Untangling the genetic secrets of retinoblastoma
Identifying all genetic culprits may improve diagnostics and prognoses
Retinoblastoma is a rare, aggressive genetic childhood cancer that results from a mutation that inactivates the RB1 gene, ie, the master gene of the disease, in the vast majority of cases.
Despite the fact that early research into retinoblastoma began more than 5 decades ago, the genetic information about the disease is far from complete. According to the authors of a current review1 of the genetics of retinoblastoma, the disease has a large spectrum of pathogenic variants, about 2,500 discovered thus far, with in excess of 500 different somatic or germline mutations resulting in RB1 gene inactivation. A complete understanding of retinoblastoma molecular genetics is crucial to the development of effective treatments to cover all variants, according to first author Leon Marković, MD, PhD.
He pointed out that remarkable advances in retinoblastoma therapy have been realised over the past decades, including intra-arterial and intraocular chemotherapy and proton-beam radiation therapy; however, these treatment options are not readily available to underdeveloped countries.
In this review, Dr Marković and colleagues provided a primer of the most recent information about retinoblastoma and discussed the importance of screening, genetic testing, treatment and how DNA is expected to shed further light on the genetic makeup of retinoblastoma. Dr Marković is from the Department of Ophthalmology, Reference Center of the Ministry of Health of the Republic of Croatia for Paediatric Ophthalmology and Strabismus, University Hospital “Sveti Duh,” Zagreb, Croatia, and the Faculty of Dental Medicine and Health Osijek, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia.
Background
Figure. The types of mutations observed in retinoblastoma.
Retinoblastoma is rare in that the incidence rate is 1 of 15,000 to 20,000 live births, or about 8,000 cases annually worldwide, but it is the most common primary intraocular tumour in children and infants.2-4 The disease presentation can vary and involve one or both eyes, with the respective patient ages at diagnosis being 24 and 12 months.
Dr Marković and colleagues explained, “Tumorigenesis begins with mutations that cause RB1 biallelic inactivation [that] prevents the production of functional RB proteins (pRB). Depending on the type of mutation, the penetrance of RB is different. However, in small percentages of tumours, additional genes may be required, such as MYCN, BCOR, and CREBBP, even without the inactivation of the RB1 gene. Additionally, epigenetic changes contribute to the progression of retinoblastoma.”
pRB also has other functions and is involved in regulating the nucleosome structure and participating in apoptosis, DNA replication and repair, cellular senescence, differentiation, angiogenesis and modulating chromatin remodelling.
Treatment availability, screening
As mentioned previously, the treatments available to patients are governed by location. “The good news is that today the survival rates in developed countries are over 95%; however, in lower- and middle-income countries, enucleation still remains the treatment of choice and the survival [rate] is much lower due to the [frequent] treatment failures.5,6 For instance, the survival rate and vision retention in one eye in the US are higher than 99% and 90%, respectively,7,8 while in African patients survival numbers are as low as 40%,9” the authors reported.
In addition, the screening programs for early disease detection in underdeveloped countries are also rudimentary and not uniform. With early detection and treatment, less radical forms of therapy can cure retinoblastoma10; with late-stage diagnosis, the chances of saving the ocular globe decrease.
Many countries now screen newborns for retinoblastoma.11,12 “The culprits for the variabilities in survival among countries are expensive treatment procedures, and the difference in the availability of specialised ophthalmologists,” the authors commented.
Importance of genetic testing
Here is where genetic testing comes into play: it provides patients and relatives with a better understanding of the nature of inheritance and their constitutional predisposition, Dr Marković explained.
A valuable resource is the National Ophthalmic Disease Genotyping and Phenotyping Network (eyeGENE) created by the National Eye Institute in 2006. The network data are from 118 participants who have undergone genetic testing for retinoblastoma (https://eyegene.nih.gov/).13,14
The accuracy of genetic testing is high when DNA can be isolated from tumour DNA after enucleation, which obviously is not the preferred end point. Acquiring DNA introduces the potential for numerous techniques to be performed, i.e., multiplex ligation-dependent probe amplification (MLPA), loss of heterozygosity, allele-specific polymerase chain reaction, promoter methylation detection, single nucleotide polymorphism (SNP) arrays and next-generation sequencing (NGS) followed by Sanger classical sequencing of the recognised regions, the authors explained.
They described how the RB1 gene can be evaluated completely using a multistep assay that was developed and includes DNA sequencing to identify mutations within coding exons and immediate flanking intronic regions plus the promoter regions, duplication/deletion analysis and methylation analysis of the RB1 promoter region.
However, the advent of newer treatments has decreased the numbers of enucleations and therefore access to DNA. This has been replaced by genetic testing of peripheral blood, but this is not without drawbacks, in that the entire RB1 locus, regulatory regions of RB1 andthe epigenetic events are not tested.15
The goals of genetic testing of the peripheral blood are to identify
germline mutations and the absence of pathogenic variants in the germline points to the absence of pre-disposition. However, the absence of detection of a RB1 pathogenic variant can lead to the erroneous conclusion that no germline RB1 pathogenic variant is present, and there is no retinoblastoma predisposition. Conventional diagnostic genetic testing does not explore the entire RB1 locus; thus, an RB1 germline predisposing pathogenic variant cannot be excluded, the authors stated.
When testing DNA from peripheral blood, investigators use a genomic hybridization technique, such as chromosomal microarray analyses. Mutations are identified by traditional Sanger sequencing of the amplified target regions or by SNP arrays. Investigators of a recent study reported that RB1 custom array–comparative genomic hybridisation and NGS methods optimised diagnostic molecular testing in patients with retinoblastoma16 and detected point mutations, macrodeletions and duplications, and narrowed down the detection of mosaicism to 1%.
Use of MLPA and direct sequencing have raised the detection frequency of gross deletions and identified genomic abnormalities including germline mosaicism.16,17
The NGS strategies are essential to treat and counsel patients. Investigations using targeted NGS using Illumina MiSeq and precision bioinformatic pipelines also have been used to identify a spectrum of pathogenic variants in
patients with retinoblastoma.15,18,19
A caveat is that direct tissue biopsy cannot be performed to identify retinoblastoma due to inaccessibility and the risk of spreading the tumour.20 A novel non-invasive alternative approach to obtain DNA for diagnostic and prognostic purposes is focusing on cell-free DNA (cfDNA) as a surrogate tumour biopsy for retinoblastoma, which can be harvested from the aqueous humour.
Dr Marković explained, “cfDNA is comprised of fragments originating from genomic DNA and circulating tumour DNA (ctDNA).21 ctDNA is shown to be representative of the tumour. It consists of small, approximately 200 base pair–long DNA fragments released into the bloodstream by various processes. When collected from the plasma, the ctDNA can be distinguished from genomic DNA based on the presence of cancer-related mutations. Jiménez and colleagues22 used high-deep NGS of the RB1 gene and reported that ctDNA wasdetected in patients with intraocular unilateral non-hereditary retinoblastoma. Using this approach, they detected 77.8% of previously reported somatic RB1 mutations. However, 15% of patients with unilateral retinoblastoma may still harbor a germline mutation, which indicates the utmost importance of genetic testing of the peripheral blood.”
ctDNA can be found in the aqueous humour and plasma and is an excellent biomarker for retinoblastoma that can also be used at later instances during patientfollow-up.23-25
In addition to ctDNA, and tumour-free DNA (tfDNA) is another novel approach with potential. The tumour genome is accessible through the tfDNA in the ocular anterior segment. The authors describe this as “a very useful source of DNA for diagnostics that can help predict which eyes can be salvaged.”
At this point in time, future investigation should identify credible biomarkers and molecular targets for improving diagnostic and treatment options.
References
1. Marković L, Bukovoc A, Varošanec AM, Šlaus N, Pećina‐Šlaus N. Genetics in ophthalmology: molecular blueprints of retinoblastoma. Hum Genomics.2023;17(1):82. doi:10.1186/s40246-023-00529-w
2. Sampieri K, Hadjistilianou T, Mari F, et al. Mutational screening of the RB1 gene in Italian patients with retinoblastoma reveals 11 novel mutations. J Hum Genet. 2006;51(3):209-216. doi:10.1007/s10038-005-0348-3
3. Broaddus E, Topham A, Singh AD. Incidence of retinoblastoma in the USA: 1975–2004. Br J Ophthalmol. 2009;93(1):21–3.
4. Cruz‐Gálvez CC, Ordaz‐Favila JC, Villar‐Calvo VM, Cancino‐Marentes ME, Bosch‐Canto V. Retinoblastoma: review and new insights. Front Oncol. 2022;12:963780. doi:10.3389/fonc.2022.963780
5. Martínez‐Sánchez M, Hernandez‐Monge J, Rangel M, Olivares‐Illana V. Retinoblastoma: from discovery to clinical management. FEBS J. 2022;289(15):4371-4382. doi:10.1111/febs.16035
6. Gündüz AK, Mirzayev I, Temel E, et al. A 20‐year audit of retinoblastoma treatment outcomes. Eye (Lond). 2020;34(10):1916-1924. doi:10.1038/s41433-020-0898-9
7. Broaddus E, Topham A, Singh AD. Survival with retinoblastoma in the USA: 1975–2004. Br J Ophthalmol. 2009;93(1):24-27. doi:10.1136/bjo.2008.143842.
8. MacCarthy A, Birch JM, Draper GJ, et al. Retinoblastoma: treatment and survival in Great Britain 1963 to 2002. Br J Ophthalmol. 2009;93(1):38-39. doi:10.1136/bjo.2008.139626
9. Canturk S, Qaddoumi I, Khetan V, et al. Survival of retinoblastoma in less‐developed countries impact of socioeconomic and health‐related indicators.
Br J Ophthalmol. 2010;94:1432–6.
10. Vempuluru VS, Kaliki S. Screening for retinoblastoma: a systematic review of current strategies. Asia Pac J Ophthalmol (Phila). 2021;10(2):192-199. doi:10.1097/APO.0000000000000378
11. Li LH, Li N, Zhao JY, et al. Findings of perinatal ocular examination performed on 3573, healthy full‐term newborns. Br J Ophthalmol. 2013;97(5):588-591. doi:10.1136/bjophthalmol-2012-302539.
12. Cagini C, Tosi G, Stracci F, Rinaldi VE, Verrotti A. Red reflex examination in neonates: evaluation of 3 years of screening. Int Ophthalmol. 2017;37:1199–204.
13. Brooks BP, Macdonald IM, Tumminia SJ, et al; National Ophthalmic Disease Genotyping Network (eyeGENE). Genomics in the era of molecular ophthalmology: reflections on the National Ophthalmic Disease Genotyping Network (eyeGENE). Arch Ophthalmol. 2008;126(3):424-425. doi:10.1001/archopht.126.3.424
14. Goetz KE, Reeves MJ, Tumminia SJ, Brooks BP. eyeGENE(R): a novel approach to combine clinical testing and researching genetic ocular disease. Curr Opin Ophthalmol. 2012;23(5):355-363. doi:10.1097/ICU.0b013e32835715c9
15. Bouchoucha Y, Matet A, Berger A, et al; European Retinoblastoma Group EuRbG. Retinoblastoma: from genes to patient care. Eur J Med Genet. 2023;66(1):104674. doi:10.1016/j.ejmg.2022.104674
16. Grotta S, D’Elia G, Scavelli R, et al. Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma. BMC Cancer. 2015;15:841. doi:10.1186/s12885-015-1854-0
17. Devarajan B, Prakash L, Kannan TR, et al. Targeted next generation sequencing of RB1 gene for the molecular diagnosis of Retinoblastoma. BMC Cancer. 2015;15:320. doi:10.1186/s12885-015-1340-8
18. Gudiseva HV, Berry JL, Polski A, Tumminia SJ, O’Brien JM. Next‐generation technologies and strategies for the management of retinoblastoma. Genes (Basel). 2019;10(12):1032. doi:10.3390/genes10121032
19. Singh AD. Retinoblastoma: what is the future? Int Ophthalmol Clin. 2019;59(2):95-99. doi:10.1097/IIO.0000000000000267
20. Galardi A, Stathopoulos C, Colletti M, et al. Proteomics of aqueous humor as a source of disease biomarkers in retinoblastoma. Int J Mol Sci. 2022;23(21):13458. doi:10.3390/ijms232113458.
21. Berry JL, Xu L, Kooi I, et al. Genomic cfDNA analysis of aqueous humor in retinoblastoma predicts eye salvage: the surrogate tumour biopsy for retinoblastoma. Mol Cancer Res. 2018;16(11):1701-1712. doi:10.1158/1541-7786.MCR-18-0369
22. Jiménez I, Frouin É, Chicard M, et al. Molecular diagnosis of retinoblastoma by circulating tumour DNA analysis. Eur J Cancer. 2023;66(1):516-528. doi:10.1159/000529207
23. Tian H, Zhao L, Li H, Huang Y, Wang Y. Circular RNA in retina: a potential biomarker and therapeutic target. Ophthalmic Res. 2023;66(1):516-528. doi:10.1159/000529207
24. He LF, Mou P, Yang CH, et al. Single‐cell sequencing in primary intraocular tumors: understanding heterogeneity, the microenvironment, and drug resistance. Front Immunol. 2023;14:1194590. doi:10.3389/fimmu.2023.1194590
25. Sun J, Xi HY, Shao Q, Liu QH. Biomarkers in retinoblastoma. Int J Ophthalmol. 13(2):325-341. doi:10.18240/ijo.2020.02.18
Leon Marković, MD, PhD | E: lemarkovic@gmail.com
Dr Marković is from the Department of Ophthalmology, Reference Center of the Ministry of Health of the Republic of Croatia for Pediatric Ophthalmology and Strabismus, University Hospital “Sveti Duh,” Zagreb, Croatia, and the Faculty of Dental Medicine and Health Osijek, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia.
He has no financial interest in this subject matter.
