4.1 Pathology
The tumor had sheets of atypical cells separated by thick fibrous septae (Fig. 2A,B). Rare areas of clear cell change were identified (Fig. 2C). Mitotic activity was prominent. Geographic necrosis, apoptotic debris, dystrophic calcifications, foci of hemorrhage and hemosiderin deposition were present. Immunohistochemistry was consistent with the diagnosis (Fig 2D). Electron microscopy demonstrated primitive intracellular junctions and rare foci of basal lamina formation, suggesting an element of epithelial differentiation. Rare primary cilia and abundant flocculent extracellular material were identified. A Nanostring-based fusion panel assay identified the YWHAE:NUTM2B fusion.
Molecular
WGTA data revealed somatic alterations in the tumour genome. Whole genome mutation burden was low at 0.4 mutations/million bases. Somatic copy number changes were found in a focal region of chromosome 17 and part of chromosome 10 long arm (Fig. 2E). WGTA data revealed 46 structural variants, including a t(10;17)(q22;p13) translocation resulting in fusion between exon 5 of YWHAE and exon 2 ofNUTM2B (Fig. 2F). No relevant cancer predisposition germline findings from 98 reviewed predisposition genes were reported, suggesting this tumor was a sporadic event, and YWHAE:NUTM2B fusion was likely a driver.
RNA-seq data revealed multiple expression aberrations indicated from high expression percentile and overexpression when compared to the TARGET CCSK cohort (TARGET_CCSK), and GTEx normal tissues (GTEx_average), respectively. High outlier expression percentile and overexpression pathways comprised receptor tyrosine kinases (RTKs) including NTRK3 , RET and KIT , and IGF signaling genes, namely IGF2 and IGF1R . Genes in MAPK and PI3K/mTor pathway, NRAS, BRAF, MAPK1, AKT1 and mTOR, showed high expression percentile compared to TARGET_CCSK. SHH, SMO, GLI1/2/3, transcription factors of SHH pathway and HES4 from NOTCH pathway demonstrated high expression percentile and overexpression. WGTA demonstrated high outlier expression percentile of cell cycle regulators in phases G1, S and M, indicating heavy dysregulation: CCND1 , CCND2 , CDK6 , CCNE2 ,CCNA2 , CCNB1 and CDK1 .
BCOR was overexpressed compared to GTEx_average. When compared to Personalized OncoGenomics (POG) Programs and TCGA sarcomas,BCOR was in the 100th percentile; however,BCOR showed average expression within TARGET CCSK cases, likely due to it being generally overexpressed in CCSK with either mutually exclusive changes, YWHAE:NUT2MB fusion or BCOR internal duplications.11
EZH2 , a gene part of polycomb repressive complex 2, had overexpression and high outlier percentile when compared to GTEx_average. TERC and TERT telomerases both showed high expression percentile and overexpression.
Discussion
Three other cases of YWHAE:NUTM2B fusion in URCS of infancy are reported, and all were aggressive tumors unresponsive to conventional chemotherapy.8,9 Our case demonstrated similar clinical and pathological features.8 As such, this infant was enrolled into the POG trial (NCT02155621) to describe the molecular landscape and identify any therapeutically actionable variants not discovered during routine work up. Targeted therapy was not pursued due to rapid clinical decline.
Based on WGTA there was limited evidence to support a targeted approach as most alterations were based on increased RNA expression data. We found mTOR transcription factors were overexpressed with high outlier expression profile. Previous literature and experience have clinically targeted mTOR in pediatric vascular tumors due to administration ease and limited side effects. Use of mTOR inhibitors had previously reduced tumor growth both in vivo and in vitro in infantile hemangiomas and angiosarcomas.12,13 mTOR inhibitors are also proven efficacious in children with Kaposiform hemangioendothelioma, where loss of tumor suppressors PTEN and TSC2 leads to abnormally activated mTOR pathway and mTOR-inhibited suppressed tumor growth.14–17 An mTOR inhibitor could have been an option in this infant and might be considered in future URCS cases.
The unique YWHAE fusion encodes a 14-3-3-ε protein which interacts with CDC25 phosphatases, RAF1, and IRS1, suggesting its role in diverse biochemical activities related to signal transduction (cell division and insulin sensitivity regulation). Since IGF2 had high outlier expression in this tumor and correlated with VEGF2 expression in infantile hemangioma, off-label use of a commercially available IGF-2 inhibitor with pediatric dosing, was considered.18–22Other targets included CDK4/CDK6 inhibitors due to increased expression of CCND1/CyclinD1 and RTK inhibitors due to high outlier expression percentile for NTRK3 , RET , and KIT .NTRK3 overexpression is reported in undifferentiated sarcomas with YWHAE/BCOR genetic modifications but may be a less efficacious target in a setting of exclusive changes in RNA expression.6,23–28
Other possibilities with very low level of evidence based on RNA expression data included targeting EZH2 ; TERT , which showed increased expression in CCSK; and TERC inhibition in clinical trials, reporting varied results in myelofibrosis, lung and breast cancer, and lymphoproliferative disorders (NCT02598661), (NCT01731951).29–33
Overall, a strongly supported targetable pathway was not identified in this infant’s tumor, but we described more precisely the genomic profile of this entity allowing better understanding of the underlying molecular changes and reinforcing the value of a detailed oncogenomic approach to confirm conventional cytogenetic findings, detect additional genetic abnormalities, and suggest alternative therapeutic strategies. Further clinical and genomic studies are needed to understand how the YWHAE:NUTM2B fusion drives tumorigenesis and to develop more effective treatments.