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

Complex cases of retinoblastoma (RB) often require integrative molecular approaches to define tumor etiology and guide clinical management.

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.

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 RB1 gene. Targeted cfDNA analysis using the Illumina TruSight Oncology 500 (TSO500) panel on AH and plasma identified a somatic RB1 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., MDM4 and ALK) and deletions (i.e., BRCA2), indicating progressive clonal tumor evolution.

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.