Fusion Sarcoma Research Articles

The Biological Significance of AFF4: Promoting Transcription Elongation, Osteogenic Differentiation and Tumor Progression.

As a member of the AF4/FMR2 (AFF) family, AFF4 is a scaffold protein in the superelongation complex (SEC). In this mini-view, we discuss the role of AFF4 as a transcription elongation factor that mediates HIV activation and replication and stem cell osteogenic differentiation. AFF4 also promotes the progression of head and neck squamous cell carcinoma, leukemia, breast cancer, bladder cancer and other malignant tumors. The biological function of AFF4 is largely achieved through SEC assembly, regulates SRY-box transcription factor 2 (SOX2), MYC, estrogen receptor alpha (ESR1), inhibitor of differentiation 1 (ID1), c-Jun and noncanonical nuclear factor-κB (NF-κB) transcription and combines with fusion in sarcoma (FUS), unique regulatory cyclins (CycT1), or mixed lineage leukemia (MLL). We explore the prospects of using AFF4 as a therapeutic in Acquired immunodeficiency syndrome (AIDS) and malignant tumors and its potential as a stemness regulator.

BCOR::CCNB3 fusion sarcoma: a clinicopathological analysis of three cases

BCOR::CCNB3 fusion sarcoma: a clinicopathological analysis of three cases

The "Other" Uterine Mesenchymal Neoplasms: Recent Developments and Emerging Entities.

Uterine mesenchymal neoplasms are a challenging group of tumors that often show overlapping morphologic features and immunohistochemical profiles. The increasing use of molecular testing in these tumors has enabled a better appreciation of their pathobiology, resulting in a wave of emerging neoplasms and improved characterization of ones previously considered exceptionally rare. Identification of specific molecular alterations has permitted targeted therapy options in tumors that were typically unresponsive to conventional therapies, as well as recognition that a subset can have a hereditary basis. This review will discuss the more "common" of the uncommon uterine mesenchymal neoplasms, including inflammatory myofibroblastic tumor, perivascular epithelioid cell tumor, uterine tumor resembling ovarian sex cord tumor, and embryonal rhabdomyosarcoma. This will be followed by an overview of emerging entities, including NTRK-rearranged uterine sarcoma, SMARCA4-deficient uterine sarcoma, KAT6B/A::KANSL1 fusion uterine sarcoma, and MEIS1::NCOA2/1 fusion sarcoma.

A Novel EPC1 :: KDM2B Fusion in High-grade Endometrial Stromal Sarcoma.

The spectrum of endometrial stromal sarcoma (ESS) has expanded substantially since the publication of the most recent World Health Organisation (WHO) Classification of Female Genital Tumours and the advent of widely available genomic testing. We describe a uterine mesenchymal tumor harboring a novel EPC1 :: KDM2B fusion, best classified within the umbrella of high-grade endometrial stromal sarcoma (HGESS). This tumor was composed of a uniform population of spindled cells with some myxoid stroma, a mitotic rate of up to 21/10 high-power fields, and a largely pushing margin with focal vascular invasion. Immunohistochemistry showed strong and diffuse cyclin D1 positivity while CD10, WT1, DOG1, CD117, CD34, CD99, S100, MelanA, SMA, desmin, and h-caldesmon were negative. The tumor was confined to the uterus and no recurrence has been detected thus far, albeit with a short follow-up interval of 9mo.

Dynamic network curvature analysis of gene expression reveals novel potential therapeutic targets in sarcoma

Network properties account for the complex relationship between genes, making it easier to identify complex patterns in their interactions. In this work, we leveraged these network properties for dual purposes. First, we clustered pediatric sarcoma tumors using network information flow as a similarity metric, computed by the Wasserstein distance. We demonstrate that this approach yields the best concordance with histological subtypes, validated against three state-of-the-art methods. Second, to identify molecular targets that would be missed by more conventional methods of analysis, we applied a novel unsupervised method to cluster gene interactomes represented as networks in pediatric sarcoma. RNA-Seq data were mapped to protein-level interactomes to construct weighted networks that were then subjected to a non-Euclidean, multi-scale geometric approach centered on a discrete notion of curvature. This provides a measure of the functional association among genes in the context of their connectivity. In confirmation of the validity of this method, hierarchical clustering revealed the characteristic EWSR1-FLI1 fusion in Ewing sarcoma. Furthermore, assessing the effects of in silico edge perturbations and simulated gene knockouts as quantified by changes in curvature, we found non-trivial gene associations not previously identified.

Successful Crizotinib-targeted Therapy of Pediatric Unresectable ERC1::ALK Fusion Sarcoma.

Anaplastic lymphoma kinase ( ALK )-fusion sarcomas are rare part of the emerging theoretically targetable tyrosine kinase RAS::MAPK pathway fusion myopericytic-ovoid sarcomas. We report our clinicopathologic and treatment experience with an ALK fusion sarcoma. A novel ELKS/RAB6-interacting/CAST family member 1 - unaligned ALK fusion infiltrative nonmetastatic low-grade sarcoma of the right hand of a 15-month-old male was treated with crizotinib, an ALK tyrosine kinase inhibitor as oral monotherapy, inducing complete radiographic and clinical resolution by 10 months and sustained response now over 12 months after elective discontinuation. Crizotinib can successfully be used to treat unresectable novel ALK fusion sarcomas.

Identification of dual STRN-NTRK2 rearrangements in a high grade sarcoma, with good clinical response to first-line larotrectinib therapy

BackgroundAmong the three NTRK genes, NTRK2 possesses a tremendous structural complexity and involves tumorigenesis of several types of tumors. To date, only STRN and RBPMS are identified in the fusion with NTRK2 in adult soft tissue tumors. More recently, the highly selective Trk tyrosine kinases inhibitors, including larotrectinib and entrectinib, have shown significant efficacy for treating tumors harboring NTRK fusions and were approved by FDA.Case presentationWe report a case of sarcoma in a 35-year-old female harboring two STRN-NTRK2 gene fusions, with a good clinical response to first-line larotrectinib treatment. Core biopsy of the 16.5 cm gluteal mass showed a high-grade mesenchymal neoplasm with features reminiscent of a solitary fibrous tumor, but negative for STAT6. In-house next-generation sequencing gene fusion panel showed two in-frame STRN-NTRK2 fusions, which contain the same 5’ partner sequence (exon 1–3) of STRN, and the 3’ fusion partner starting from either the exon 15 or the exon 16 of NTRK2. Due to the large size and location of the tumor, first-line neoadjuvant therapy with larotrectinib was initiated. The patient has an excellent clinical response with an 83% tumor size reduction by imaging. The tumor was subsequently completely resected. After 130 days, larotrectinib was reinitiated for lung metastasis (up to 7 cm), and a complete resolution was achieved. When compared with NTRK1 and NTRK3, NTRK2 fusions are the least common. Of note, the only other report in the literature on NRTK2 fusion-positive sarcoma also showed solitary fibrous tumor (SFT)-like morphology, and the patient responded well to larotrectinib as the second line adjuvant therapy.ConclusionsIn conclusion, the identification of NTRK2 fusions in patients with soft tissue tumors could significantly improve the clinical outcome through selective NTRK inhibitor therapy, especially in the first-line setting. Prompt RNA-based NGS testing at initial diagnosis may benefit these patients. Our case is among the first few in the literature on NTRK2 fusion sarcoma with first-line larotrectinib therapy in the primary and metastatic setting, with good clinical response and minimal side effects.

Abstract LB078: pbfusion: Detecting gene-fusion and other transcriptional abnormalities using PacBio HiFi data

Abstract Sarcomas are a broad group of soft tissue and bone cancers that can be difficult to treat leading to a high mortality rate. Sarcomas comprise two broad genomic classes: (1) simple karyotypes, where a single oncogenic structural variant (SV) clonally expands a subtype that is diagnostic and relevant to tumor burden tracking; and (2) complex karyotypes, genomic instability, where SVs continuously arise throughout tumor evolution resulting in heterogeneous cellular subtypes. Class two sarcomas are harder to characterize using genome sequencing because there may be multiple low-frequency mutations. In both genomic classes, accurate and sensitive detection of fusion transcripts is needed to interpret functional consequences, to understand tumor biology and evolution, and potentially identify new targets for therapy. Many fusions have complex structures that cannot be uniquely resolved using short reads due to a lack of exon connectivity. PacBio full-length RNA isoform sequencing resolves complex fusions, providing more accurate breakpoints, and a complete sequence readout of the associated fusion transcript. To date, long-read fusion detection software was designed for high-error sequencing. PacBio HiFi data provides both full-length transcripts and accurate base calls. Here we present a fusion detection tool, pbfusion, specifically designed for HiFi sequence data, and apply it to sarcoma patients from both classes. pbfusion converts mapped sequences (either HiFi reads or Iso-Seq isoforms) into transcript objects that are annotated with reference gene models. Annotations determine whether transcripts are discordantly mapped, overlap differing genes, strand swap, transcriptional readthrough, or contain novel exons. The discordant exonic boundaries are treated as breakpoints between two genomic locations. All breakpoints are clustered with a multi-directional chaining algorithm and annotated with exonic information, gene names, and quality information. To test our method, we applied pbfusion to twelve samples from 8 sarcoma patients from both genomic classes. We discovered the known and novel fusions, including validated driver events in the fusion-driven samples (e.g. ASPSCR1-TFE3 in alveolar soft part sarcoma and SS18-SSX2/1 fusion in synovial sarcoma). This approach demonstrates the utility of HiFi sequence data for identification of fusion transcripts in patient samples, and the use of pbfusion in quantifying and annotating these events. pbfusion provides a user-friendly interface, can process a sample in a few minutes, and is freely available to the research community on Bioconda. Citation Format: Roger Volden, Zev Kronenberg, Aaron Gillmor, Ted Verhey, Michael Monument, Donna Senger, Harsharan Dhillon, Jason Underwood, Elizabeth Tseng, Daniel Baker, Primo Baybayan, Michael A. Eberle, Jonas Korlach, Sorana Morrissy. pbfusion: Detecting gene-fusion and other transcriptional abnormalities using PacBio HiFi data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB078.

Abstract 4545: Integrative immune-genomic comparison of canonical Ewing sarcoma with Ewing-like mimics identifies potential targets for personalized therapies

Abstract Introduction: Ewing sarcoma (ES) is one of the most common types of small round cell sarcoma (SRCS) from the skeletal origin, characterized by small round blue cell morphology and FET-ETS fusions. However, recent NGS-based technology has dramatically increased the characterization of other SRCS entities that share morphological, pathological, and clinical similarity with canonical ES. Currently, these sarcomas are often treated with similar regimens derived from ES. Whether any subsets of these sarcomas might benefit from personalized anti-cancer strategies remains largely unknown. Methods: To explore potential therapeutic vulnerabilities, 51 FFPE samples from 49 patients (median age 31 years, 65.3% male) diagnosed as ES or its mimics by conventional pathology review were sent to DNA (OncoScreen®Plus) and RNA sequencing (OncoRNA, Burning Rock Biotech) for genomic and immune signature profiling. Tumor samples were classified into 5 groups based on their similarity with classic ES: (a) Canonical ES (n=12), defined as ES with EWSR1-FLI1fusion and a primary bone origin; (b) Non-canonical ES, which were found with either non-EWSR1-FLI1 FET-ETS fusion, or ES in extra-skeletal sites (n=11); (c) Non-FET-ETS fusion SRCS (n=10), defined as SRCS with gene fusions other than FET-ETS family; (d) Fusion-negative SRCS (n=4); and (e) non-SRCS, where the diagnosis was primarily suspected as ES but finally re-diagnosed as another histology (n=12). Results: The NGS-based diagnosis rectified the pathological diagnosis in 9/51 samples, including ES misdiagnosed as other (n=3), other misdiagnosed as ES (n=4), Non-FET-ETS fusion SRCS misdiagnosed as ES (n=1) and ALK fusion sarcoma misdiagnosed as SRCS (n=1). In the remaining 42 samples, 23 diagnoses were confirmed by both NGS and FISH, while 11 SRCS were negative in both NGS and FISH assay, yielding a concordance of 81.0% (34/42). In the 8 samples with discordant diagnosis, a definitive diagnosis was achieved by FISH but not NGS in 1 of 8 (12.5%), and vice versa in 7 of 8 (87.5%). Interestingly, genomic profiling revealed that Non-FET-ETS fusion SRCS had high frequencies of TP53 mutations (40%) and copy number loss (including 30% in CD274 and JAK2; 20% in PTCH1, CDKN2A/B, and MTAP) and featured higher levels of homologous recombination deficiency (HRD) (p=0.024) and chromosome instability (p=0.022) than other groups. Remarkably, non-canonical ES demonstrated upregulated PDCD1 expression (p=0.034). The distribution of immune hot and cold subtypes was comparable among all groups (p=0.334). Conclusion: Our study shows that NGS could efficiently facilitate the definitive diagnosis of ES and its mimics. We also revealed a diverse immune-genomic landscape of these SRCS, indicative of potential therapeutic opportunities targeting HRD and immune check-point in specific subtypes of these sarcoma entities. Citation Format: Zhusheng Zhang, Qiyuan Bao, Junxiang Wen, Zhuochao Liu, Qi Liu, Yuhui Shen, Lin Shao, Bing Li, Song-An Chen, Weibin Zhang. Integrative immune-genomic comparison of canonical Ewing sarcoma with Ewing-like mimics identifies potential targets for personalized therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4545.

Han family with essential tremor caused by the P421L variant of the TENM4 gene in China.

Essential tremor (ET) is an autosomal dominant inheritance disorder. Mutations in fusion sarcoma (FUS), mitochondrial serine peptidase 2 (HTRA2), teneurin transmembrane protein 4 (TENM4), sortilin1 (SORT1), SCN11A, and notch2N-terminal-like (NOTCH2NLC) genes are associated with familial ET. A proband with ET was tested using whole-exome sequencing and repeat-primed polymerase chain reaction. Subsequently, the family members were screened for the suspected mutation, and the results were verified using Sanger sequencing. The relationship between pedigree and phenotype was also analyzed, and structural and functional changes in the variants were predicted using bioinformatics analysis. In a family with ET, the proband (III4) and the proband's father (II1), grandfather (I1), uncle (II2), and cousin (III5) all presented with involuntary tremors of both upper limbs. The responsible mutation was identified as TENM4 c.1262C > T (p.P421L), which showed genetic co-segregation in the family survey. AlphaFold predicted a change in the spatial position of TENM4 after the P421L mutation, which may have affected its stability. AlphaFold also predicted P421L to be a deleterious variation, which would lead to lower degrees of freedom of the TENM4 protein, thereby affecting the protein's structure and stability. According to the bioinformatics analysis, TENM4 (p.P421L) may reduce the signal reaching the nucleus by affecting the expression of TENM4 messenger RNA (mRNA), thereby impairing the normal oligodendrocyte differentiation process and leading to impaired myelination. This study revealed that the TENM4 (p.P421L) pathogenic missense variation was responsible for ET in the proband.

ETMR-02. NOVEL CIC-LEUTX FUSION IN CNS EMBRYONAL TUMOR: A CASE REPORT AND REVIEW OF THE LITERATURE

INTRODUCTIONCentral nervous system (CNS) embryonal tumor is a group of rare, poorly differentiated neuroepithelial malignant neoplasm predominantly occurs in pediatrics. Herein, we firstly report a CNS embryonal tumor harboring pathogenic CIC-LEUTX fusion.METHODSImmunohistochemistry (IHC), Fluorescence in Situ Hybridization (FISH) and Next Generation sequencing (NGS).RESULTSA 2-year-old male was found to have solid and cystic mass in left temporal lobe-basal ganglia and left parietal lobe (maximum diameter=75mm). The pathological diagnosis was CNS embryonal tumor (NEC) after totally resection. The tumor was poorly differentiated embryonal neoplasms of neuroectodermal origin that lacked the specific features and rosettes. IHC showed Syn was strongly/diffusely positive and Ki67 proliferation index was high (50%+), and copy number at the 19q13.42 C19MC locus showed no alterations. NGS showed pathogenic mutations including a brand new CIC-LEUTX fusion, heterozygous germline NBN c.C127T mutation and somatic TSC2 c.G2714A mutation. One month after operation, intracranial tumor recurred (maximum diameter=55mm) and spinal cord implantation metastasis occurred, and then the patient received chemotherapy (CTX+CBP+VCR/DDP+VP-16) and had significant improvement in symptoms and tumor shrinkage (maximum diameter=31mm). Literature review revealed CIC fusion predominantly presented in sarcomas, such as CIC-NUTM1 fusion in rare CNS sarcoma, CIC-LEUTX fusion in epithelioid angiosarcoma and CIC-DUX4 fusion in Ewing-like sarcoma. Hitherto, apart from this case, there were only two cases which had CIC-LEUTX fusion in CNS, including a case of CNS angiosarcoma and a case of anaplastic ganglioglioma.CONCLUSIONSWe firstly found a specific new type in CNS embryonal tumor with distinct molecular-pathological characteristics of CIC-LEUTX fusion.

Clinicopathological findings of pediatric NTRK fusion mesenchymal tumors

BackgroundWhile ETV6- NTRK3 fusion is common in infantile fibrosarcoma, NTRK1/3 fusion in pediatric tumors is scarce and, consequently, not well known. Herein, we evaluated for the presence of NTRK1/3 fusion in pediatric mesenchymal tumors, clinicopathologically and immunophenotypically.MethodsWe reviewed nine NTRK fusion-positive pediatric sarcomas confirmed by fluorescence in situ hybridization and/or next-generation sequencing from Seoul National University Hospital between 2002 and 2020.ResultsOne case of TPR-NTRK1 fusion-positive intracranial, extra-axial, high-grade undifferentiated sarcoma (12-year-old boy), one case of LMNA-NTRK1 fusion-positive low-grade infantile fibrosarcoma of the forehead (3-year-old boy), one case of ETV6-NTRK3 fusion-positive inflammatory myofibroblastic tumor (IMT) (3-months-old girl), and six cases of ETV6-NTRK3 fusion-positive infantile fibrosarcoma (median age: 2.6 months, range: 1.6–5.6 months, M: F = 5:1) were reviewed. The Trk immunopositivity patterns were distinct, depending on what fusion genes were present. We observed nuclear positivity in TPR-NTRK1 fusion-positive sarcoma, nuclear membrane positivity in LMNA-NTRK1 fusion-positive sarcoma, and both cytoplasmic and nuclear positivity in ETV6-NTRK3 fusion-positive IMT and infantile fibrosarcomas. Also, the TPR-NTRK1 fusion-positive sarcoma showed robust positivity for CD34/nestin, and also showed high mitotic rate. The LMNA-NTRK1 fusion-positive sarcoma revealed CD34/S100 protein/nestin/CD10 coexpression, and a low mitotic rate. The IMT with ETV6-NTRK3 fusion expressed SMA. Six infantile fibrosarcomas with ETV6-NTRK3 fusion showed variable coexpression of nestin (6/6)/CD10 (4/5)/ S100 protein (3/6).ConclusionsAll cases of NTRK1 and NTRK3 fusion-positive pediatric tumors robustly expressed the Trk protein. A Trk immunopositive pattern and CD34/S100/nestin/CD10/SMA immunohistochemical expression may suggest the presence of NTRK fusion partner genes. LMNA-NTRK1 fusion sarcoma might be a low-grade subtype of infantile fibrosarcoma. Interestingly, more than half of the infantile fibrosarcoma cases were positive for S100 protein and CD10. The follow-up period of TPR-NTRK1 and LMNA-NTRK1 fusion-positive tumors are not enough to predict prognosis. However, ETV6-NTRK3 fusion-positive infantile fibrosarcomas showed an excellent prognosis with no evidence of disease for an average of 11.7 years, after gross total resection of the tumor.

Abstract B67: EGFR as a target in pediatric solid tumors

Abstract The epidermal growth factor receptor (EGFR or HER1) is a cell surface tyrosine kinase receptor that is expressed in a diverse group of epithelial and nonepithelial tissues and broadly associated with cell proliferation and differentiation. Expression of wild-type EGFR and activating mutations are described in many malignancies, including a variety of pediatric solid tumors. In the setting of malignancy, EGFR has been associated with aggressive disease, chemotherapy resistance, and increased metastatic potential. Employment of anti-EGFR agents has yielded some promising results, but they are not always effective against all cancer-associated aberrant EGFR expression and are frequently associated with notable off-tumor toxicities due to the expression of EGFR in normal tissue. The unique EGFR monoclonal antibody (mAb) 806 selectively binds to an epitope on the extracellular portion of human EGFR expressed on the surface of tumor cells. This antibody recognizes the deletion mutant EGFRvIII commonly present in glioblastomas and some other solid tumors as well as wild-type EGFR. We designed a phase I trial for pediatric and young adult patients with recurrent or refractory solid tumors expressing epidermal growth factor (EGFR) to examine the safety and feasibility of administering autologous, peripheral blood-derived T cells that have been genetically modified to express a second-generation (2G) EGFR806-specific chimeric antigen receptor (CAR). Literature review and evaluation of EGFR immunohistochemistry (IHC) performed on several tissue microarrays were used to estimate the percentage of EGFR positivity among common subtypes of pediatric cancer. Using these tools, we hypothesized that 15-40% of otherwise eligible patients would meet the eligibility criteria of EGFR positivity. The trial opened in August of 2018. To date, a total of 33 patient tumors have undergone EGFR IHC to determine eligibility for the trial. 9/33 (27.3%) have been EGFR+. EGFR+ histologies include BCOR fusion sarcoma (3), germ cell tumor (2), osteosarcoma, Ewing sarcoma, desmoplastic small round cell tumor, and synovial sarcoma. Our eligibility testing thus far demonstrates EGFR expression by IHC in a variety of histologies. Two rare diseases, recurrent BCOR fusion sarcoma and refractory germ cell tumors, have demonstrated a significantly higher rate of EGFR IHC positivity, (3/3)100% and (2/3) 66.7%, respectively. These early results indicate that this trial and other anti-EGFR therapies may be most effectively targeted to certain subgroups of pediatric cancer patients. Citation Format: Catherine M. Albert, Navin R. Pinto, Erin R. Rudzinski, Julie R. Park. EGFR as a target in pediatric solid tumors [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B67.

EWSR1 fusions: Ewing sarcoma and beyond.

EWSR1 fusions: Ewing sarcoma and beyond.

Clinical, pathological, and genomic features of EWSR1-PATZ1 fusion sarcoma

Molecular diagnostics of sarcoma subtypes commonly involve the identification of characteristic oncogenic fusions. EWSR1-PATZ1 is a rare fusion partnering in sarcoma, with few cases reported in the literature. In the current study, a series of 11 cases of EWSR1-PATZ1 fusion positive malignancies are described. EWSR1-PATZ1-related sarcomas occur across a wide age range and have a strong predilection for chest wall primary site. Secondary driver mutations in cell-cycle genes, and in particular CDKN2A (71%), are common in EWSR1-PATZ1 sarcomas in this series. In a subset of cases, an extended clinical and histopathological review was performed, as was confirmation and characterization of the fusion breakpoint revealing a novel intronic pseudoexon sequence insertion. Unified by a shared gene fusion, EWSR1-PATZ1 sarcomas otherwise appear to exhibit divergent morphology, a polyphenotypic immunoprofile, and variable clinical behavior posing challenges for precise classification.

Detecting disease-defining gene fusions in unclassified round cell sarcomas using anchored multiplex PCR/targeted RNA next-generation sequencing-Molecular and clinicopathological characterization of 16 cases.

Detection of disease-defining gene fusions in sarcoma has led to refining their classification, as well as to discover several new entities. The advent of anchored multiplex PCR/targeted RNA next-generation sequencing (AMP/RNA-seq) has allowed for the development of scalable platforms that can simultaneously examine multiple fusion transcripts without prior knowledge of specific fusion partners.In this study, we assess the utility of a FusionPlex sarcoma panel analysis by AMP/RNA-seq to detect disease-defining gene fusions in 16 cases of undifferentiated round cell sarcoma in which prior diagnostic work-up could not establish a definitive diagnosis. The clinical and pathologic features of these cases were correlated with the molecular findings. Validation of the method using 41 cases with known diagnoses showed analytic sensitivity and specificity of 98% and 100%, respectively. Of the 16 cases of undifferentiated round cell sarcoma, gene fusions were found in 9 (56%). These included three cases with CIC-DUX4 fusion, two cases with BCOR-CCNB3, and four single cases with CIC-NUTM2A, HEY1-NCOA2, EWSR1-NFATC2, and NUT-MGA1 fusions. Overall, despite some degree of morphologic overlap, all fusion-positive cases had distinct morphologic features, which can be helpful for their histologic classification. We also describe the first adult case of MGA-NUTM1 fusion sarcoma, as well as cartilaginous differentiation in a BCOR-CCNB3 fusion sarcoma, which has not been previously reported. Our study demonstrated that FusionPlex sarcoma panel analysis, in the appropriate morphologic context, is a sensitive and precise ancillary method for the detection of disease-defining gene fusions in undifferentiated round cell sarcomas, aiding in their definitive classification.

New fusion sarcomas: histopathology and clinical significance of selected entities

Many sarcomas contain gene fusions that can be pathogenetic mechanisms and diagnostic markers. In this article we review selected fusion sarcomas and techniques for their detection. CIC-DUX4 fusion sarcoma is a round cell tumor now considered an entity separate from Ewing sarcoma with a more aggressive clinical course, occurrence in older age, and predilection to soft tissues. It is composed of larger cells than Ewing sarcoma and often has prominent necrosis. Nuclear DUX4 expression is a promising immuno histochemical marker. BCOR-CCNB3 fusion sarcoma is cyclin B3-positive, usually occurs in bone or soft tissue of children, and may mimic a poorly differentiated synovial sarcoma. EWSR1-NFATC2 sarcoma may present in bone or soft tissue. It is typically composed of small round cells in a trabecular pattern in a myxoid matrix resembling myoepithelioma. ACTB-GLI1 fusion sarcoma may mimic a skin adnexal carcinoma, showing focal expression of epithelial markers and S100 protein. NTRK-fusion sarcomas include, in addition to infantile fibrosarcoma with ETV6-NTRK3 fusion, LMNA-NTRK1 fusion sarcoma, a low-grade spindle cell sarcoma seen in peripheral soft tissues in children and young adults. Methods to detect gene fusions include next-generation sequencing panels, anchored multiplex polymerase chain reaction systems to detect partner for a known fusion gene, and comprehensive RNA sequencing to detect virtually all gene fusions. In situ hybridization testing using probes for both fusion partners can be used as an alternative confirmation technique, especially in the absence of satisfactory RNA yield. In addition, fusion protein-related and other immunohistochemical markers can have a high specificity for fusion sarcomas.

Targeted degradation of BRD9 reverses oncogenic gene expression in synovial sarcoma

Synovial sarcoma tumours contain a characteristic fusion protein, SS18-SSX, which drives disease development. Targeting oncogenic fusion proteins presents an attractive therapeutic opportunity. However, SS18-SSX has proven intractable for therapeutic intervention. Using a domain-focused CRISPR screen we identified the bromodomain of BRD9 as a critical functional dependency in synovial sarcoma. BRD9 is a component of SS18-SSX containing BAF complexes in synovial sarcoma cells; and integration of BRD9 into these complexes is critical for cell growth. Moreover BRD9 and SS18-SSX co-localize extensively on the synovial sarcoma genome. Remarkably, synovial sarcoma cells are highly sensitive to a novel small molecule degrader of BRD9, while other sarcoma subtypes are unaffected. Degradation of BRD9 induces downregulation of oncogenic transcriptional programs and inhibits tumour progression in vivo. We demonstrate that BRD9 supports oncogenic mechanisms underlying the SS18-SSX fusion in synovial sarcoma and highlight targeted degradation of BRD9 as a potential therapeutic opportunity in this disease.

Epithelioid Hemangioendothelioma as a Model of YAP/TAZ-Driven Cancer: Insights from a Rare Fusion Sarcoma.

Epithelioid hemangioendothelioma (EHE) is a rare soft-tissue sarcoma involving cells with histologic markers that suggest an endothelial origin. Around 90% of EHEs are caused by the fusion of Transcriptional Co-activator with a PDZ-motif (TAZ) with Calmodulin Binding Transcription Activator 1 (CAMTA1), a central nervous system-specific transcription activator. The 10% of EHEs that lack the TAZ–CAMTA1 fusion instead have a fusion of Yes-associated Protein (YAP) and Transcription Factor E3 (TFE3) genes (YAP-TFE3). YAP and TAZ are well-defined downstream effectors in the Hippo pathway that promote cell growth when translocated to the nucleus. The TAZ–CAMTA1 fusion transcript is insensitive to the Hippo inhibitory signals that normally prevent this process and thus constitutively activates the TAZ transcriptome. In EHE, this causes tumors to form in a variety of organs and tissue types, most commonly the liver, lung, and bone. Its clinical course is unpredictable and highly variable. TAZ activation is known to contribute to key aspects of the cancer phenotype, including metastasis and fibrosis, and increased expression of TAZ is thought to be causally related to the progression of many cancers, including breast, lung, and liver. Therefore, understanding TAZ biology and the molecular mechanisms by which it promotes unregulated cell proliferation will yield insights and possibly improved treatments for both EHE as well as much more common cancers.

Abstract B24: Molecular and functional dissection of the CIC-DUX4 fusion in undifferentiated round cell sarcoma

Abstract CIC-DUX4 soft-tissue tumors are an aggressive subset of undifferentiated round cell sarcomas that arise in children and young adults. The cytogenetic hallmark, (t(4;19) or t(10;19)), of this tumor is a chromosomal translocation that fuses the transcription factor, Capicua (CIC), with the double homeobox 4 protein, DUX4. Despite morphologic similarities to Ewing sarcoma, CIC-DUX4 sarcomas are clinically distinct with rapid development of lethal metastatic disease and chemoinsensitivity. The unique cytogenetic and clinical features that distinguish CIC-DUX4 sarcomas from other small round cell tumors provide a unique opportunity to identify and rationally target this population to improve clinical outcomes. To meet this need, we are taking a systematic functional approach to identify critical CIC-DUX4 targets that promote tumor progression and metastasis. Leveraging a comparative transcriptional analysis between cytogenetically distinct small round cell sarcomas, we reveal that the CIC-DUX4 fusion activates highly conserved molecular networks to control tumor growth and metastatic capacity. Using an orthotopic soft-tissue sarcoma model, we show that CIC-DUX4 induces ETV4 expression to drive invasion and metastasis. Genetic silencing of ETV4 in CIC-DUX4 expressing tumors limits tumor dissemination without a profound impact on tumor cell growth. These findings suggest that CIC-DUX4 enhances metastasis and tumor growth through distinct transcriptional repertoires. Consistent with this, we identify multiple components of the cell cycle machinery that are transcriptionally regulated by CIC-DUX4 expression. Specifically, the CIC-DUX4 fusion co-opts native CIC-binding specificity to transcriptionally activate these cell cycle target genes. Altogether, our molecular and functional dissection of the CIC-DUX4 fusion reveals highly conserved CIC-regulated transcriptional targets that promote tumor growth and metastasis. We aim to pharmacologically exploit these key CIC-DUX4-dependent transcriptional nodes to improve outcomes for this population with few therapeutic options. Citation Format: Ross A. Okimoto, Wei Wu, Trever G. Bivona. Molecular and functional dissection of the CIC-DUX4 fusion in undifferentiated round cell sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B24.