Diagnostic genomics in retinoblastoma: Aqueous humor outperforms blood for tumor-specific insights

Purpose: There remains controversy in ocular oncology over whether blood or AH is a superior liquid biopsy for retinoblastoma (RB). Herein, we compare their diagnostic utility. Methods: Matched AH and plasma were collected at diagnosis from 13 patients (16 eyes) with RB. Cell-free DNA was sequenced to assess copy number alteration (CNA) amplitude; ichorCNA software was used to estimate tumor fraction (TFx). Paired results were compared using the Wilcoxon test. Results: SCNAs were detectable in 93.8% of AH samples versus 53.8% of plasma samples. Plasma TFx was significantly lower (4.33-7.84%; average 7.65%) than AH TFx (4.69-99.37%; average 34.19%; p =0.004), with a median difference of 16.73%. Plasma CNAs were detected only in patients with Group D/E eyes, two-thirds of which were enucleated. By contrast, AH enabled CNA detection across all groups. In 75% of patients positive in both substrates, AH identified additional alterations absent in plasma. In bilateral cases (23%), AH revealed eye-specific alterations indistinguishable in plasma. No evidence of metastasis was observed at average follow-up of 4 months. Conclusion: While blood CNA detection may correlate with advanced disease, AH demonstrates superior sensitivity for intraocular diagnosis. Clinical Implication: Validated by a CLIA-certified assay (LBSeq4Kids), AH consistently outperforms plasma as a biopsy surrogate.