Fig. 6: Potential impact of tiered multiplexed ddPCR in the clinical care of patients with fusion-positive sarcomas.

Clinical timelines illustrating radiologic, diagnostic, pathologic, and treatment milestones in example patients with fusion-positive pediatric sarcomas. Triple green arrows indicate the predicted time to tiered multiplexed ddPCR results if the assay had been implemented in the clinical pathology lab with return of results. A The initial clinical diagnosis and treatment for patient MRD0124 was osteosarcoma. Tiered multiplexed ddPCR from the initial diagnostic procedures was obtained and identified the presence of a BCOR-CCNB3 translocation, contradicting the histologic diagnosis. The diagnosis of a BCOR-CCNB3 sarcoma was later made at relapse (week 321) by FISH. After responding to relapse therapy, the patient then developed pleural effusions that were negative for cancer during cytologic examination but tested positive for the BCOR-CCNB3 fusion by tiered multiplexed ddPCR in cells isolated from the same effusion sample. This was later confirmed to be a site of further disease progression. B The diagnosis of Ewing sarcoma for patient MRD0176 was made by histology after 3 weeks and chemotherapy was started without detection of a Ewing-specific fusion. Tiered multiplexed ddPCR identified an EWS-FLI1 fusion from the initial biopsy tissue and from a pre-treatment blood sample containing CTCs. Later, the patient developed pleural effusions that were negative for cancer cells by cytologic examination but tested positive for the EWS-FLI1 fusion by tiered multiplexed ddPCR in cells extracted from the same effusion sample. This was later confirmed to be a site of further disease progression. Clinically, the EWSR1-FLI1 fusion was identified by NGS performed on tumor tissue only after the patient died from disease. C The histologic diagnosis of DSRCT for patient MRD0270 was made 4 weeks after initial diagnostic biopsy was performed. Detection of an EWS-WT1 fusion by NGS was preliminary at the time of treatment initiation and later finalized six weeks after biopsy. Tiered multiplexed ddPCR detected the pathognomonic EWS-WT1 fusion in the diagnostic sample. TMA tiered multiplexed ddPCR assay, NGS next-generation sequencing, CTCs circulating tumor cells, DSRCT desmoplastic small round cell tumor. ǂ The chemotherapy used was not standard of care for the patient’s final diagnosis. * Cytology did not find any malignant cells.