Decoding oncogene amplification structures from retinoblastoma aqueous humor cfDNA as a model for chromothripsis and ecDNA biology

Purpose: Complex oncogene amplifications-including MDM4 1q gains and MYCN high-level amplification-frequently arise through mechanisms such as chromothripsis, tandem-duplication cycles, and extrachromosomal DNA (ecDNA). These structural drivers shape tumor aggression and therapy resistance across cancers, yet they remain largely inaccessible in plasma ctDNA due to low tumor fraction and fragment constraints. Retinoblastoma aqueous humor (AH) contains predominantly tumor-derived cfDNA with a genomically simple background, providing a uniquely powerful human model system to benchmark the technical feasibility, data characteristics, and analytical requirements for cfDNA-based amplification profiling. Methods: Deep whole-genome sequencing (>20×) was performed on AH cfDNA from four RB eyes having MDM4 focal gain or MYCN high-level amplification. Matched long-read nanopore WGS was generated for canonical subtype cell lines: WERI-Rb1 (MDM4) and Y79 (MYCN) to serve as high-confidence reference amplification profiles. We evaluated copy-number resolution, breakpoint features, and junction-spanning fragment representation in cfDNA relative to long-read tumor DNA. Results: All AH cfDNA samples achieved >20× genome-wide coverage with >90% inferred tumor fraction from Illumina PE150 NGS setting, enabling high-resolution focal amplification assessment. MDM4-positive AH samples demonstrated multi-segment focal 1q gain with moderate copy elevation (~3-8× over baseline) and sharp regional boundaries, while MYCN-amplified samples exhibited ultra-high amplification (>50× copies) with dense, localized breakpoint clustering. Long-read tumor DNA (>1.5× whole-genome coverage) provided a structural reference sufficient to confirm amplification topology and breakpoint positions. Junction-spanning cfDNA fragments aligned to predicted boundaries, supporting direct focal amplification recovery from liquid biopsy. Conclusions: Aqueous humor cfDNA provides a high-performance testbed for characterizing focal amplification signatures and defining technical performance thresholds relevant to liquid biopsy assay development. These results establish analytical parameters-minimum sequencing depth, copy-number resolution limits, and junction-fragment detectability-that may inform future platform optimization for lower-tumor-fraction biofluids such as plasma and CSF.