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Original research
Cell-free RB1 DNA not detected in the blood of pseudoretinoblastoma patients
  1. David H Abramson1,
  2. Diana Mandelker2,
  3. A Rose Brannon2,
  4. Michael F Berger2,
  5. Melissa Robbins1,
  6. Ira J Dunkel3,
  7. Jasmine H Francis1
  1. 1Surgery, Memorial Sloan Kettering Cancer Center, New York, New York, USA
  2. 2Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
  3. 3Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
  1. Correspondence to Dr David H Abramson; abramsod{at}


Cell-free DNA (cfDNA) is commonly found in the blood (plasma) of patients with cancer. When analysing cfDNA for a specific cancer-causing mutation, it is referred to as ctDNA. RB1 ctDNA is commonly present in the blood of retinoblastoma patients. We examined RB1 ctDNA from blood of 40 children with retinoblastoma look alike lesions (‘pseudoretinoblastoma’) to determine if any RB1 abnormalities could be identified.

Objectives Because retinoblastoma diagnosis is usually made with the indirect ophthalmoscope without biopsy clinical errors continue to occur worldwide. Because cf RB1 is detectible in plasma of children with retinoblastoma, we wondered if it was present in the blood of pseudoretinoblastomas with the hope of ultimately developing a blood based test to aid clinicians in the diagnosis of retinoblastoma. The goal of this project was to see if circulating plasma RB1 cfDNA could be detected in the blood of patients with pseudoretinoblastoma.

Methods and analysis Plasma cfDNA for circulating RB1 cfDNA was assayed with MSKCC’s next generation sequencing, N.Y. State Approved assay called ACCESS to evaluate somaticmutations in 40 patients with pseudoretinoblastoma.

Results No plasma cfDNA RB1 was detected in the blood (plasma) of 40 patients with pseudoretinoblastoma.

Conclusion Plasma cfDNA RB1 is commonly detectible in retinoblastoma patients but not in patients with a diverse group of pseudoretinoblastomas.

  • Child health (paediatrics)
  • Diagnostic tests/Investigation
  • Eye (Globe)
  • Genetics
  • Retina

Data availability statement

Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as online supplemental information.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See:

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  • Mutant RB1 is commonly detectable in the plasma of children with newly diagnosed retinoblastoma. Few cases of pseudoretinoblastoma have been similarly studied.


  • No mutant RB1 cell free DNA (cfDNA) was detected in a diverse group of pseudoretinoblastomas.


  • Analysis of RB1 cfDNA in plasma may be useful in the non-invasive differential diagnosis of retinoblastoma. Additional research is needed to determine the true sensitivity and specificity of cfDNA for the diagnosis of retinoblastoma.


Cell-free DNA (CfDNA) is now a clinical tool used to aid the diagnosis, prognosis and management of diverse cancers.1 We have demonstrated that RB1 cfDNA is commonly detected in the plasma of retinoblastoma patients before treatment and in the one patient studied the level increased after a month without treatment.2 Following enucleation or intraarterial chemotherapy, it becomes undetectable.2 3

The diagnosis of retinoblastoma is usually made with the indirect ophthalmoscope and sometimes aided with ultrasound and MRI.4 Biopsy is not done because of the concern for spreading the cancer. A diverse group of intraocular lesions that resemble retinoblastoma (but are not cancer) simulate retinoblastoma and accurate diagnosis at times is very difficult and errors continue to be made resulting in inappropriate surgery and even removal of eyes in children who do not have cancer.5–7 In addition, RB1 abnormalities have been reported in a variety of other conditions including rhabdomyosarcoma.8

Tumour-specific plasma cfDNA is now being used in adult and paediatric solid cancers for diagnosis, differential diagnosis, prognosis, monitoring of response and detection of tumour mutations. We have shown that RB1 fragments (ctDNA) are commonly (60%–80%) present in the blood (plasma) of children with newly diagnosed retinoblastoma.2 9 10 The purpose of this study was to see if RB1 fragments are detectible in the plasma of patients with pseudoretinoblastoma.


The cfDNA was analysed with hybridisation capture and next-generation sequencing in blood (plasma) using MSKCC’s analysis of circulating cfDNA to evaluate somatic status11 in 40 patients with diverse pseudoretinoblastoma lesions. This technique interrogates 129 established cancer mutations and because the buffy coat is simultaneously analysed germline defects and clonal haematopoiesis are filtered out in the results. Children with pseudoretinoblastoma like lesions that had been referred to our centre for a possible diagnosis of retinoblastoma were included.

Patient involvement

Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research.


Table 1 depicts/shows that for 40 patients with pseudoretinoblastoma, plasma RB1 cfDNA was not detectable in any patient.

Table 1

Detection of plasma RB1 cell-free DNA (cfDNA) in pseudoretinoblastoma lesions


The diagnosis of retinoblastoma is usually made with the indirect ophthalmoscope and often ultrasound and MRI are performed. There are more than 30 conditions that have been described that mimic the appearance of retinoblastoma and they are referred to as ‘pseudoretinoblastomas.’ The most common ‘pseudoretinoblastomas’ are Coats’ disease, persistent fetal vasculature syndrome, retinal detachment, ocular toxocariasis and other infectious, genetic and even traumatic lesions (table 1).1 Despite the assistance of ultrasound and MRI, the differential diagnosis of retinoblastoma remains a challenge and errors are still made with children’s eyes receiving inappropriate surgery including vitrectomy, retinal detachment repair and enucleation.5–7 In addition, some eyes with retinoblastoma are not enucleated because the clinician misdiagnosed them as benign conditions. The true, modern incidence of these errors is not known because few centres are eager to publish on their errors in diagnosis, but recent publications suggest an inappropriate enucleation rate in some centres of 11%–24%.5–7

MSKCC developed a next generation sequencing assay for 129 cancer-related genetic abnormalities and one of these genes is RB1. All exons of RB1 are interrogated.

In this study, we assayed for cfDNA of RB1 and in all cases no RB1 fragments were detectable. These assays were done simultaneously with true retinoblastoma samples (which did detect RB1 abnormalities) and occasionally other, non-RB1 abnormalities giving us additional confidence that the assay was accurate. This suggests that finding RB1 cfDNA in the blood of a child with suspected retinoblastoma is not a pseudoretinoblastoma. Whether this test can be used as an aid in the differential diagnosis of retinoblastoma will depend on additional analyses of sensitivity and specificity in a larger cohort of both retinoblastoma and pseudoretinoblastoma eyes, but neither this study nor any prior published studies have demonstrated RB1 abnormalities in pseudoretinoblastomas.

Data availability statement

Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as online supplemental information.

Ethics statements

Patient consent for publication

Ethics approval

This study involves human participants and was approved by an Ethics Committee or Institutional Board. Informed consent was obtained from all subjects (or their legal guardian) using MSKCC Institutional Review Board Number 12-245. Participants gave informed consent to participate in the study before taking part.



  • Contributors Conceptualisation, DHA and JHF; Data curation, DHA, MR and JHF; Formal analysis, DHA, DM, MR and JHF; Funding acquisition, DHA and JHF; Investigation, DHA, IJD, MFB and JHF; Methodology, DHA, DM, ARB, IJD and JHF; Project administration, DHA, ARB and JHF; Resources, DM and ARB; Software, DM and MFB; Supervision, DM and ARB; Validation, DM, ARB and MFB; Visualisation, DHA and JHF; Writing—original draft, DHA, MR and JHF; Writing—review and editing, DHA, DM, ARB, IJD, MFB, MR and JHF. All authors have read and agreed to the published version of the manuscript. Guarantor, DHA.

  • Funding This work was supported in part by grants from the Gerber Foundation (DHA and JHF), Fund for Ophthalmic Knowledge (DHA and JHF), Cancer Center Support Grant (grant no.: P30 CA008748) (DHA, DM, ARB, MFB, MR, IJD and JHF).

  • Disclaimer The sponsors or funding organisations had no role in the design or conduct of this research.

  • Competing interests IJD is a consultant or advisory board member for Apexigen, Astra-Zeneca, Bristol-Myers Squibb/Celgene, Day One, Fennec, QED and Roche. MFB declares research funding from Grail; personal fees from Roche and PetDX; and a provisional patent for systems and methods for detecting cancer via cfDNA screening (PCT/US2019/027487).

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

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