Validation of an aqueous humor liquid biopsy for molecular prognostication and monitoring of children with retinoblastoma. - Project Summary There is a significant body of research into the genetic, genomic and epigenomic alterations of retinoblastoma (RB), a primary eye cancer that forms in the developing retina in young children. However, these studies were done on tumor tissues from surgically removed (enucleated) eyes with advanced RB, as tumor biopsy is not possible due to the real risk of tumor extraocular dissemination. As a result, RB tumor DNA was never previously accessible aside from these enucleated specimens, and there is limited understanding of the molecular alterations that may drive tumor behavior. Furthermore, any application of molecular diagnostic or use of prognostic biomarkers for personalized medicine in vivo is limited by the lack of tumor tissue at diagnosis or during therapy. Thus, a liquid biopsy approach, which overcomes this critical lack of tumor tissue, was needed for this disease. With support of an NCI K08, we demonstrated that the aqueous humor (AH), an intraocular fluid, is an enriched source of tumor-derived cell-free DNA (cfDNA). We developed a liquid biopsy assay to detect somatic copy number alterations (SCNAs) and pathogenic variants in the RB1 tumor suppressor gene from a single 100 l sample of AH. We identified that the genomic alterations from the AH are highly concordant (>94%) with those found in the tumor of enucleated eyes. We identified potential candidate biomarkers, chromosome 6p gain and/or focal MYCNa in the AH cfDNA that are associated with a 16.5-fold increased risk of treatment failure requiring surgical removal of the eye. We demonstrated that changes in AH cfDNA tumor fraction (TFx) correlate with treatment response, with increases in TFx indicative of recurrence or minimal residual intraocular disease. This suggests that TFx alone may serve as a reliable real-time biomarker for treatment response. We also demonstrated the feasibility of evaluating tumor methylation profiles using the AH, thereby facilitating a better understanding of tumor biomarkers that may predict tumor behavior and potentially treatment response. Based in these results, we hypothesize that AH cfDNA can be used for molecular characterization of in vivo RB tumors to inform diagnosis and prognosis for eye salvage based on validated genomic and epigenomic biomarkers. To test this hypothesis, we now propose a multi-center, multi-omics, prospective study to characterize prognostic AH biomarkers prospectively and longitudinally to determine treatment outcomes. Benefits from this study will include advancing knowledge about the course of the disease at any stage, providing biomarkers to guide treatment, and forming the basis for future molecular-based, precision medicine clinical trials for RB.
