 locus and encodes a protein with putative transcriptional factor function. AFF2 gene is associated with folate sensitive Fragile XE locus on chromosome X 13 , 14 . Loss of AFF2 expression is correlated with expansion of Fragile XE locus and is implicated in mental and intellectual disability, particularly Fragile X E syndrome 13 – 18 . It has been suggested that AFF2 may be involved in controlled gene expression and regulation of RNA splicing through an interaction with G-quartet RNA structure 19 , 20 . Biased expression of AFF2 is seen in bone marrow, brain and placental tissue. Fusions of AFF2 are uncommon in solid tumors. A novel AFF2::RET fusion has been identified in a single case of lung adenocarcinoma, with the breakpoint occurring between intron 7 of AFF2 and exon 11 of RET 21 , with no other oncogenic drivers being present. Other recently reported fusions demonstrated AFF2 as the 3’ partner, similar to our current case, including recurrent DEK::AFF2 fusions in non-keratinizing squamous cell carcinomas of the sinonasal tract 22 – 24 . Thus far fewer than 20 of these head and neck squamous cell carcinomas have been identified, being characterized by the first 7 exons of DEK fused to exons 4, 6 or 9 of AFF2 , respectively. Lastly, a Japanese study reported a S TAG2::AFF2 fusion in a single case of pediatric EBV-negative peripheral T-cell lymphoma 25 . Morphologically, the differential diagnosis entertained in our case was quite broad and included leiomyosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, gastrointestinal stromal tumor, inflammatory myofibroblastic tumor and endometrial stromal sarcoma (ESS). Strong desmin expression prompts consideration of tumors of smooth muscle origin, namely leiomyosarcoma. In contrast to bona fide leiomyosarcoma, marked cytological atypia and pleomorphism were absent. Areas of tumor necrosis or brisk mitotic activity were not appreciated and immunostains for SMA and caldesmon were negative. Synovial sarcoma is characterized by expression of epithelial markers and more importantly harbor SS18 gene fusions. The architecture of long intersecting fascicles also brought up malignant peripheral nerve sheath tumor (MPNST). However, there was no association with a nerve, neural markers were negative, and the tumor did not show significant atypia often seen in MPNST. The tumor’s inflammatory background and areas of loose edematous stroma were somewhat suggestive of an inflammatory myofibroblastic tumor (IMT). IMT is a kinase-fusion positive mesenchymal neoplasm, with more than half of the cases showing alteration in ALK 26 , 27 , while remaining harbor alternative fusions involving ROS1 28 , 29 , RET 28 , PDGFRβ 29 and NTRK3 30 genes. Immunostain for ALK was negative and no ALK or other kinase fusion was identified by molecular studies. The intra-abdominal location of the tumor prompted consideration of a gastrointestinal stromal tumor, however CD34, DOG1 and CD117 were all negative. Presence of endometriosis in the close proximity to the tumor also raised the possibility of an endometrial stromal sarcoma (ESS). ESS resembles proliferative phase endometrial stroma and is divided into low grade, high grade and not otherwise specified group. ESS with smooth muscle differentiation have been documented, showing expression of desmin and caldesmon and ESS with sex cord-like elements may be additionally positive for WT-1. However, diffuse expression of desmin and WT-1 is rather unusual for an ESS diagnosis. Despite ER and PR diffuse positivity, the CD10 was entirely negative. With exception of YWHAE::NUTM2A/B fusion positive high grade ESS, most low grade ESS typically show some level of CD10 expression, either focal or diffuse. Furthermore, no connection to GYN tract was identified and subsequent TAHBSO revealed no evidence of tumor. Rare examples of extrauterine ESS, also known as endometrioid stromal sarcomas, have been reported in ovaries, bowel wall, abdomen/peritoneum, pelvis and vagina and are predominantly low grade 31 , 32 . Up to two thirds of low grade ESS are characterized by recurrent fusions, including: JAZF1::PHF1 , MBTD1-EZHIP , EPC1::PHF1 , EPC2::PHF1 , MEAF6::PHF1 , BRD8::PHF1 and EPC1::SUZ12 33 – 40 . In contrast, high grade ESS harbor YWHAE::NUTM2A/B , ZC3H7B::BCOR fusions 41 , 42 . Rare cases may reveal alternative fusions, such as EPC1::BCOR , JAZF1::BCORL1 and BRD8::PHF1 39 , 43 , 44 and a minor subset is characterized by BCOR ITD 45 – 47 . No genetic alterations involving either WWTR1 or AFF2 genes have been reported so far in ESS. In summary, we describe a unique case of an intra-abdominal spindle cell sarcoma seen adjacent to areas of endometriosis and co-expressing WT1, desmin and hormonal receptors that by RNA sequencing displayed a novel WWTR1::AFF2 fusion. Arguably the behavior of this unique and novel tumor is uncertain, however, given the deep location, striking cellularity, prominent fascicular growth, epithelioid morphology and large vesicular nuclei with conspicuous nucleoli, we find the morphology more akin to a low grade sarcoma. Additional studies are warranted to clarify its recurrent potential as well as its histogenesis and molecular kinship to other sarcomas for optimal subclassification.

Introduction

The molecular landscape of surgical pathology in general and sarcoma pathology in particular has expanded exponentially in recent years. Application of molecular studies such as RNA-based sequencing has led to identification of new classes of mesenchymal neoplasms/sarcomas and more accurate diagnosis in clinical practice. In this report, we document the first description of a soft tissue sarcoma harboring a novel WWTR1::AFF2 fusion.

Supplementary Material

Supplementary Figure 1 . Fluorescence in situ hybridization showing WWTR1 break-apart signals consistent with gene rearrangement (red: centromeric; green: telomeric).

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endometriosis

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Abdominal Cavity Abdominal Cavity Abdominal Cavity Abdominal Cavity Abdominal Cavity Abdominal Cavity Abdominal Cavity Abdominal Cavity Endometrial Neoplasms Endometrial Neoplasms Endometrial Neoplasms Endometrial Neoplasms Endometrial Neoplasms Endometrial Neoplasms Endometrial Neoplasms Endometrial Neoplasms Endometriosis Endometriosis Endometriosis Endometriosis

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