Retinoblastoma was one of the first malignant tumors to be described as a genetic disease and its development occurs from the loss of function of the retinoblastoma gene (RB1). The difficulty in accessing the tumor during diagnosis highlights the need for non-invasive diagnostic methods. Studies have shown that liquid biopsy, obtained from any fluid material in the body, for example blood, contains free tumor cells and free and circulating DNA or RNA, making it a convenient tool for diagnosis and prognosis during cancer treatment without the need for invasive procedures. Taking advantage of these events, given this situation, we investigated molecular alterations in samples from retinoblastoma cases, using the NGS strategy as a powerful tool for characterization and aid in diagnosis and prognosis. Genomic data from 76 patients diagnosed with retinoblastoma, comprising 162 samples, tumor (TU), aqueous humor (AH), and peripheral blood (PB), were analyzed using the Oncomine Childhood Cancer Research Panel (OCCRA^®). A total of 22 altered genes were detected, and 54 variants. Of the 76 cases, 29 included paired tumor (TU), aqueous humor (AH), and peripheral blood (PB) samples from the same patient. Alterations in the RB1 gene were detected in 16 of these 29 cases, with concordant alterations identified across all three sample types in three patients. In 12 out of 29 patients, the same genetic alteration was found in both TU and AH. In conclusion, the OCCRA panel enabled the detection, in different samples, of molecular alterations in the RB1 gene, as well as CNAs in the MYCN, ABL2, and MDM4 genes. Limitations of AH were observed, primarily due to the small volume of material available and the consequently low concentration of cell-free DNA (cfDNA). However, as AH provides a viable alternative for analyzing tumors, inaccessible to traditional biopsy methods, liquid biopsy holds significant potential to improve diagnostic accuracy and guide treatment strategies in retinoblastoma cases.

Retinoblastoma (RB) is the most common intraocular malignancy of childhood, yet molecular assessment of disease dissemination and minimal residual disease (MRD) remains challenging due to the contraindication of intraocular biopsy. Here, we evaluate the feasibility of cell-free RNA (cfRNA)- and circulating tumor cell RNA (ctcRNA)-based liquid biopsy for the sensitive detection of disseminated retinoblastoma using digital PCR (dPCR) targeting the retina-specific markers CRX and RBP3. We analyzed 433 bone marrow (BM), peripheral blood (PB) and cerebrospinal fluid (CSF) samples collected longitudinally from 50 patients with RB. dPCR assays demonstrated high analytical sensitivity. cfRNA detection showed complete sensitivity and negative predictive value in bone marrow compared with myelogram analysis, frequently identifying molecular positivity in cytologically negative samples. In cerebrospinal fluid, cfRNA detection was highly specific but less sensitive, reflecting compartment-specific biological constraints. Longitudinal analysis revealed that changes in CRX and RBP3 ctcRNA levels closely tracked treatment response, preceded cytological evidence of bone marrow involvement in several cases, and identified molecular persistence or re-emergence during follow-up, including after hematopoietic stem cell transplantation. Together, these findings demonstrate that cfRNA- and ctcRNA-based liquid biopsy using CRX and RBP3 enables sensitive and dynamic detection of disseminated retinoblastoma, particularly in bone marrow, and supports its potential utility for MRD monitoring. Longitudinal patient analyses will be required to define prognostic thresholds and establish the clinical role of this approach in risk stratification and long-term surveillance.