A Complex Case of Retinoblastoma Solved by the Combined Approach of Humor/Plasma cfDNA-NGS and LR-WGS

<h4>Background</h4>Complex cases of retinoblastoma (RB) often require integrative molecular approaches to define tumor etiology and guide clinical management.<h4>Purpose</h4>Our aim was to evaluate the usefulness of combining aqueous humor (AH)/plasma cell-free DNA next-generation sequencing (cfDNA-NGS) and long-read-whole-genome sequencing (LR-WGS) to resolve diagnostically challenging RB cases.<h4>Case description</h4>We report the case of a 3-year-old Caucasian girl, conceived by heterologous assisted reproductive technology (ART), presenting with unilateral, widely infiltrative RB in the right eye. She exhibited limited verbal communication, a glabellar angioma extending to the nasal bridge and philtrum, and mild hypertelorism. Standard blood testing revealed no pathogenic SNVs, CNVs, or methylation abnormalities in the <i>RB1</i> gene. Targeted cfDNA analysis using the Illumina TruSight Oncology 500 (TSO500) panel on AH and plasma identified a somatic <i>RB1</i> splice-site variant (c.1498+2T>C) with a variant allele frequency (VAF) of 98.5%, consistent with biallelic inactivation. Additional gains (fold change > 1.5) were found in AH and confirmed in plasma, suggesting a germline 13q duplication. Third-generation LR-WGS, performed with Oxford Nanopore Technology (ONT), on blood confirmed a 24.6 Mb duplication on chromosome 13, compatible with the rare 13q duplication syndrome characterized by psychomotor delay, craniofacial dysmorphism, and hemangiomas. AH-cfDNA revealed additional somatic copy-number alterations, including amplifications (i.e., <i>MDM4</i> and <i>ALK</i>) and deletions (i.e., <i>BRCA2</i>), indicating progressive clonal tumor evolution.<h4>Conclusions</h4>This experience tells us that a combined approach with TSO500 Illumina NGS on cfDNA, along with LR-WGS, is able to help solve complex cases and define the appropriate treatment and surveillance strategy.