Comprehensive profiling of pediatric chordoma：poorly differentiated and conventional subtypes

Chordoma in children is rare and inadequately characterized. This study aims to provide a comprehensive characterization of the clinicopathological and (epi)genetic features of chordomas in children, particularly the poorly differentiated chordomas (PDC). We established a cohort of seven PDCs and 28 conventional chordomas (CC). Besides characterizing the clinical and histopathological features, we performed comprehensive immunohistochemical staining for markers enabling diagnosis of chordoma and members of the sonic hedgehog signaling pathway. Using DNA methylation analysis, we characterized the molecular subtypes among chordomas in children and the SMARCB1 (INI1)-deficient tumors, which was further complemented by fluorescence in situ hybridization and targeted exome sequencing. Pediatric patients with PDC showed poorer prognosis compared with pediatric patients with CC. PDC tumors occurred more frequently in younger children and typically located at the clivus. Histologically, PDC tumors exhibited large polygonal/epithelioid cells or chubby spindle cells, with positive staining for cytokeratins and brachyury, but loss of expression of SMARCB1 (INI1) protein, and a high Ki-67 index. PDC samples showed promiscuous staining of sonic hedgehog pathway members, which contrasts the homogenous staining of sonic hedgehog pathway members in CC samples. Unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding analyses of DNA-methylation data showed that pediatric PDCs formed a distinct methylation cluster in comparison with the samples of CC, atypical teratoid/rhabdoid tumors and bone fide extracranial proximal epithelioid sarcoma. Whereas PDCs harbored relatively stable karyotype, frequent chromosomal gains or losses were observed in CCs. Mutational profiling identified different sets of genes in PDCs versus CCs. Loss of SMARCB1 (INI1) expression in PDCs was predominantly due to locus deletion. In summary, our findings support the differential diagnosis between pediatric PDC and CC. Further, our findings suggest that besides distinct methylome profiles, pediatric PDC and CC are likely driven by distinct pathogenic pathways.