Genomic cfDNA Analysis of Aqueous Humor in Retinoblastoma Predicts Eye Salvage: The Surrogate Tumor Biopsy for Retinoblastoma

Abstract

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. .

Preclinical studies reveal MLN4924 is a promising new retinoblastoma therapy

“…Therefore, assessing multiple injections or combining MLN4924 with standard-of-care chemotherapies are attractive strategies to achieve CR. It would also be of interest to test MLN4924 efficacy in metastatic RB or on more aggressive primary tumors that lead to enucleation, which can now be predicted from somatic chromosomal copy-number alterations (SCNA) detected in aqueous humor extracts 38 .…”

Next-Generation Technologies and Strategies for the Management of Retinoblastoma

“…AH is extracted from the affected eye at the time of diagnosis. The Berry et al [50] study also demonstrated several parameters that have the potential to be critically important to the future of molecular diagnostics and prognostics in retinoblastoma. First, rare focal genomic alterations, such as MYCN amplification, can be detected, which can be additionally impactful for prognosis of this disease.…”

“…The authors found that gain of 6p was the most commonly seen SCNA in the AH, with a statistically significant difference for this biomarker in enucleated eyes (77%) versus salvaged eyes (25%) (p = 0.0092). Notably, if gain of 6p was present in the AH of an eye with retinoblastoma, it was associated with 10x increased odds of that eye requiring enucleation (OR 10, 95% CI 1.8-55.6) [50]. This was the first study to suggest that genetic and genomic evaluation of the AH could be used for prognostication of clinical outcomes, and to describe gain of 6p in the AH as a biomarker (Figure 1).…”

“…Together, these are termed RB SCNAs. The gain of 6p is particularly common in RB tumors [49] and has been associated with significantly lower rates of ocular salvage [50]. Although the specific role of these chromosomal alterations in RB development is not entirely known, it is thought that RB SCNAs influence the activity and expression of oncogenes and/or tumor suppressor genes to cause malignant progression of disease [49].…”

“…For the first time, this opened the door to obtaining tumor-specific molecular biomarkers in vivo-either at diagnosis or during globe-salvaging treatment. The authors went on to analyze 63 AH samples from 29 eyes of 26 patients; 13 eyes were enucleated and 16 were saved [50]. This approach allowed for a direct comparison of the genomic profiles between the eyes that responded to therapy and were saved versus those that subsequently required enucleation.…”