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Non-invasive diagnosis of retinoblastoma using cell-free DNA from aqueous humour
  1. Amy Gerrish1,
  2. Edward Stone1,
  3. Samuel Clokie1,
  4. John R Ainsworth2,
  5. Helen Jenkinson2,
  6. Maureen McCalla2,
  7. Carol Hitchcott2,
  8. Isabel Colmenero3,
  9. Stephanie Allen1,
  10. Manoj Parulekar2,
  11. Trevor Cole1
  1. 1 West Midlands Regional Genetics Service, Birmingham Women's and Children's NHS Foundation Trust, Birmingham, UK
  2. 2 Birmingham Children’s Hospital Eye Department, Birmingham Women's and Children's NHS Foundation Trust, Birmingham, UK
  3. 3 Department of Histopathology, Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, UK
  1. Correspondence to Dr Amy Gerrish; amy.gerrish{at}


Retinoblastoma is the most common eye malignancy in childhood caused by mutations in the RB1 gene. Both alleles of the RB1 gene must be mutated for tumour development. The initial RB1 mutation may be constitutional germline or somatic (originating in one retinal cell only). Distinguishing between these alternative mechanisms is crucial, with wider implications for management of the patient and family members. Bilateral retinoblastoma is nearly always due to a constitutional mutation; however, approximately 15% of unilateral cases also carry a germline mutation, and identifying these cases is important. This can be achieved by identifying both mutation types in tumour tissue and excluding their presence in blood. Modern eye-saving chemotherapy treatment (systemic, intra-arterial and intravitreal) has resulted in fewer enucleations. As a result, tumour tissue required to identify sporadic RB1 mutation(s) is not always available. Modern intravitreal chemotherapeutic techniques for retinoblastoma involve aspiration of aqueous humour (AH), providing a novel sample source for analysis. By analysing cell-free DNA present in the AH fluid of eyes affected with retinoblastoma, we have developed a screening test capable of detecting somatic RB1 mutations that is comparable to current tests on enucleated tumour tissue. The results obtained with fluid from enucleated eyes were concordant with tumour tissue in all 10 cases analysed. In addition, AH analysis from two patients undergoing intravitreal chemotherapy successfully identified somatic variants in both cases. Our findings suggest that AH fluid is a promising source of tumour-derived DNA in retinoblastoma for analysis.

  • genetics
  • diagnostic tests/investigation
  • aqueous humour

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See:

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  • Contributors AG, SA, ES, SC and TC provided experimental design and execution and drafting of the manuscript. MP, JA and TC provided clinical input, recruitment and drafting/ editing of the manuscript.

  • Funding This work was funded by a Wellcome trust Pathfinder Award Ref. 201073/Z/16/Z.

  • Competing interests None declared.

  • Patient consent Not required.

  • Ethics approval This study has REC and HRA approval 16/EE/0528.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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