Recent advances have expanded the role of genetic testing in retinoblastoma beyond the detection of germline mutations. Genetic testing may now contribute to diagnosis and may also provide prognostic information regarding local recurrence and systemic metastasis. For ophthalmologists, understanding these emerging applications is essential for counseling patients and families, identifying children at risk, and ensuring appropriate early intervention.

Retinoblastoma Genetics

Retinoblastoma can present as a heritable (germline) form, in which a constitutional pathogenic variant is present on one RB1 allele in all cells of the body (the “first hit”). Tumor development occurs when a second somatic mutation (the “second hit”) inactivates the remaining normal RB1 allele within a retinal cell. Germline retinoblastoma can also occur in the setting of mosaicism, where only a subset of the body’s cells carry a pathogenic RB1 mutation.¹

The second clinical form is nonheritable (somatic) retinoblastoma, in which both RB1 mutations (“first and second hit”) occur exclusively within a retinal cell. Because these mutations are not present in the germline, they are not transmitted to offspring and typically result in a unilateral, unifocal tumor.¹

It is important to note that RB1 inactivation is necessary but not sufficient for the development of retinoblastoma. Additional somatic genetic alterations are typically required for tumor progression. For example, recurrent somatic copy number alterations (SCNAs), including gains of chromosome arms 1q, 2p, and 6p and losses of 13q and 16q, are frequently observed in retinoblastoma and may serve as tumor biomarkers.²

Sources of DNA

Tumor DNA can be isolated not only from tumor tissue, but also from aqueous humor and plasma. Cell-free DNA (cfDNA) consists of fragmented extracellular DNA released from both normal and tumor cells. Circulating tumor DNA (ctDNA) represents the tumor-derived fraction of cfDNA that can be detected in body fluids such as plasma.

Pioneering work from Children’s Hospital Los Angeles (CHLA) and Memorial Sloan Kettering Cancer Center (MSKCC) has demonstrated that retinoblastoma-derived cfDNA can be safely obtained from the anterior chamber³ and detected in both aqueous humor and peripheral blood.⁴ Importantly, genomic analysis of aqueous humor cfDNA accurately reflects the SCNAs present within the corresponding tumor, providing a minimally invasive approach to tumor genotyping.

Germline Testing

With the development of globe-salvaging treatments such as intra-arterial chemotherapy and intravitreal chemotherapy, primary enucleation is no longer performed in all cases. When the eye is preserved, direct tumor biopsy is contraindicated because of safety concerns. In this setting, liquid biopsy—particularly cfDNA analysis of aqueous humor or blood—may serve as a surrogate for tumor tissue.^5-8 By defining the tumor mutation profile, liquid biopsy may help guide targeted germline testing, thereby improving diagnostic sensitivity, particularly in cases of mosaicism.^4,9-11

Diagnostic Biopsy

The diagnosis of retinoblastoma is primarily clinical and is based on ophthalmic examination and ancillary imaging. However, diagnosis can be challenging in certain situations, particularly in the presence of media opacity that limits fundus visualization or when distinguishing retinoblastoma from simulating conditions such as Coats disease or familial exudative vitreoretinopathy.⁴

A recent prospective study from CHLA evaluated aqueous humor liquid biopsy for the diagnosis and management of retinoblastoma.¹² The assay demonstrated a feasibility rate of 98%, with a sensitivity of 98% for detecting active disease (95% CI, 87%-100%) and a specificity of 100% (95% CI, 81%-100%). None of the 14 patients with retinoblastoma-mimicking conditions had a positive result. False-negative results may occur in Group A tumors measuring less than 1 mm, likely reflecting insufficient tumor DNA shedding into the aqueous humor. These findings support aqueous humor liquid biopsy as a highly accurate adjunctive tool for the diagnosis of retinoblastoma.

Circulating tumor DNA (ctDNA) can be detected in peripheral blood in up to 85% of patients with untreated unilateral retinoblastoma and seems to reflect tumor burden.¹³ False-negative results may occur in patients with small tumors, low levels of tumor DNA shedding, or in the rare subset of retinoblastomas that retain an intact RB1 gene and are driven by MYCN amplification.

Prognostic Biopsy

Aqueous humor biopsy may also provide prognostic information regarding the likelihood of globe salvage with conservative therapy or the need for secondary enucleation. Although larger prospective studies are needed to validate these findings, chromosome 6p gain, 7q gain, and MYCN amplification have been associated with poorer globe salvage outcomes and an increased risk of secondary enucleation.^5,14-16 Epigenetic alterations may also help stratify retinoblastoma tumors according to their biological aggressiveness.¹⁷

Blood ctDNA level appears to also correlate with ocular disease progression¹⁸ and regression¹⁹ following intra-arterial chemotherapy.

Plasma ctDNA analysis may also have value for metastatic risk assessment.⁸ Although the number of reported patients remains small and the optimal sensitivity threshold is still under investigation, treatment-naïve patients with a variant allele frequency greater than 8.1% subsequently developed metastasis (n=3), whereas patients with a variant allele frequency less than 3.2% did not develop metastasis (n=127).^4,8

Conclusions

In the era of personalized medicine, genetic testing in retinoblastoma is evolving beyond the detection of germline RB1 mutations. Emerging approaches, including analysis of cfDNA and molecular profiling of tumor-derived biomarkers, have the potential to improve germline mutation detection while also providing valuable prognostic information regarding local tumor control, globe salvage, and metastasis. These advances enable individualized management and surveillance strategies for patients with retinoblastoma. RP

Soumaya Bouhout, MD, is an ocular oncology fellow at the Cole Eye Institute of Cleveland Clinic in Cleveland, Ohio. She reports no disclosures.

Arun D. Singh, MD, is a professor of ophthalmology and director of the department of ophthalmic oncology at the Cole Eye Institute and Taussig Cancer Center of Cleveland Clinic. He reports consulting fees from Aura Biosciences, Castle Biosciences, Ideaya Biosciences,  Immunocore, and Isoaid. Reach him at singha@ccf.org.

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