Generation of human embryonic stem cell models to exploit the EWSR1-CREB fusion promiscuity as a common pathway of transformation in human tumors.

Chromosomal translocations constitute driver mutations in solid tumors and leukemias. The mechanisms of how related or even identical gene fusions drive the pathogenesis of various tumor types remain elusive. One remarkable example is the presence of EWSR1 fusions with CREB1 and ATF1, members of the CREB family of transcription factors, in a variety of sarcomas, carcinomas and mesotheliomas. To address this, we have developed in vitro models of oncogenic fusions, in particular, EWSR1-CREB1 and EWSR1-ATF1, in human embryonic stem (hES) cells, which are capable of multipotent differentiation, using CRISPR-Cas9 technology and HDR together with conditional fusion gene expression that allows investigation into the early steps of cellular transformation. We show that expression of EWSR1-CREB1/ATF1 fusion in hES cells recapitulates the core gene signatures, respectively, of angiomatoid fibrous histiocytoma (AFH) and gastrointestinal clear cell sarcoma (GI-CCS), although both fusions lead to cell lethality. Conversely, expression of the fusions in hES cells differentiated to mesenchymal progenitors is compatible with prolonged viability while maintaining the core gene signatures. Moreover, in the context of a mesenchymal lineage, the proliferation of cells expressing the EWSR1-CREB1 fusion is further extended by deletion of the tumor suppressor TP53. We expect the generation of isogenic lines carrying oncogenic fusions in various cell lineages to expand our general understanding of how those single genetic events drive tumorigenesis while providing valuable resources for drug discovery.

Undifferentiated round cell sarcoma with BCOR internal tandem duplications (ITD) or YWHAE fusions: a clinicopathologic and molecular study.

Until recently, undifferentiated round cell sarcomas (URCS) in infants have been considered a wastebasket diagnosis, composed of various pathologic entities and lacking consistent genetic alterations. The recent identification of recurrent BCOR internal tandem duplications (ITD) and less common alternative YWHAE-NUTM2B/E fusions in half of infantile URCS and the majority of so-called primitive myxoid mesenchymal tumors of infancy (PMMTI) suggests a common pathogenesis with clear cell sarcoma of the kidney which also harbors the same genetic alterations. These tumors also share a similar morphology and immunoprofile, including positivity for BCOR, cyclin D1, and SATB2. In this study, we investigate the largest cohort to date of genetically confirmed URCS and PMMTI with BCOR ITD or YWHAE fusions to better define their morphologic spectrum and clinical behavior. Twenty-eight cases harbored BCOR ITD and five YWHAE fusions, occurring in 29 infants and 4 children, 19 males and 14 females. Microscopically, 20 were classified as URCS and 13 as PMMTI. Follow-up was available in 25 patients, with 14 (56%) succumbing to their diseases at a mean duration of 18-months follow-up (range: 2-62). Six patients remained with no evidence of disease at a mean follow-up of 63 months (range: 4-192), four patients were still alive with disease (mean follow-up: 46 months, range: 4-120), and one died of other causes. Local recurrence and distant metastasis were each observed in 11/25 (44%) of the patients. The overall survival was 42% at 3 years and 34% at 5 years (median survival: 26 months). There was no statistically significant survival difference between cases diagnosed as URCS and PMMTI and between those with BCOR ITD and YWHAE fusions.

MYOD1-mutant spindle cell and sclerosing rhabdomyosarcoma: an aggressive subtype irrespective of age. A reappraisal for molecular classification and risk stratification.

Sclerosing and spindle cell rhabdomyosarcoma is a rare histologic subtype, designated in the latest WHO classification as a stand-alone pathologic entity. Three genomic groups have been defined: an infantile subset of spindle cell rhabdomyosarcoma harboring VGLL2-related gene fusions, a MYOD1-mutant subset commonly associated with sclerosing morphology, and a subset lacking recurrent genetic abnormalities. In this study, we focus on MYOD1-mutant rhabdomyosarcoma to further define their clinicopathologic characteristics and behavior in a larger patient cohort. We investigated 30 cases of MYOD1-mutant rhabdomyosarcoma (12 previously reported and 18 newly diagnosed) with an age range of 2-94 years, including 15 children. All cases showed morphology within the spectrum of spindle cell/sclerosing rhabdomyosarcoma (8 cases showing pure sclerosing morphology, 8 cases  showing pure spindle cell morphology and 14 cases showing a hybrid phenotype of spindle, sclerosing and primitive undifferentiated areas). All tumors harbored either homozygous or heterozygous MYOD1 (p.L122R) exon 1 mutations. In 10 (33%) cases, a co-existent PIK3CA mutation was identified. Hot-spot mutations in NRAS and HRAS were each identified in a single case, respectively. Follow-up was available on 22 (73%) patients with a median duration of 28 months. Local recurrence was seen in 12 (55%) and distant recurrence in 12 (55%) cases, despite multimodality chemoradiation therapy. At last follow-up, 15 (68%) patients died of the disease, one patient was alive with disease and five had no evidence of disease. The prognosis was equally poor in pediatric and adult patients. In conclusion, MYOD1 mutation defines an aggressive rhabdomyosarcoma subset, with poor outcome and response to therapy, irrespective of age. Given that this distinct molecular subtype is characterized by an aggressive biologic behavior compared to other genetic subtypes of spindle and sclerosing rhabdomyosarcoma, the MYOD1 genotype should be used as a molecular marker in both subclassification and prognostication of rhabdomyosarcoma.

BCOR-CCNB3 Fusion Positive Sarcomas: A Clinicopathologic and Molecular Analysis of 36 Cases With Comparison to Morphologic Spectrum and Clinical Behavior of Other Round Cell Sarcomas.

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.