Untangling the genetic secrets of retinoblastoma

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Retinoblastoma is a rare, aggressive genetic childhood cancer that results from a mutation that inactivates the RB1 gene, 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 2500 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.

Marković pointed out that remarkable advances in retinoblastoma therapy have been realized 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, 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. 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

Retinoblastoma is rare in that the incidence rate is 1 in 15,000 to 20,000 live births, or about 8000 cases annually worldwide, but it is the most common primary intraocular tumor in children and infants.^2-4 The disease presentation can vary and involve 1 or both eyes, with the respective patient ages at diagnosis being 24 and 12 months.

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.”

Moreover, 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 remodeling.

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, 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.^13,14

The accuracy of genetic testing is high when DNA can be isolated from tumor DNA after enucleation, which obviously is not the preferred end point. Acquiring DNA introduces the potential for numerous techniques to be performed, ie, 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 recognized 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, and the 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 predisposition. 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 hybridization and NGS methods optimized 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 has 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 tumor.²⁰ A novel noninvasive alternative approach to obtain DNA for diagnostic and prognostic purposes is focusing on cell-free DNA (cfDNA) as a surrogate tumor biopsy for retinoblastoma, which can be harvested from the aqueous humor.

Marković explained, “cfDNA is comprised of fragments originating from genomic DNA and circulating tumor DNA (ctDNA).²¹ ctDNA is shown to be representative of the tumor. 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 was detected in patients with intraocular unilateral nonhereditary 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,” he said.

ctDNA can be found in the aqueous humor and plasma and is an excellent biomarker for retinoblastoma that can also be used at later instances during patient follow-up.^23-25

In addition to ctDNA, tumor-free DNA (tfDNA) is another novel approach with potential. The tumor 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, future investigation should identify credible biomarkers and molecular targets for improving diagnostic and treatment options.

Leon Marković, MD, PhD

E: lemarkovic@gmail.com

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.

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