(P 152) NOVEL PAX3::MAML3 FUSION IDENTIFIED IN ALVEOLAR RHABDOMYOSARCOMA: USING DNA METHYLATION TO EXPANDE THE GENETIC SPECTRUM OF "FUSION POSITIVE" CASES

Objective: Alveolar rhabdomyosarcoma (ARMS) with PAX3/PAX7::FOXO1 gene rearrangement is an aggressive pediatric rhabdomyosarcoma subtype associated with a worse prognosis compared to embryonal rhabdomyosarcoma (ERMS) and fusion-negative ARMS. Even though rare cases of ARMS with alternate PAX3 fusion partners, such as NCOA1 or INO80D, have been reported, only FOXO1 fusion status is used as a risk stratification biomarker to determine eligibility for inclusion in the Children’s Oncology Group (COG) RMS clinical trials. DNA methylation profiling has been previously shown to clearly distinguish rhabdomyosarcomas with characteristic FOXO1 rearrangements from those without these rearrangements. In this study, we utilize DNA methylation profiling to help inform classification of two cases of ARMS with PAX3::MAML3 fusions. As biphenotypic sinonasal sarcoma (BSNS), a low grade mesenchymal neoplasm with neural and myogenic differentiation, are similarly characterized by PAX3::MAML3 fusions, we sought to compare differentially methylated genes between ARMS and BSNS.

Methods: Clinical data, including age, sex, anatomic site and treatment information were retrieved from clinical chart review. Hematoxylin and eosin–stained slides from resection specimens were rereviewed. PAX3::MAML3 fusion status was performed using FOXO1 FISH and Anchored Multiplex PCR-based targeted RNA sequencing. Methylation profiling was performed on the two cases with PAX3::MAML3 fusions. We used several external, publicly available methylation data sets, including ARMS, BCOR altered sarcoma, ERMS, desmoplastic small round cell tumor and Ewing sarcoma, to enhance the analysis. Unsupervised analysis of genome-wide DNA methylation patterns was performed using the t-distributed stochastic neighbor embedding (t-SNE) method to compare the methylation profiles of the PAX3::MAML3-fused tumors with those of ARMS, BSNS, ERMS, and other small round blue cell tumors. Differential methylation analysis between ARMS and BSNS was performed, and differentially methylated CpG sites were computed using the empirical Bayes statistics.

Results: The two cases of PAX3::MAML3 rhabdomyosarcoma arose in an infant and an adolescent as stage IV metastatic disease (by Children’s Oncology Group staging system). Histologically, both cases were small round blue cell tumors arranged in vague nests and solid sheets that were diffusely positive for desmin and myogenin. By methylation profiling and unsupervised clustering analysis, the tumors clustered with ARMS with classic FOXO1 rearrangements and ARMS with variant PAX3::NCOA1/INO80D fusions, but not with biphenotypic sinonasal sarcoma (BSNS) with PAX3::MAML3/NCOA2/FOXO1/YAP1 fusions, nor with other small round blue cell tumors, including embryonal rhabdomyosarcoma. The differentially methylated genes between ARMS and BSNS were highly enriched in genes involved in myogenesis, and 21% of these genes overlap with target genes of the PAX3::FOXO1 fusion transcription factor. On follow-up after initiation of vincristine/actinomycin/cyclophosphamide chemotherapy, the tumors showed partial and complete clinical response, consistent with typical upfront chemotherapy responsiveness of ARMS with the classic FOXO1 rearrangement.

Conclusion: We conclude that PAX3::MAML3 is a novel variant fusion of ARMS, which displays a methylation signature distinct from BSNS despite sharing similar PAX3 fusions. These findings highlight the utility of methylation profiling in classifying ARMS with non-canonical fusions.