Soft tissue undifferentiated round cell sarcoma (URCS) occurring in infants is a heterogeneous group of tumors, often lacking known genetic abnormalities. Based on a t(10;17;14) karyotype in a pelvic URCS of a 4-month-old boy showing similar breakpoints with clear cell sarcoma of kidney (CCSK), we have investigated the possibility of shared genetic abnormalities in CCSK and soft tissue URCS. Most CCSK are characterized by BCOR exon 16 internal tandem duplications (ITD), while a smaller subset shows YWHAE-NUTM2B/E fusions. Due to overlapping clinicopathologic features, we have also investigated these genetic alterations in the so-called primitive myxoid mesenchymal tumor of infancy (PMMTI). Among the 22 infantile URCS and 7 PMMTI selected, RNA sequencing (RNAseq) was performed in 5 and 2 cases, with frozen tissue, respectively. The remaining cases with archival material were tested for YWHAE-NUTM2B/E by FISH or RT-PCR, and BCOR ITD by PCR. A control group of 4 CCSK, 14 URCS in older children or adults without known gene fusion, and 20 other sarcomas with similar histomorphology or age at presentation were also tested. A YWHAE-NUTM2B fusion was confirmed in the index case by FISH and RT-PCR, while lacking BCOR ITD. An identical YWHAE-NUTM2B fusion was found in another URCS case of a 5-month-old girl from the back. The remaining cases and control group lacked YWHAE gene rearrangements; instead, consistent BCOR ITD, similar to CCSK, were found in 15/29 (52%) infantile sarcoma cases (9/22 infantile URCS and 6/7 PMMTI). In the control cohort, BCOR ITD was found only in 3 CCSK but not in the other sarcomas. Histologically, URCS with both genotypes and PMMTI shared significant histologic overlap, with uniform small blue round cells with fine chromatin and indistinct nucleoli. A prominent capillary network similar to CCSK, rosette structures and varying degree of myxoid change were occasionally seen. BCOR ITD positive tumors occurred preferentially in the somatic soft tissue of trunk, abdomen and head and neck, sparing the extremities. RNAseq showed high BCOR mRNA levels in BCOR ITD-positive cases, compared to other URCSs. In summary, we report recurrent BCOR exon 16 ITD and YWHAE-NUTM2B fusions in half of infantile soft tissue URCS and most PMMTI, but not in other pediatric sarcomas. These findings suggest a significant overlap between infantile URCS and CCSK, such as age at presentation, histologic features and genetic signature; thus raising the possibility of a soft tissue counterpart to CCSK.

With the advent of next-generation sequencing, an increasing number of novel gene fusions and other abnormalities have emerged recently in the spectrum of EWSR1-negative small blue round cell tumors (SBRCTs). In this regard, a subset of SBRCTs harboring either BCOR gene fusions (BCOR-CCNB3, BCOR-MAML3), BCOR internal tandem duplications (ITD), or YWHAE-NUTM2B share a transcriptional signature including high BCOR mRNA expression, as well as similar histologic features. Furthermore, other tumors such as clear cell sarcoma of kidney (CCSK) and primitive myxoid mesenchymal tumor of infancy (PMMTI) also demonstrate BCOR ITDs and high BCOR gene expression. The molecular diagnosis of these various BCOR genetic alterations requires an elaborate methodology including custom BAC FISH probes and RT-PCR assays. As these tumors show high level of BCOR overexpression regardless of the genetic mechanism involved, either conventional gene fusion or ITD, we sought to investigate the performance of an anti-BCOR monoclonal antibody clone C-10 (sc-514576) as an immunohistochemical marker for sarcomas with BCOR gene abnormalities. Thus we assessed the BCOR expression in a pathologically and genetically well-characterized cohort of 25 SBRCTs, spanning various BCOR-related fusions and ITDs and YWHAE-NUTM2B fusion. In addition we included related pathologic entities such as 8 CCSKs and other sarcomas with BCOR gene fusions. As a control group we included 20 SBRCT with various (non-BCOR) genetic abnormalities, 10 fusion-negative SBRCT, 74 synovial sarcomas, 29 rhabdomyosarcoma and other sarcoma types. Additionally we evaluated the same study group for SATB2 immunoreactivity, as these tumors also showed SATB2 mRNA up-regulation. All SBRCTs with BCOR-MAML3 and BCOR-CCNB3 fusions, as well as most with BCOR ITD (93%), and all CCSKs showed strong and diffuse nuclear BCOR immunoreactivity. Furthermore, all SBRCTs with YWHAE-NUTM2B also were positive. SATB2 stain was also positive in tumors with YWHAE-NUTM2B, BCOR-MAML3, BCOR ITD (75%), BCOR-CCNB3 (71%), and a subset of CCSKs (33%). In conclusion, BCOR immunohistochemical stain is a highly sensitive marker for SBRCTs and CCSKs with BCOR abnormalities and YWHAE rearrangements and can be used as a useful diagnostic marker in these various molecular subsets. SATB2 immunoreactivity is also present in the majority of this group of tumors.

Sclerosing and spindle cell rhabdomyosarcoma (SRMS) have been recently re-classified as a stand-alone pathologic entity, separate from embryonal RMS (ERMS). Genetically a subset of the congenital cases display NCOA2 gene rearrangements, while tumors occurring in older children or adults harbor MYOD1 gene mutations with or without coexisting PIK3CA mutations. Despite these recent advances, a significant number of tumors lack known genetic alterations. In this study we sought to investigate a large group of pediatric spindle cell/sclerosing RMS, spanning a diverse clinical and pathologic spectrum, by using a combined FISH, targeted DNA and whole transcriptome sequencing methodology for a more definitive molecular classification. A total of 26 spindle and sclerosing RMS cases were selected from the two participating Institutions for the molecular analysis. Ten of the 11 congenital/infantile SRMS showed recurrent fusion genes: with novel VGLL2 rearrangements seen in 7 (63%), including VGLL2-CITED2 fusion in 4 and VGLL2-NCOA2 in 2 cases. Three (27%) cases harbored the previously described NCOA2 gene fusions, including TEAD1-NCOA2 in 2, and SRF-NCOA2 in 1. All fusion positive congenital/infantile SRMS patients with available long term follow-up were alive and well, none developing distant metastases. Among the remaining 15 SRMS patients older than age of one, 10 (67%) showed MYOD1 L122R mutations, most of them following a fatal outcome despite an aggressive multi-modality treatment. All 4 cases harboring co-existing MYOD1/PIK3CA mutations shared sclerosing morphology. All 5 fusion/mutation-negative SRMS cases presented as intra-abdominal or para-testicular lesions.

Fusion status and tumor invasiveness appear to have a strong impact on prognosis in patients with aRMS/N1. Fusion status will be used to stratify these patients in the next EpSSG RMS study, and treatment will be intensified in patients with fusion-positive tumors. Cancer 2018. © 2018 American Cancer Society.

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 5 YWHAE fusions, occurring in 29 infants and 4 children, 19 males and 14 females. Microscopically, 20 were classified as URCSs 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), 4 patients were still alive with disease (mean follow-up: 46 months, range: 4–120), and 1 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.