Non-invasive diagnosis of retinoblastoma using cell-free DNA from aqueous humour - 11th International Symposium on Circulating Nucleic Acids in Plasma and Serum (CNAPS)

Amy Gerrish ¹ Edward Stone ¹ Samuel Clokie ¹ John Ainsworth ² Helen Jenkinson ² Maureen McCalla ² Carol Hitchcott ² Isabel Colmenero ³ Stephanie Allen ¹ Manoj Parulekar ² Trevor Cole ¹

¹West Midlands Regional Genetics Service, Birmingham Women’s and Children’s NHS Foundation Trust, UK
²Birmingham Children’s Hospital Eye Department, Birmingham Women’s and Children’s NHS Foundation Trust, UK
³Department of Histopathology, Birmingham Women’s and Children’s NHS Foundation Trust, UK

Retinoblastoma is the most common eye malignancy in childhood caused by mutations in the RB1 gene. The initial RB1 mutation may be germline or somatic. Distinguishing between these alternative mechanisms is crucial, with wider implications for management of the patient and family members. Modern eye-saving chemotherapy treatment has resulted in fewer enucleations. As a result, tumour tissue required to identify sporadic RB1 mutation(s) is not always available. Intravitreal chemotherapy (IViC) techniques for retinoblastoma involve prior aspiration of aqueous humour (AH), providing a novel sample source for analysis. Using hybridization-based next generation sequencing, we have designed a test capable of detecting somatic mutations of the RB1 gene in cfDNA extracted from the AH of retinoblastoma patients. The assay captures sequences across the whole of chromosome 13 with a higher density of probes across 6.5Mb surrounding the RB1 gene. We have developed an in house bioinformatic pipeline to detect a variety of genetic mutations observed in retinoblastoma patients including single nucleotide variants, INDELs, loss of heterozygosity and copy number variants. The results obtained with fluid from enucleated eyes were concordant with tumour tissue in all 10 cases analysed. While we observed much lower cfDNA concentrations in the AH from patients undergoing IViC, we successfully identified previously unknown somatic variants in 3 cases including c.751C>T p.Arg251* and two LOH regions. Detection of these variants was aided by the fact that the majority of cfDNA in the AH originates from the tumour, likely due to the compartmentalised nature of ocular fluids. This proof of principle study indicates that cfDNA from the eye fluid of retinoblastoma patients could be used for somatic mutation studies where tumour samples are unavailable. We plan to further develop this assay using our collection of over 40 additional AH samples obtained from a cohort of 11 patients undergoing IViC treatment.