Expression profiles of Ewing family of tumors authenticate distinct molecular entities
ABSTRACT: The concept of Ewing family of tumors (EFT), characterized by FET-ETS fusions, has been recently challenged by the description of Ewing-like tumors with different gene fusions. Here we investigate the similarities and differences of FET-ETS, BCOR-CCNB3, CIC-DUX4 and EWSR1-NFATc2 tumors samples with a number of other sarcomas. Unsupervised clustering of gene expression profiles fully discriminates these four molecular entities. Specific gene signatures and pathways were further validated in model cell lines. While a clear inflammatory signature characterizes EWSR1-NFATc2 tumors, BCOR-CCNB3 and CIC-DUX4 exhibit high expression of homeobox and ETS protein families, respectively. We strongly suggest that abnormalities of chromatin remodeling may gather CIC-DUX4 and BCOR-CCNB3 tumors with rhabdoid tumors and synovial sarcomas. This dataset conains 14 CIC-DUX4 and 7 EWSR1-NFATc2 tumor samples as well as Human mesenchymal cell line expressing EWSR1-NFATc2 (duplicates) or mock treated (duplicates) and IB120 CIC-DUX4 cell line expressing an shRNA directed against CIC-DUX4 (4 replicates) or mock treated (duplicates).
Project description:Ewing sarcoma is an undifferentiated small-round-cell sarcoma. Although molecular detection of pathognomonic EWSR1-ETS fusions such as EWSR1-FLI1 enables definitive diagnosis, substantial confusion can arise if molecular diagnostics are unavailable. Diagnosis based on the conventional immunohistochemical marker CD99 is unreliable due to its abundant expression in morphological mimics. To identify novel diagnostic immunohistochemical markers for Ewing sarcoma, we performed comparative expression analyses in 768 tumors representing 21 entities including Ewing-like sarcomas, which confirmed that CIC-DUX4-, BCOR-CCNB3-, EWSR1-NFATc2-, and EWSR1-ETS-translocated sarcomas are distinct entities, and revealed that ATP1A1, BCL11B, and GLG1 constitute specific markers for Ewing sarcoma. Their high expression was validated by immunohistochemistry and proved to depend on EWSR1-FLI1-binding to highly active proximal super-enhancers. Automated cut-off-finding and combination-testing in a tissue-microarray comprising 174 samples demonstrated that detection of high BCL11B and/or GLG1 expression is sufficient to reach 96% specificity for Ewing sarcoma. While 88% of tested Ewing-like sarcomas displayed strong CD99-immunoreactivity, none displayed combined strong BCL11B- and GLG1-immunoreactivity. Collectively, we show that ATP1A1, BCL11B, and GLG1 are EWSR1-FLI1 targets, of which BCL11B and GLG1 offer a fast, simple, and cost-efficient way to diagnose Ewing sarcoma by immunohistochemistry. These markers may significantly reduce the number of misdiagnosed patients, and thus improve patient care.
Project description:PURPOSE:Recent studies revealed divergent gene expression patterns in Ewing sarcoma (EwS) with canonical EWSR1-ETS gene fusions and undifferentiated round cell sarcomas (URCS) with EWSR1 rearrangements fused to the non-ETS gene NFATc2. Thus, the question arises whether the latter tumors really belong to EwS. METHODS:We collected five cases matching the group of URCS with EWSR1-NFATc2 fusion and performed DNA methylation and copy number profiling. Results were compared to methylation data of 30 EwS with various EWSR1-ETS fusions and one EwS with FUS-ERG fusion, 16 URCS with CIC rearrangement and 10 URCS with BCOR alteration and a total of 81 EWSR1-associated soft tissue sarcomas including 7 angiomatoid fibrous histiocytomas, 7 clear cell sarcomas of the soft tissue, 28 desmoplastic small round cell tumors, 10 extraskeletal myxoid chondrosarcomas and 29 myxoid liposarcomas. RESULTS:Unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding analysis of DNA methylation data revealed a homogeneous methylation cluster for URCS with EWSR1-NFATc2 fusion, which clearly segregated from EwS and the other subtypes. Copy number profiles of EWSR1-NFATc2 cases showed recurrent losses on chromosome 9q and segmental gains on 20q13 and 22q12 involving the EWSR1 and NFATc2 loci, respectively. CONCLUSION:In summary, URCS with EWSR1-NFATc2 fusion share a distinct DNA methylation signature and carry characteristic copy number alterations, which emphasizes that these sarcomas should be considered separately from EwS.
Project description:Small blue round cell tumors (SBRCTs) are a heterogenous group of tumors that are difficult to diagnose because of overlapping morphologic, immunohistochemical, and clinical features. About two-thirds of EWSR1-negative SBRCTs are associated with CIC-DUX4-related fusions, whereas another small subset shows BCOR-CCNB3 X-chromosomal paracentric inversion. Applying paired-end RNA sequencing to an SBRCT index case of a 44-year-old man, we identified a novel BCOR-MAML3 chimeric fusion, which was validated by reverse transcription polymerase chain reaction and fluorescence in situ hybridization techniques. We then screened a total of 75 SBRCTs lacking EWSR1, FUS, SYT, CIC, and BCOR-CCNB3 abnormalities for BCOR break-apart probes by fluorescence in situ hybridization to detect potential recurrent BCOR gene rearrangements outside the typical X-chromosomal inversion. Indeed, 8/75 (11%) SBRCTs showed distinct BCOR gene rearrangements, with 2 cases each showing either a BCOR-MAML3 or the alternative ZC3H7B-BCOR fusion, whereas no fusion partner was detected in the remaining 4 cases. Gene expression of the BCOR-MAML3-positive index case showed a distinct transcriptional profile with upregulation of HOX-gene signature, compared with classic Ewing's sarcoma or CIC-DUX4-positive SBRCTs. The clinicopathologic features of the SBRCTs with alternative BCOR rearrangements were also compared with a group of BCOR-CCNB3 inversion-positive cases, combining 11 from our files with a meta-analysis of 42 published cases. The BCOR-CCNB3-positive tumors occurred preferentially in children and in bone, in contrast to alternative BCOR-rearranged SBRCTs, which presented in young adults, with a variable anatomic distribution. Furthermore, BCOR-rearranged tumors often displayed spindle cell areas, either well defined in intersecting fascicles or blending with the round cell component, which appears distinct from most other fusion-positive SBRCTs and shares histologic overlap with poorly differentiated synovial sarcoma.
Project description:CIC-DUX4 gene fusion, resulting from either a t(4;19) or t(10;19) translocation, is the most common genetic abnormality detected in EWSR1-negative small blue round cell tumors. Following their discovery it was debated if these tumors should be classified as variants of Ewing sarcoma (ie, atypical Ewing sarcoma) or as a stand-alone pathologic entity. As such the WHO classification temporarily grouped the CIC-rearranged tumors under undifferentiated sarcomas with round cell phenotype, until further clinical evidence was available. However, most studies reported so far include small series with limited follow-up information, which preclude a more definitive assessment. The present work investigates the clinicopathologic features of a large cohort of sarcomas with CIC gene rearrangement, to define their clinical presentation, morphologic spectrum, and outcome. Our study further examines the overall survival of the CIC-positive cohort compared with a control group of EWSR1-rearranged Ewing sarcoma matched for age and stage. The study cohort included 115 patients, with a mean age of 32 years and a slight male predominance. Most tumors occurred in the soft tissue (86%), predominantly deep-seated and equally divided among trunk and extremity, followed by visceral locations (12%) and rarely in the bone (3%). Microscopically, most tumors showed round to ovoid cytomorphology but half of the cases showed also focal areas of spindling and epithelioid/rhabdoid phenotype, with frequent myxoid stromal changes. Variable CD99 reactivity was seen in 84% cases, with a diffuse pattern only in 23% of cases, whereas nuclear WT1 was seen in 92%. A CIC-DUX4 fusion was detected in 57% of cases, with either DUX4 on 4q35 (35%) or on 10q26 in 25 (22%) cases. No FOXO4 gene rearrangements were present in 39 cases tested. Clinical follow-up was available in 57 patients, with a 5-year survival of 43%, which was significantly lower than the 77% 5-year survival in the control Ewing sarcoma group (P=0.002). Our findings show that CIC-DUX4 sarcomas occur most commonly in young adults within the somatic soft tissues, having a wide spectrum of morphology including round, epithelioid and spindle cells, and associated with an aggressive clinical course, with an inferior overall survival compared with Ewing sarcoma. The results support the classification of CIC-rearranged tumors as an independent molecular and clinical subset of small blue round cell tumors distinct from Ewing sarcoma.
Project description:Primitive round- or spindle-cell EWSR1-negative undifferentiated sarcomas harboring CIC-DUX4 gene fusion are the most common form of Ewing-like sarcomas. These tumors primarily occur in peripheral soft tissues, but examples have been described within viscera and the brain. As far as we are aware, CIC-DUX4 positive primary epidural spinal sarcoma has not been reported. Herein, we describe a T5-T6 epidural tumor in a 15-year-old girl in which many neoplastic cells had moderate and focally abundant cytoplasm, including plasmacytoid or rhabdoid cells, rather than the more common Ewing-like morphology described in the majority of such tumors. The diagnosis was confirmed by fluorescent in situ hybridization after the tumor was found to be WT-1 positive, and comprehensive genomic profiling demonstrated breakpoints in exon 20 and exon 1 of the CIC and DUX4 genes, respectively. After treatment with local radiation and systemic chemotherapy, resected recurrent tumor demonstrated more pleomorphic neoplastic cells as well as intracytoplasmic eosinophilic globules and nuclear pseudoinclusions which may reflect therapy-related changes. Unfortunately, there was further progression of tumor including the development of intracranial lesions, and the patient succumbed to her tumor 22 months after the original resection.
Project description:Primitive round cell sarcomas of childhood and young adults have been problematic to diagnose and classify. Our goal was to investigate the pathologic and molecular characteristics of small blue round cell tumors (SBRCT) that remained unclassified after exhaustive immunohistochemistry and molecular screening to exclude known sarcoma-related translocations. As rare examples of EWSR1-negative SBRCT have been shown to carry rearrangements for FUS and CIC genes, we undertook a systematic screening for these two genes. CIC rearrangements by FISH were detected in 15/22 (68%), while none showed FUS abnormalities. RACE, RT-PCR, and/or long-range DNA PCR performed in two cases with frozen material showed that CIC was fused to copies of the DUX4 gene on either 4q35 or 10q26.3. Subsequent FISH analysis confirmed fused signals of CIC with either 4q35 or 10q26.3 region in six cases each. Tumors positive for CIC-DUX4 fusion occurred mainly in male young adult patients (median age: 29 years), with the extremities being the most frequent location. Microscopically, tumors displayed a primitive, round to oval cell morphology with prominent nucleoli, high mitotic count, and areas of necrosis. O13 expression was variable, being either diffuse or patchy and tumors mostly lacked other markers of differentiation. Although CIC-DUX4 resulting in a t(4;19) translocation has been previously described in primitive sarcomas, this is the first report implicating the related DUX4 on 10q26 in oncogenesis. These results suggest the possibility of a newly defined subgroup of primitive round cell sarcomas characterized by CIC rearrangements, distinct from Ewing sarcoma family of tumors.
Project description:The spectrum of mesenchymal tumors associated with rearrangements of the EWSR1 gene has been growing in recent years due to progress in molecular detection techniques. Originally identified as the gene involved in the pathogenesis of Ewing sarcoma, the EWSR1 gene is now known to be rearranged in diverse clinical and histopathological entities. The NFATC2 gene is one of the many translocation partners of EWSR1 in gene fusions in a morphologically typical, albeit rare, subgroup of mesenchymal tumors. Little is known about the clinical characteristics of tumors containing NFATC2 gene rearrangements since most of the few reports published describe molecular rather than clinical aspects. In the current study, we report three patients with tumors carrying the EWSR1-NFATC2 gene translocation, including one rare primary tumor of soft tissues. Another patient with a benign-appearing bone tumor with a unique FUS-NFATC2 gene translocation is described. In various mesenchymal tumors (e.g., myxoid/round cell liposarcoma, low-grade fibromyxoid sarcoma, or angiomatoid fibrous histiocytoma), the FUS gene, as a member of the TET family, may be alternatively rearranged instead of the EWSR1 gene without any noticeable influence on the microscopical appearance or clinical outcome. This fact seems not to apply to mesenchymal tumors with the involvement of the NFATC2 gene because both in our experience and according to the extensive literature review, they have different properties on the morphological and molecular level. Both ESWSR1-NFATC2 and FUS-NFATC2 fusion-carrying tumors do not show microscopical or clinical features of Ewing sarcoma.
Project description:BCOR-CCNB3 sarcoma (BCS) is a recently defined genetic entity among undifferentiated round cell sarcomas, which was initially classified as and treated similarly to the Ewing sarcoma (ES) family of tumors. In contrast to ES, BCS shows consistent BCOR overexpression, and preliminary evidence suggests that these tumors share morphologic features with other tumors harboring BCOR genetic alterations, including BCOR internal tandem duplication (ITD) and BCOR-MAML3. To further investigate the pathologic features, clinical behavior, and their relationship to other round cell sarcomas, we collected 36 molecularly confirmed BCSs for a detailed histologic and immunohistochemical analysis. Four of the cases were also analyzed by RNA sequencing (RNAseq). An additional case with BCOR overexpression but negative CCNB3 abnormality showed a novel KMT2D-BCOR fusion by targeted RNAseq. The patients ranged in age from 2 to 44 years old (mean and median, 15), with striking male predominance (M:F=31:5). The tumor locations were slightly more common in bone (n=20) than soft tissue (n=14), with rare visceral (kidney, n=2) involvement. Histologically, BCS showed a spectrum of round to spindle cells with variable cellularity, monomorphic nuclei and fine chromatin pattern, delicate capillary network, and varying amounts of myxoid or collagenous stroma. The morphologic features and immunoprofile showed considerable overlap with other round cell sarcomas with BCOR oncogenic upregulation, that is, BCOR-MAML3 and BCOR ITD. Follow-up available in 22 patients showed a 5-year overall survival of 72%, which was relatively similar to ES (79%, P=0.738) and significantly better than CIC-DUX4 sarcomas (43%, P=0.005) control groups. Local recurrences occurred in 6 patients and distant metastases (lung, soft tissue/bone, pancreas) in 4. Seven of 9 cases treated with an ES chemotherapy regimen with evaluable histologic response showed >60% necrosis in posttherapy resections. Unsupervised clustering by RNAseq data revealed that tumors with BCOR genetic alterations, including BCOR-CCNB3, BCOR-MAML3, and BCOR ITD, formed a tight genomic group distinct from ES and CIC-rearranged sarcomas.
Project description:CIC rearrangements have been reported in two-thirds of EWSR1-negative small blue round cell tumors (SBRCTs). However, a number of SBRCTs remain unclassified despite exhaustive analysis. Fourteen SBRCTs lacking driver genetic events by RNA sequencing (RNAseq) analysis were collected. Unsupervised hierarchical clustering was performed using samples from our RNAseq database, including 13 SBRCTs with non-CIC genetic abnormalities and 2 CIC-rearranged angiosarcomas among others. Remarkably, all 14 study cases showed high mRNA levels of ETV1/4/5, and by unsupervised clustering most grouped into a distinct cluster, separate from other tumors. Based on these results indicating a close relationship with CIC-rearranged tumors, we manually inspected CIC reads in RNAseq data. FISH for CIC and DUX4 abnormalities and immunohistochemical stains for ETV4 were also performed. In the control group, only 2 CIC-rearranged angiosarcomas had high ETV1/4/5 expression. Upon manual inspection of CIC traces, 7 of 14 cases showed CIC-DUX4 fusion reads, 2 cases had DUX4-CIC reads, while the remaining 5 were negative. FISH showed CIC break-apart in 7 cases, including 5 cases lacking CIC-DUX4 or DUX4-CIC fusion reads on RNAseq manual inspection. However, no CIC abnormalities were detected by FISH in 6 cases with CIC-DUX4 or DUX4-CIC reads. ETV4 immunoreactivity was positive in 7 of 11 cases. Our results highlight the underperformance of FISH and RNAseq methods in diagnosing SBRCTs with CIC gene abnormalities. The downstream ETV1/4/5 transcriptional up-regulation appears highly sensitive and specific and can be used as a reliable molecular signature and diagnostic method for CIC fusion positive SBRCTs.