Fusion of EWSR1 with the DUX4 facioscapulohumeral muscular dystrophy region resulting from t(4;22)(q35;q12) in a case of embryonal rhabdomyosarcoma

doi:10.1016/j.cancergencyto.2009.06.011

Cancer Genetics and Cytogenetics

Volume 195, Issue 1, November 2009, Pages 12-18

https://doi.org/10.1016/j.cancergencyto.2009.06.011 Get rights and content

Abstract

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and rarely occurs in adults. There are six main subtypes, each histologically, clinically, and cytogenetically distinct. Embryonal RMS is characterized by chromosomal gains, usually not associated with any consistent structural anomaly. We describe here a case of embryonal RMS in a 19-year-old female patient. The conventional cytogenetic analysis showed a t(4;22)(q35;q12) translocation as the sole cytogenetic change. Complementary fluorescence in situ hybridization analysis showed that the translocation breakpoints were located in the EWSR1 gene at 22q12 and the region of the DUX4 and FSHMD1A at 4q35. This constitutes a novel example of the high frequency of EWSR1 rearrangements in various types of sarcomas as well as of its ability to fuse with a large variety of partner genes. Because DUX4 is involved in myogenic differentiation and cell-cycle control, the striated muscle differentiation observed in the present case might be a direct consequence of the alteration of the DUX4 region generated by the t(4;22). The involvement of the DUX4 region might represent the genetic hallmark of a novel subclass of small round cell tumors.

Introduction

Rhabdomyosarcoma (RMS) belongs to a unique entity characterized by morphologic, immunohistochemical and ultrastructural features of embryonic skeletal muscle differentiation. Although RMS is the most common pediatric soft tissue sarcoma, it rarely occurs in adults. Six main subtypes of RMS have been established: embryonal, botryoid, spindle cell, alveolar, anaplastic, and undifferentiated. In addition, a category of sarcoma not otherwise specified (NOS) was created for tumors that could not be classified into a specific subtype. Finally, pleomorphic RMS was individualized as aggressive lesions with a poor outcome similar to other pleomorphic sarcomas of adults. The two major histological subtypes of RMS contains distinct genetic alterations that are presumed to play a role in the pathogenesis of these tumors [1].

Alveolar RMS is characterized by a t(2;13)(q35;q14), resulting in the fusion of the FOXO1 gene (alias FKHR) at 13q14 with PAX3, or less commonly a t(1;13)(p36;q14) resulting in fusion of FOXO1 with PAX7[2], [3]. The presence of the PAX7–FOXO1 fusion is correlated with a better prognosis than PAX3–FOXO1 for patients presenting with metastatic alveolar RMS [4]. The few cytogenetic reports of pleomorphic RMS have described complex karyotypes with numerous rearrangements and evidence of gene amplification, as is usual in pleomorphic sarcomas [5]. Most embryonal RMS cases are cytogenetically characterized by numerical chromosome alterations, such as gains of chromosomes 2, 7, 8, 9, 11, 12, 13, 17, 18, 19, or 20 [6]. In addition, loss of heterozygosity at the 11p15 locus has been described [7]. The chromosomal region 11p15 is the location of the IGF2 gene, which codes for a growth factor potentially involved in the pathogenesis of embryonal RMS [8]. Amplification of 15q25∼q26, the locus for the insulin-like growth factor receptor gene (IGF1R) has been described in some cases of embryonal RMS with anaplasia [9]. In addition, simple structural rearrangements as the sole anomalies have been documented in a few pediatric cases [10], [11], [12], [13], [14].

Here we describe a case of embryonal RMS in a young adult, with a novel structural aberration t(4;22)(q35;q12) involving the EWSR1 gene (alias EWS) at 22q12 and the region at 4q35 with the genes DUX4 and FSHMD1 (facioscapulohumeral muscular dystrophy 1A; previously FSHD).

Section snippets

Case report

A 19-year-old female patient presented with a 3-month history of progressive enlargement of her right thigh. Clinical examination revealed a tender, mobile, subcutaneous mass of the thigh posterior face. The patient was otherwise in good health, and her past medical history was unremarkable. A computed tomography scan showed a well-circumscribed and hypodense mass of 12 cm in size arising within the sartorius muscle. An intense enhancement of the whole tumor was noted after contrast

Histologic and immunohistochemical analysis

The tumor material was fixed in 4% phosphate-buffered formalin and embedded in paraffin, and histological sections were stained with hematoxylin–eosin–saffron (HES). Immunohistochemical analysis was performed on paraffin-embedded tissue according to the avidin–biotin complex standard method in a Techmat device (Dako, Glostrup, Denmark; Trappes, France). The following monoclonal and polyclonal antibodies were used: AE1/AE3 (monoclonal, clone AE1/AE3, 1/100; Dako), epithelial membrane antigen

Pathological and immunohistochemical findings

The surgical specimen showed a well-defined intramuscular mass measuring 9×4 cm with tan-yellow appearance on cut surface. No hemorrhagic or necrotic area was noted. Histologically, the proliferation showed varying degrees of cellularity with densely cellular areas and alternating abundant fibromyxoid stroma. It was composed predominantly of small ovoid or spindle-shaped cells with slightly irregular nuclei dense chromatin and indistinct cytoplasm. Occasionally, spindled cells were arranged in

Discussion

We have described a case of embryonal RMS in a 19-year-old woman. Because RMS in adults is uncommon, and is represented mainly by the pleomorphic subtype, this tumor was initially misclassified as a pleomorphic RMS. According to its predominant histological features, however, the final diagnosis was embryonal RMS. Both the excellent clinical evolution and the simple cytogenetic features were also more compatible with an embryonal than with a pleomorphic RMS. Indeed, pleomorphic RMSs are

Acknowledgments

This work was supported by the Alpes-Maritimes committee of the French National League Against Cancer (Comité des Alpes-Maritimes de la Ligue Nationale Contre le Cancer) and by the French National Cancer Institute (Institut National du Cancer).

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Cited by (31)

Control of DUX4 Expression in Facioscapulohumeral Muscular Dystrophy and Cancer
2021, Trends in Molecular Medicine
Citation Excerpt :
Aberrant DUX4 expression is considered to be one of the major causes of facioscapulohumeral dystrophy (FSHD), an autosomal-dominant disorder characterized by progressive and asymmetric muscle weakness [9]. Recently, chromosomal DUX4 rearrangements were implicated in the pathogenesis of rhabdomyosarcoma (RMS) [10], Ewing-like sarcoma [11], and B cell precursor acute lymphoblastic leukemia (BCP-ALL) [12]. All of these translocations result in the production of putative chimeric proteins implicated in oncogenesis (reviewed in [13]) (Figure 1, Key Figure).
DUX4, a gene encoding a transcription factor involved in early embryogenesis, is located within the D4Z4 subtelomeric repeat on chromosome 4q35. In most healthy somatic tissues, DUX4 is heavily repressed by multiple genetic and epigenetic mechanisms, and its aberrant expression is linked to facioscapulohumeral muscular dystrophy (FSHD) where it has been extensively studied. Recently, DUX4 expression has been implicated in oncogenesis, although this is much less explored. In this review, we discuss multiple levels of control of DUX4 expression, including enhancer–promoter interactions, DNA methylation, histone modifications, noncoding RNAs, and telomere positioning effect. We also connect disparate data on intrachromosomal contacts involving DUX4 and emphasize the feedback loops in DUX4 regulation. Finally, we bridge data on DUX4 in FSHD and cancer and discuss prospective approaches for future FSHD therapies and the potential outcomes of DUX4 inhibition in cancer.
DUX4 Pathological Expression: Causes and Consequences in Cancer
2019, Trends in Cancer
DUX4, a double homeobox transcription factor, has been mostly studied in facioscapulohumeral dystrophy (FSHD), a pathology linked to a deletion of subtelomeric repeats on chromosome 4q. More recently, however, the gene has been associated with various sarcomas and haematological malignancies. Drugs developed for FSHD could be tested on cancer cells to develop efficient treatment strategies for both pathologies.
A t(4;19) pediatric undifferentiated sarcoma with a novel variant of the CIC-DUX4 fusion transcript
2017, Pathology Research and Practice
We report cytogenetic and molecular genetic analysis of a pediatric tumor positive for the CIC-DUX4 fusion. The tumor belongs to a rare, diagnostically challenging subgroup of undifferentiated small round cell sarcomas. A balanced t(4;19)(q35;q13.1-2) was identified by G-banding, as a sole cytogenetic finding. The translocation was also identified by the M-FISH technique. After RT-PCR, the tumor sample was positive for the CIC-DUX4 fusion. The PCR product contains a novel, so far unreported variant of the CIC-DUX4 fusion transcript, with a fusion of the exon 20 from the CIC gene and the exon 1 from the DUX4 gene.
Defining Ewing and Ewing-like small round cell tumors (SRCT): The need for molecular techniques in their categorization and differential diagnosis. A study of 200 cases
2016, Annals of Diagnostic Pathology
Citation Excerpt :
So far, despite the extensive IHC and molecular assays, a small proportion of SRCS with Ewing morphology remain undifferentiated due to negative or non-informative molecular results, leading to uncertainty in diagnosis and treatment options [2–5,8–22]. At present, tumors with Ewing-like morphology displaying FUS-ETS partners (ERG, FEV) are very infrequent [2–4,8,10,17–22], however a new group of Ewing-like sarcomas is emerging which reveals the CIC-DUX4 [2,3,23–37], BCOR-CCNB3 [2,3,37–43] or CIC-FOX04 gene fusions [44,45]. Recent data suggest that CIC-DUX4 and BCOR-CCNB3 fusions are more prevalent than CIC-FOX04 [2,3,23–45].
Differentiation of Ewing sarcoma family of tumors (ESFT) and Ewing-like tumors remains problematic. Certain ESFT with morphological and immunohistochemical (IHC) profiles lack the EWSR1-ETS transcript. To improve diagnostic accuracy we investigated the presence of several specific transcripts in 200 small round cell tumors (SRCT) displaying ESFT morphology and immunophenotype in which EWSR1 FISH analysis was non-informative or negative.
200 tumors (formalin-fixed, paraffin-embedded) were analyzed by RT-PCR. All tumors were tested for EWSR1-ETS, EWSR1/WT1, PAX3/7-FOX01 or SYT/SSX transcripts, and the negative tumors were subsequently analyzed for CIC/DUX4, BCOR/CCNB3 and CIC/FOX04 transcripts.
133 (66.5%) ESFT displayed one of the above EWSR1-ETS translocations. Three cases (1.5%) revealed the SYT-SSX transcript for Synovial sarcoma, and one (0.5%) a EWSR1-WT1 transcript for Desmoplastic Small Round Cell tumor. The CIC-DUX4 translocation was found in six Ewing-like tumors (3%) with CD99 positivity. The BCOR-CCNB3 gene fusion was observed in 5 tumors (2.5%) displaying round or spindle cells with strong CCNB3 IHC expression in 3 tumors. Moreover, RT-PCR failed to detect any gene fusion transcripts in 19 tumors (9.5%) and were considered “undifferentiated small round cell sarcoma” (SRCS). Molecular biology results were non-informative in 33 SRCTs (16.5%) due to RNA degradation through inadequate fixation and/or decalcification.
Our analysis of 200 SRCTs confirms the molecular heterogeneity of neoplasms with ESFT morphology and highlight that molecular studies with RT-PCR including new emerging gene fusion transcripts are mandatory for the diagnosis when EWSR1 FISH is negative or non-informative. The incidence of CIC-DUX4, BCOR-CCNB3 and CIC-FOX04 transcripts was relatively low. A small group of Ewing-like sarcomas or undifferentiated SRCS remains unclassified. Adopting appropriate tissue fixation and processing protocols is important to avoid degradation of fixed/embedded tissue when no frozen tumor is available.
Ewing/PNET sarcoma family of tumors: Towards a new paradigm?
2015, Annales de Pathologie
Les tumeurs de la famille Ewing/PNET sont des sarcomes agressifs majoritairement osseux mais survenant également au niveau des tissus mous, qui présentent des caractéristiques communes : morphologiques sous la forme de tumeurs à cellules rondes basophiles, immunophénotypiques avec une positivité membranaire du CD99, et génétiques avec la présence d’une translocation impliquant les gènes EWS et FLI1 dans environ 90 % des cas. C’est la découverte de cette anomalie moléculaire qui a permis d’unifier en une seule entité des lésions jusqu’alors décrites séparément, telles que les PNET, neuroépithéliomes, tumeurs d’Askin, sarcomes d’Ewing… Depuis, l’utilisation extensive de techniques de biologie moléculaire toujours plus performantes a contribué à la découverte d’un nombre croissant d’anomalies moléculaires dans des sarcomes à cellules rondes inclassés jusqu’alors sans transcrit connu, souvent atypiques morphologiquement et plus rarement CD99+ : outre les translocations entre la famille de gènes FET (EWS ou FUS) et la grande famille de gènes ETS (FLI1, ERG, FEV, ETV…), sont progressivement identifiés de nouveaux gènes partenaires de translocations. On compte de rarissimes cas à transcrit EWS-non ETS mais aussi des groupes plus importants que sont les sarcomes à translocation CIC-DUX4 et BCOR-CCNB3. Ces découvertes posent le problème des limites nosologiques de l’entité Ewing/PNET et de ses liens avec ces nouveaux groupes, ainsi que le problème de leur prise en charge thérapeutique. L’identification prospective de ces nouveaux sarcomes à cellules rondes devrait permettre l’étude de plus larges séries pour tenter de répondre à ces questions.
Ewing sarcoma family of tumors are mainly aggressive sarcomas of bone and also arising in soft tissues, which share common features: morphological features of basophilic round cell tumors, immunohistochemical features by expression of membrane CD99 protein, and genetic features with a translocation involving EWS and FLI1 in approximately 90% of cases. The discovery of this translocation has made it possible to unify in a single entity several lesions such as PNET, neuropitheliomas, Askin tumors, Ewing sarcomas… Since then, the extensive use of molecular/genetic methods has helped to identify an increasing number of molecular anomalies in unclassified round cell sarcomas, these sarcomas often harboring an atypical morphology and a less frequent CD99 positivity. Besides the rearrangements between the FET family of genes (EWS or FUS) and the wide ETS family of genes (FLI1, ERG, FEV, ETV…), new partner genes are gradually identified: cases with EWS-non ETS partners are extremely rare, but there are more important groups which are CIC-DUX4 and BCOR-CCNB3 translocation-positive sarcomas. These findings raise the problem of the nosological borders of the Ewing/PNET entity and its links with new “Ewing-like” groups of tumors, and raise the therapeutic problems. The forward-looking identification of new round cell sarcomas should enable studies of wider series to try to answer these questions.
The CIC-DUX4 fusion transcript is present in a subgroup of pediatric primitive round cell sarcomas
2012, Human Pathology
Citation Excerpt :
DUX4 is located within the tandem repeat locus D4Z4 on chromosome 4 and contains 2 DNA-binding homeoboxes at its N-terminus [30]. DUX4 has been implicated in the pathogenesis of facioscapulohumeral muscular dystrophy, in which the D4Z4 copy number is significantly reduced [31,32], and in embryonal RMS where DUX4 is involved in a chromosomal rearrangement with the EWSR1 gene [33]. Under normal conditions, DUX4 is believed to be involved in myogenic differentiation and cell-cycle control [33], and overexpression of this gene in cultured primary myoblasts has been shown to induce caspase activity and promote cell death [34].
Pediatric undifferentiated soft tissue sarcomas are a group of diagnostically challenging tumors. Recent studies have identified a subgroup of undifferentiated soft tissue sarcomas with primitive round to plump spindle cell morphology and a t(4;19)(q35;q13.1) translocation resulting in the expression of a CIC-DUX4 fusion transcript, including 2 tumors previously reported by our laboratory (Cancer Genet Cytogenet 2009;195:1). In the present study, reverse transcriptase polymerase chain reaction assays developed for both frozen and paraffin-based tissues were applied to a series of 19 pediatric undifferentiated soft tissue sarcomas using a combination of primer sets covering the CIC-DUX4 fusion transcript. Of the 19 undifferentiated soft tissue sarcomas, 16 had primitive round to plump spindle cell morphology, and 3 had pure spindle cell morphology. Three of the 16 undifferentiated soft tissue sarcomas with primitive round cell morphology were found to harbor the CIC-DUX4 fusion transcript by reverse transcriptase polymerase chain reaction. Automated DNA sequencing of the polymerase chain reaction products identified 2 distinct transcript variants. One CIC-DUX4–positive tumor showed membranous CD99 positivity, 2 showed focal S100 positivity, and 1 showed focal CD57 positivity. With the 2 previously reported cases, the total number of CIC-DUX4–positive primitive round cell sarcomas identified at our institution has been brought to 5 (28%) of 18. Given the consistent involvement of the CIC-DUX4 fusion in a subset of primitive round cell undifferentiated soft tissue sarcomas, these findings suggest a central role for the fusion transcript in such tumors. The current findings further define a novel genetic subset of pediatric primitive round cell sarcomas and provide an additional diagnostic tool for their characterization and diagnosis.

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Original articleFusion of EWSR1 with the DUX4 facioscapulohumeral muscular dystrophy region resulting from t(4;22)(q35;q12) in a case of embryonal rhabdomyosarcoma

Abstract

Introduction

Section snippets

Case report

Histologic and immunohistochemical analysis

Pathological and immunohistochemical findings

Discussion

Acknowledgments

Neoplasia

Cancer Genet Cytogenet

Cancer Genet Cytogenet

Cancer Genet Cytogenet

Cancer Genet Cytogenet

Cancer Genet Cytogenet

Gene

Cancer Cell

Rhabdomyosarcomas in adults and children: an update

Arch Pathol Lab Med

Fusion of PAX3 to a member of the forkhead family of transcription factors in human alveolar rhabdomyosarcoma

Cancer Res

Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma

Cancer Res

PAX3–FKHR and PAX7–FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report of the Children's Oncology Group

J Clin Oncol

Cytogenetic analysis of 46 pleomorphic soft tissue sarcomas and correlation with morphologic and clinical features

Genes Chromosomes Cancer

Genes, chromosomes and rhabdomyosarcoma

Genes Chromosomes Cancer

Molecular differential pathology of rhabdomyosarcoma

Genes Chromosomes Cancer

Original article
Fusion of EWSR1 with the DUX4 facioscapulohumeral muscular dystrophy region resulting from t(4;22)(q35;q12) in a case of embryonal rhabdomyosarcoma