Classification of pediatric soft and bone sarcomas using DNA methylation-based profiling

Abstract

Pediatric sarcomas present heterogeneous morphology, genetics and clinical behavior posing a challenge for an accurate diagnosis. DNA methylation is an epigenetic modification that coordinates chromatin structure and regulates gene expression, determining cell type and function. DNA methylation-based tumor profiling classifier for sarcomas (known as sarcoma classifier) from the German Cancer Research Center (Deutsches Krebsforschungszentrum) was applied to 122 pediatric sarcomas referred to a reference pediatric oncology hospital. The classifiers reported 88.5% of agreement between histopathological and molecular classification confirming the initial diagnosis of all osteosarcomas and Ewing sarcomas. The Ewing-like sarcomas were reclassified into sarcomas with BCOR or CIC alterations, later confirmed by orthogonal diagnostic techniques. Regarding the CNAs profile, osteosarcomas had several chromosomal gains and losses as well as chromothripsis, whereas Ewing sarcomas had few large events, such as amplifications of chromosomes 8 and 12. The molecular classification together with clinical and histopathological assessment could improve the diagnosis of pediatric sarcomas although there are limitations to deal with more rare classes. This study provides an increase in the number of sarcomas evaluated for DNA methylation profiling in the pediatric population.

Keywords: Copy number alteration; DNA methylation; Molecular classification; Pediatric sarcomas; Soft and bone sarcomas.

Fig. 1

Molecular classification of pediatric sarcomas. A Sankey plot showing molecular classification (right) compared to histopathological diagnosis (left) Altered. B Unsupervised hierarchical clustering baked on the 10 thousand most variable probes (Red: methylated, blue: unmethylated CpG site) Individual samples are color-coded labeled in the respective molecular class. C Unsupervised hierarchical clustering of samples with cluster names. D Survival analysis showing the impact of osteosarcoma methylation subgroups (Overall survival by Kaplan–Meier analysis). SARC_CIC, CIC rearranged sarcoma (Brain classifier); OB Osteoblastoma, EWING Ewing sarcoma, OS_HG High-grade osteosarcoma, SBRCT_BCOR BCOR rearranged sarcoma, CSA_MES Mesenchymal chondrosarcoma, ERMS Embryonal rhabdomyosarcoma, RMS_EMB Embryonal rhabdomyosarcoma, ARMS Alveolar rhabdomyosarcoma, CSA_CC Clear cell chondrosarcoma, SPIND_SARC spindle cell sarcoma, USARC Undifferentiated sarcoma, IMT Inflammatory myofibroblastic tumor, LCH Langerhans cell histiocytosis, CSA_IDH_GROUP_B Chondrosarcoma IDH group B, ATRT_MYC Atypical teratoid rhabdoid tumor, MYC activated SYSA, Synovial sarcoma CB Chondroblastoma, AS Alveolar sarcoma, ASPS Alveolar soft part sarcoma, CCSK Clear cell sarcoma of the kidney, CTRL_MUS Control tissue: muscle, DSRCT Desmoplastic small round cell tumors, LIPO Lipoma, SEF Sclerosing epithelioid fibrosarcoma. Created with BioRender.com (o99u352)

Fig. 2

Identification of the translocation pointed by the methylation-based classification. Example of 4 cases with diagnostic fusion genes, representing A Ewing sarcoma, B PNET, C and D as original diagnostic that changed to BCOR-rearranged sarcoma and high-grade neuroepithelial tumor with MN1:CXXC5 fusion, respectively. Circus plot showing 22 autosomes and 2 sexual chromosomes as well as the mitochondrial genome, all chromosomes with cytoband information. Fusion events as shown as links between locations in chromosomes (with gene names), red and blue lines indicate intrachromosomal and interchromosomal fusions, respectively. The width of each line varies according to how many reads support the fusion event. Created with BioRender.com (z83l932)

Fig. 3

Copy number profiles of pediatric sarcomas. A Recurrent copy number alterations (%) in all pediatric sarcomas and among the more common molecular classes. Gray line means a chromosome and dashed gray lines centromeric regions dividing p and q arms. Zero means two copies, above zero gain and below zero loss. B Number of altered chromosomes and number of copy number alterations among the more common molecular classes, including benign samples. C Percentage of copy number alterations among the more common molecular classes. Orange: gain, Blue: loss. Created with BioRender.com (x24n652)

Fig. 4

A Tumor infiltrating leukocytes, red line highlighting samples with higher content of neutrophils, classified as inflammatory environment, top panel showing percentage of tumor cell purity in the assayed samples and B Correlation between tumor purity and calibrated score. NET_CXXC5, high-grade neuroepithelial tumor with MN1:CXXC5 fusion; SARC_CIC, CIC rearranged sarcoma (Brain classifier); OB, Osteoblastoma; EWING, Ewing sarcoma; OS_HG, High grade osteosarcoma; SBRCT_BCOR, BCOR rearranged sarcoma; CSA_MES, Mesenchymal chondrosarcoma; ERMS, Embryonal rhabdomyosarcoma; RMS_EMB, Embryonal rhabdomyosarcoma; ARMS, Alveolar rhabdomyosarcoma; CSA_CC, Clear cell chondrosarcoma; USARC, Undifferentiated sarcoma; IMT, Inflammatory myofibroblastic tumor; LCH, Langerhans cell histiocytosis; CSA_IDH_GROUP_B, Chondrosarcoma IDH group B; ATRT_MYC, Atypical teratoid rhabdoid tumor, MYC activated; SYSA, Synovial sarcoma; CB, Chondroblastoma. Other, classes represented by one sample (AS, Alveolar sarcoma; ASPS, Alveolar soft part sarcoma; CCSK, Clear cell sarcoma of the kidney; CTRL_MUS, Control tissue: muscle; DSRCT, Desmoplastic small round cell tumors; LIPO, Lipoma; SEF, Sclerosing epithelioid fibrosarcoma; respectively). Created with BioRender.com (v43j268)