Tumor-derived cell-free DNA (cfDNA) has biomarker potential; therefore, this study aimed to identify cfDNA in the aqueous humor (AH) of retinoblastoma eyes and correlate somatic chromosomal copy-number alterations (SCNA) with clinical outcomes, specifically eye salvage. AH was extracted via paracentesis during intravitreal injection of chemotherapy or enucleation. Shallow whole-genome sequencing was performed using isolated cfDNA to assess for highly recurrent SCNAs in retinoblastoma including gain of 1q, 2p, 6p, loss of 13q, 16q, and focal amplification. Sixty-three clinical specimens of AH from 29 eyes of 26 patients were evaluated; 13 eyes were enucleated and 16 were salvaged (e.g., saved). The presence of detectable SCNAs was 92% in enucleated eyes versus 38% in salvaged eyes ( = 0.006). Gain of chromosome 6p was the most common SCNA found in 77% of enucleated eyes, compared with 25% of salvaged eyes ( = 0.0092), and associated with a 10-fold increased odds of enucleation (OR, 10; 95% CI, 1.8-55.6). The median amplitude of 6p gain was 1.47 in enucleated versus 1.07 in salvaged eyes ( = 0.001). The presence of AH SCNAs was correlated retrospectively with eye salvage. The probability of ocular salvage was higher in eyes without detectable SCNAs in the AH ( = 0.0028), specifically 6p gain. This is the first study to correlate clinical outcomes with SCNAs in the AH from retinoblastoma eyes, as such these findings indicate that 6p gain in the aqueous humor is a potential prognostic biomarker for poor clinical response to therapy. The correlation of clinical outcomes and SCNAs in the AH identified in the current study requires prospective studies to validate these finding before SCNAs, like 6p gain, can be used to predict clinical outcomes at diagnosis. .

◥Aqueous humor contains tumor-derived cell-free DNA (cfDNA) and can serve as a liquid biopsy for retinoblastoma. We previously associated somatic copy-number alteration (SCNA) 6p gain with a 10-fold increased risk of enucleation. Here we provide a 2-year update to further explore 6p gain as a prognostic biomarker for ocular survival. Patients diagnosed with retinoblastoma from December 2014 to July 2019 from whom aqueous humor was sampled were included. cfDNA was extracted and shallow whole-genome sequencing performed to identify highly recurrent retinoblastoma SCNAs (gain of 1q, 2p, 6p, loss of 13q, 16q). 116 aqueous humor samples from 50 eyes of 46 patients were included: 27 eyes were salvaged, 23 were enucleated. Highly recurrent retinoblastoma SCNAs were found in 66% eyes. 6p gain was the most prevalent SCNA (50% eyes). It was particularly more prevalent in enucleated eyes (73.9%) than in salvaged eyes (29.6%; P ¼ 0.004). 6p gain in aqueous humor cfDNA portended nearly 10-fold increased odds of enucleation (OR ¼ 9.87; 95% confidence interval ¼ 1.75-55.65; P ¼ 0.009). In the enucleated eyes, 6p gain was associated with aggressive histopathologic features, including necrosis, higher degrees of anaplasia, and focal invasion of ocular structures. With extended follow-up and nearly double the aqueous humor samples, we continue to demonstrate 6p gain as a potential prognostic biomarker for retinoblastoma.Implications: Aqueous humor is a high-yield source of tumorderived DNA in retinoblastoma eyes. Detection of 6p gain in the aqueous humor allows for targeted, patient-centered therapies based on this molecular prognostic marker. Prospective, multicenter studies with aqueous humor sampled from all eyes at diagnosis are warranted to validate these findings.

Because direct tumor biopsy is prohibited for retinoblastoma (RB), eye-specific molecular biomarkers are not used in clinical practice for RB. Recently, we demonstrated that the aqueous humor (AH) is a rich liquid biopsy source of cell-free tumor DNA. Herein, we detail clinically-relevant molecular biomarkers from the first year of prospective validation data. Seven eyes from 6 RB patients who had AH sampled at diagnosis and throughout therapy with ≥12 months of follow-up were included. Cell-free DNA (cfDNA) from each sample was isolated and sequenced to assess genome-wide somatic copy number alterations (SCNAs), followed by targeted resequencing for pathogenic variants using a RB1 and MYCN custom hybridization panel. Tumoral genomic information was detected in 100% of diagnostic AH samples. Of the seven diagnostic AH samples, 5/7 were positive for RB SCNAs. Mutational analysis identified RB1 variants in 5/7 AH samples, including the 2 samples in which no SCNAs were detected. Two eyes failed therapy and required enucleation; both had poor prognostic biomarkers (chromosome 6p gain or MYCN amplification) present in the AH at the time of diagnosis. In the context of previously established pre-analytical, analytical, and clinical validity, this provides evidence for larger, prospective studies to further establish the clinical utility of the AH liquid biopsy and its applications to precision oncology for RB.

Retinoblastoma (RB) is a childhood intraocular cancer initiated by biallelic inactivation of the RB tumor suppressor gene (RB1−/−). RB can be hereditary (germline RB1 pathogenic allele is present) or non‐hereditary. Somatic copy number alterations (SCNAs) contribute to subsequent tumorigenesis. Previous studies of only enucleated RB eyes have reported associations between heritability status and the prevalence of SCNAs. Herein, we use an aqueous humor (AH) liquid biopsy to investigate RB genomic profiles in the context of germline RB1 status, age, and International Intraocular Retinoblastoma Classification (IIRC) clinical grouping for both enucleated and salvaged eyes. Between 2014 and 2019, AH was sampled from a total of 54 eyes of 50 patients. Germline RB1 status was determined from clinical blood testing, and cell‐free DNA from AH was analyzed for SCNAs. Of the 50 patients, 23 (46.0%; 27 eyes) had hereditary RB, and 27 (54.0%, 27 eyes) had non‐hereditary RB. Median age at diagnosis was comparable between hereditary (13 ± 10 months) and non‐hereditary (13 ± 8 months) eyes (P = 0.818). There was no significant difference in the prevalence or number of SCNAs based on (1) hereditary status (P > 0.56) or (2) IIRC grouping (P > 0.47). There was, however, a significant correlation between patient age at diagnosis, and (1) number of total SCNAs (r[52] = 0.672, P < 0.00001) and (2) number of highly‐recurrent RB SCNAs (r[52] = 0.616, P < 0.00001). This evidence does not support the theory that specific molecular or genomic subtypes exist between hereditary and non‐hereditary RB; rather, the prevalence of genomic alterations in RB eyes is strongly related to patient age at diagnosis.

The aqueous humor (AH) liquid biopsy enables in vivo evaluation of tumorderived cell-free DNA (cfDNA) from retinoblastoma (RB) eyes. Herein, we test our hypothesis that longitudinal dynamics of AH cfDNA-including tumor fraction (TFx) and somatic copy number alteration (SCNA) amplitude-correspond to therapeutic response. Methods: Eyes with ≥3 AH extractions during intravitreal chemotherapy (IVM) or at secondary enucleation between 2015 to 2019 were included. AH cfDNA was sequenced to assess RB SCNA amplitude; ichorCNA software was used to estimate TFx. Eyes without SCNAs or with TFx < 0.10 across all samples were excluded. Therapeutic responses for each eye were determined from clinical records. Statistical analyses included Mann-Whitney U and Pearson correlation tests. Results: Twenty eyes of 20 patients underwent ≥3 AH extractions; 6 eyes lacked SCNAs or had TFx < 0.10 throughout sampling and were excluded. Clinical progression was associated with significantly higher SCNA amplitudes and TFx values than regression (P ≤ 0.04). Relative increases in TFx (TFx 1.86 ± 2.22) were associated with disease progression, whereas relative decreases in TFx (TFx 0.53 ± 0.36) were associated with disease regression (P < 0.00001). A ≥15% increase in TFx relative to baseline during treatment was associated with an over 90-fold increased likelihood of clinical progression (odds ratio = 90.67, 95% confidence interval = 8.30-990.16, P = 0.0002). TFx and SCNA amplitude were significantly positively correlated throughout sampling (P ≤ 0.002). Conclusions: Longitudinal changes in AH-derived cfDNA TFx and SCNA amplitude are concordant with clinical responses of intraocular RB during active therapy. Translational Relevance: Longitudinal evaluation of AH cfDNA may provide an objective, quantitative way to monitor therapeutic response and disease burden in RB patients.