To the Editor Recent advances in sequencing technologies have generated extensive interest and research in cell-free DNA as a cancer biomarker.1 In their study on the use of aqueous humor as a surrogate tumor biopsy for the diagnosis of retinoblastoma, Berry et al2 pioneer the use of cell-free DNA from the aqueous humor (AH) to characterize the genetics of retinoblastoma tumors in 3 pediatric patients. Given the lack of feasibility of tissue biopsy in this disease, the clinical utility of a liquid biopsy for assessing RB genetic status is clear. While these results lay the foundation for AH liquid biopsy development, questions on the study and on the best path forward remain.
First, careful consideration of the platform used for genetic analysis is necessary. Whole-genome sequencing is likely to be prohibitively shallow in this context. This raises the question of what the depth, DNA input, and limit of detection were for these samples in the study by Berry et al.2 Deeper platforms, such as droplet digital polymerase chain reaction or targeted next-generation sequencing, have higher sensitivities (limit of detection, <0.5% variant allele frequency) than whole-genome sequencing.3,4 Considering the small quantities of cell-free DNA in AH, the use of a targeted, deep assay is especially important. In particular, patients with early-stage retinoblastoma in which the AH contains low total variant DNA or low variant allele frequency would benefit from deep sequencing. Fortunately, the well-characterized genetics of retinoblastoma are amenable to coverage via a small next-generation sequencing panel.5
Second, it is necessary to assess cell-free DNA in AH against other noninvasive biomarkers in future trials. Collection of AH is more invasive and carries higher complication rates than simple blood draws do. To this end, did the authors collect plasma from these patients? Were the alterations found in tumor or cell-free DNA in AH also found in plasma? Furthermore, assessing cell-free DNA sampled from plasma may help with diagnosis and/or prognosis of other ocular conditions arising from somatic mutations (eg, ocular melanoma).
Third, defining the most promising clinical situations in which to use this assay is imperative. Given the relative ease of diagnosing retinoblastoma based on clinical features alone, it is likely to have greater utility for providing prognostic information than for making or confirming a diagnosis.
Overall, Berry and coauthors2 have broadened the use of liquid biopsy assays to retinoblastoma tumors, an indication that derives obvious benefit from noninvasive tumor genotyping. Further development is necessary, but this laudable work brings the field closer to noninvasive options for genetic analysis of retinoblastoma.
Footnotes
Conflict of Interest Disclosures:
All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. No disclosures were reported.
References
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