To quantify copy number heterogeneity of ecDNA in medulloblastoma, we established an automated image analysis pipeline to estimate the distributions of copy number per cell in interphase FISH microscopy imaging and applied it to four primary medulloblastoma tumors harboring ecDNA: MB036 (MYCN), MB177 (MYCN), MB268 (MDM4) and RCMB56 (DNTTIP2, KMT2E, ETV1). This evidence concerns the gene MDM4 and medulloblastoma.