Array-based DNA-methylation profiling in sarcomas with small blue round cell histology provides valuable diagnostic information

Abstract

Undifferentiated solid tumors with small blue round cell histology and expression of CD99 mostly resemble Ewing sarcoma. However, they also may include other tumors such as mesenchymal chondrosarcoma, synovial sarcoma, or small cell osteosarcoma. Definitive classification usually requires detection of entity-specific mutations. While this approach identifies the majority of Ewing sarcomas, a subset of lesions remains unclassified and, therefore, has been termed "Ewing-like sarcomas" or small blue round cell tumors not otherwise specified. We developed an approach for further characterization of small blue round cell tumors not otherwise specified using an array-based DNA-methylation profiling approach. Data were analyzed by unsupervised clustering and t-distributed stochastic neighbor embedding analysis and compared with a reference methylation data set of 460 well-characterized prototypical sarcomas encompassing 18 subtypes. Verification was performed by additional FISH analyses, RNA sequencing from formalin-fixed paraffin-embedded material or immunohistochemical marker analyses. In a cohort of more than 1,000 tumors assumed to represent Ewing sarcomas, 30 failed to exhibit the typical EWS translocation. These tumors were subjected to methylation profiling and could be assigned to Ewing sarcoma in 14 (47%), to small blue round cell tumors with CIC alteration in 6 (20%), to small blue round cell tumors with BCOR alteration in 4 (13%), to synovial sarcoma and to malignant rhabdoid tumor in 2 cases each. One single case each was allotted to mesenchymal chondrosarcoma and adamantinoma. 12/14 tumors classified as Ewing sarcoma could be verified by demonstrating either a canonical EWS translocation evading initial testing, by identifying rare breakpoints or fusion partners. The methylation-based assignment of the remaining small blue round cell tumors not otherwise specified also could be verified by entity-specific molecular alterations in 13/16 cases. In conclusion, array-based DNA-methylation analysis of undifferentiated tumors with small blue round cell histology is a powerful tool for precisely classifying this diagnostically challenging tumor group.

Figure 1.. Assignment of 30 small blue round cell tumors not otherwise specified to methylation groups of reference sarcoma sets.

(A) - unsupervised hierarchical clustering analysis, and (B) - t-distributed stochastic neighbor embedding using the 10,000 most variable DNA-methylation probes of array-generated DNA-methylation profiles from the Illumina Infinium HumanMethylation450 or EPIC BeadChip (Illumina, San Diego, USA). Black bars/circles indicate the positions of the 30 small blue round cell tumors not otherwise specified. Abbreviations: Ada = adamantinoma (* suspect); ASPS = alveolar soft part sarcoma; mCS = mesenchymal chondrosarcoma; CCSK = clear cell sarcoma of the kidney; CT = control tissue of non-neoplastic inflammatory origin; OS = conventional osteosarcoma; DFSP - dermatofibrosarcoma protuberans ; DSRCT = desmoplastic small round cell tumor; ES = epithelioid sarcoma; EwS = Ewing sarcoma; IFS = infantile fibrosarcoma; MPNST = malignant peripheral nerve sheath tumor; MRT = malignant rhabdoid tumor; eRMS = embryonal rhabdomyosarcoma; aRMS = alveolar rhabdomyosarcoma; SBRCT (BCOR) = small blue round cell tumor with BCOR alteration (BCOR-CCNB3 fusion = yellow; internal tandem duplication = yellow with greyish contour); SBRCT (CIC) = small blue round cell tumor with CIC alteration; SFT = solitary fibrous tumor; SySa = synovial sarcoma

Figure 2.. BCOR internal tandem duplication in a skull tumor of an infant.

The genomic footprint of BCOR is depicted (A). The duplicated 66 bp sequence in Exon 15, which encodes for the PCGF Ub-like fold discriminator (PUFD) domain at the C-terminus of BCOR, is recognizable by a sharply demarcated doubling of the coverage rate. The tumor cells focally present with a vacuolated cytoplasm (B). Rosette formations are prominent in some areas (C).

Figure 3.. Histologic phenotypes of two representative Ewing-like sarcomas.

Case 83172 (A-C) represents a poorly differentiated synovial sarcoma exhibiting a small blue round cell phenotype (A). FISH analysis revealed a SS18 break-apart signal indicated by two separated green and red signals (B). Interestingly, some tumor cells express CD99 (C). Case 94172 (D, E) exhibits an epithelioid to rhabdoid phenotype (D). Nuclear INI-1 expression is lost (D; inlet). Copy number analysis demonstrated a loss on chromosome arm 19q involving the SMARCB1 locus (E).

Figure 4.. Histological phenotype of an unusual mesenchymal chondrosarcoma.

Case 95322 shows an organoid growth pattern at low-power view (A). The small round tumor cells are arranged in sheets (B). In less cellular parts the tumor shows a vague reticulated growth pattern (C). The tumor is almost negative for S100 (D) and CD99 (E). A copy number analysis demonstrates several whole-chromosome gains (F).

Figure 5.. Histologic phenotype of case with an EWSR1-NFATC2 gene fusion.

Case 97480 is a highly cellular round cell tumor with an indistinctive growth pattern (A). The tumor cells faintly express CD99 (B). A copy number analysis demonstrates complex chromosomal alterations, e.g. on chromosome 22, on an otherwise relatively balanced background (C).