Retinoblastoma is the most common childhood intraocular malignancy that affects one or both eyes.1 Because of the strong links between clinical care and genetic causation,2 retinoblastoma is considered the prototype of heritable cancers.3
Worldwide, about 8000 children are newly diagnosed with retinoblastoma every year [1/16,000 live births]1,4 but most have no access to knowledge of the important role genetics plays in many aspects of retinoblastoma: clinical presentation, choice of treatment modalities, and follow-up for both child and family. We now highlight the genetic etiology of retinoblastoma in the context of individual children and families, led by the questions
commonly asked by parents. Retinoblastoma is a cancer that arises because both copies of the RB1 gene that normally suppresses retinoblastoma are lost from a developing retinal cell in fetuses, babies, and young children. Retinoblastoma can affect one [unilateral] or both eyes [bilateral] and, in
5% of children with heritable retinoblastoma [H1],5 is associated with a midline brain tumor [trilateral].6 Without timely and effective treatment, retinoblastoma may spread through the optic nerve to the brain, or via blood, particularly to bone marrow, and result in death. The cell of origin of retinoblastoma is most likely a developing cone photoreceptor precursor cell that has lost both alleles of the RB1 tumor suppressor gene and remains in the inner nuclear layer of the retina [Fig. 1], perhaps because it is unable to migrate to the outer retina and function
normally.1,7,8 The cell susceptible to developing cancer is present in the retinas of young children, from before birth, up to around 7 years of age. Rarely, retinoblastoma is first diagnosed in older persons, who likely previously had an undetected small tumor [retinoma] present from childhood that later became active.9,10 In high-income countries, the mean age at presentation is 12
months in bilateral disease and 24 months in unilateral disease, whereas significant delay in low-income countries impacts negatively on the children.11 Early awareness of risk for retinoblastoma optimizes therapy and outcomes. This child was examined because his sibling [triplets] presented with
retinoblastoma. His right eye appeared normal, but optical coherence tomography [OCT] revealed 2 invisible tumors [* and ↓] [A-C], which were treated with laser therapy only; OCT after laser shows coagulation of tumors, visible in the retinal image [D-F]. No one knows what really causes the genomic damage to the
RB1 gene, but retinoblastoma arises at a constant rate in all races irrespective of local environment. In nearly 50% of patients, the first copy of the RB1 gene is damaged in most, or all, normal cells of the patient. A retinal tumor develops when the second copy of the RB1 gene is also damaged in a developing retinal
cell.1 The RB1 gene resides on chromosome 13q14 and encodes the retinoblastoma protein [pRB], an important regulator of cell division cycle in most cell types, and the first tumor suppressor gene discovered.12 Normally, cell division is inhibited by hypophosphorylated pRB binding to E2F molecules
and blocking their transactivation of RB1, E2F, and other promoters of molecules that support cell division.13-15 To resume cell division, cyclindependent kinases re-phosphorylate pRB, activating promoters of key proteins important in cell division.16 Loss of pRB
therefore allows uncontrolled cell division. In many cell types, loss of the RB1 gene is compensated by increased expression of other related proteins. However, in susceptible cells such as retinal cone cell precursors, compensatory mechanisms are insufficient, allowing uncontrolled cell division to initiate cancer.8 In heritable retinoblastoma [also called germline retinoblastoma], the first RB1 allele is mutated [M1] in nearly all cells, including germline reproductive cells, whereas the second allele is mutated [M2] in the retinal cells that become cancer, usually in both eyes
[Figs. 2A, B]. The most common M2 event is loss of the normal RB1 allele and duplication of the mutated M1 allele, in that 2 copies of the mutated RB1 gene remain; a mutational event referred to as loss of heterozygosity [LOH].17,18
Heritable retinoblastoma encompasses 45% of all reported cases19-21 with bilateral [80%], unilateral [15%], or trilateral [5%] tumors.1 Germline RB1 mutations carry the risk of second primary cancers, most commonly leiomyosarcoma, osteosarcoma, and melanoma.22 These patients may
benefit from regular surveillance for such cancers over their lifetime. Pedigrees illustrating inheritance patterns for RB1 mutations. A, Full penetrance and expressivity for null mutation: father [bilaterally enucleated] and 2/2 bilaterally affected offspring [I-1 unilateral enucleation vision 1.0 remaining eye]; all H1 with 11 base pair deletion in exon 12; DER = 2. B, No family
history, new null mutation: triplets1 diagnosed with bilateral retinoblastoma at age 2.5 months due to c.1345G>T [p.Gly449Ter] RB1 missense mutation resulting in no pRB; parents showed no evidence of the mutation but were considered H0* because there remains a less than 1% risk of mosaicism in either parent; older sibling is negative for the mutation, therefore H0; DER = 2. C, 100%
penetrance and variable expressivity: grandfather [I-1] was diagnosed with bilateral retinoma when his daughter [II-1] was diagnosed with bilateral retinoblastoma due to c.1960G>T [p.Val654Leu] RB1 missense mutation; she [II-1] later developed meningioma in the radiation field and breast cancer; her brother [II-3] and daughter [III-2] inherited the mutation and developed unilateral retinoblastoma; DER = 1.5. D, Parent of origin low
penetrance62: c.607+1G>T RB1 splice mutation that shows higher penetrance when inherited from the father [ie, II-1, III-1, III-3; DER = 1] than from the mother [ie, III-1, III-3; DER = 0], likely due to increased expression from the maternal than the paternal mutant RB1 allele60
[overall DER = 0.7]; IV-1 had a small unilateral tumor but died at 11 years of age due to radiation-induced secondary malignancies; IV-5 has not been tested but developed thyroid cancer. Of nonheritable retinoblastoma, 98% have both RB1 M1 and M2 events within a retinal cell. In the remaining 2%, retinoblastoma is induced by somatic amplification of the
MYCN oncogene, in the presence of normal RB1 genes.23 No one is to blame for the mutations causing retinoblastoma. Many environmental forces induce DNA damage,
including cosmic rays, X-rays, DNA viruses, ultraviolet irradiation, and smoking. The DNA damage may be point mutations, small and large deletions, promotor methylation shutting down RB1 expression, and rarely, chromothripsis.24,25 The majority of RB1 mutations arise de novo, unique to a specific patient or family
[Fig. 2B]. However, some recurrent mutations are found in unrelated individuals, such as those that affect 11 hyper-mutable CpG DNA sequence sites, which make up 22% of all RB1 mutations.26,27 A de novo RB1 germline
mutation may arise either pre- or post-conception. Pre-conception mutagenesis of RB1 usually occurs during spermatogenesis, perhaps because cell division [an opportunity for mutation] is very active during spermatogenesis but not during oogenesis.28,29 Advanced paternal age increases risk for retinoblastoma,30 suggesting that genomic
errors may increase in aging men. The affected child carries the de novo RB1 mutation in every cell, typically presenting with 4-5 tumors and bilateral retinoblastoma. In contrast, if mutagenesis occurs postconception, during embryogenesis, only a portion of cells will carry the RB1 mutation [ie, mosaicism].1 There are 2 answers to this question: [i] loss of both RB1 alleles only causes retinoma, a benign precursor to retinoblastoma, and other genes are modified to cause
progression to cancer;10 and [ii] 2% of unilateral retinoblastoma have normal RB1 and are caused by a different gene. [i] Retinoma is a premalignant precursor to retinoblastoma with characteristic clinical features: translucent white mass, reactive retinal pigment epithelial proliferation, and calcific foci.9 Retinoma may be
found retrospectively after a child develops retinoblastoma [Figs. 2C, 3]. Pathology of retinoma reveals nonproliferative fleurettes.10,31 Comparison of adjacent normal retina, retinoma, and retinoblastoma showed loss of both RB1 alleles and early genomic copy number changes in retinoma that were
amplified further in the adjacent retinoblastoma.10 Many retinoblastomas have underlying elements of retinoma. Retinoma can transform to retinoblastoma even after many years of stability, so they need lifelong monitoring. The alternate, confusing term “retinocytoma” was proposed 1 year later32 and is inappropriate because it had been used for several different entities, including active tumors with
Flexner-Wintersteiner rosettes.33-36 In addition to loss of RB1, specific alterations in copy number of other genes are common in RB1-/- retinoblastoma. There are gains [4-10 copies] in oncogenes MDM4,
KIF14 [1q32], MYCN [2p24], DEK, and E2F3 [6p22] and loss of the tumor suppressor gene CDH11 [16q22-24].3,37 Other less common genomic alterations in
retinoblastoma tumors include differential expression of specific microRNAs,38 recurrent single nucleotide variants/insertion-deletions in the genes BCOR and CREBBP,39 and upregulation of spleen tyrosine kinase
[SYK].40 In comparison with the genomic landscape of other cancers, retinoblastoma is one of the least mutated, and epigenetic modification of gene expression may play an important role in retinoblastoma progression.39-41SYK is a postulated methylating proto-oncogene
required for retinoblastoma cell survival. It was found to be upregulated in retinoblastoma and suggested as a potential target of therapy.40,42 It was present in 100% of retinoblastomas and 0% of normal retina histologically by immunohistochemical staining of pathological specimens of enucleated eyes with retinoblastoma.43 Retinoma, the benign precursor of retinoblastoma. Bilateral stable translucent retinal masses with calcification and associate retinal pigment epithelial disturbance were diagnosed to be retinoma in the grandparent I-1 [Fig. 2C], only when his daughter [II-1] was diagnosed with bilateral retinoblastoma. He never received
treatment and was alive and well at age 90 at last follow-up. [ii] There is a newly recognized form of retinoblastoma with normal RB1 genes. Two percent of unilateral patients have RB1+/+ MYCNA tumors, in which the MYCN oncogene is amplified [28-121
DNA copies instead of the normal 2 copies].23 These children are diagnosed at a median age of 4.5 months compared with 24 months for nonheritable unilateral RB-/- patients, and the tumors are histologically distinct with advanced features at diagnosis. The only way to find retinoblastoma tumor early is to examine the eye with specific expertise, which we cannot do for every child. The earliest signs of retinoblastoma detectable by parents are leukocoria [white pupil], either directly or in photographs [photo-leukocoria], and strabismus when the macula is involved by tumor. Facial features and various degrees of hypotony and mental retardation in 13q deletion syndrome can lead to discovery of
retinoblastoma.44,45 If we examine the retina of patients with positive familial history early, we may discover the smallest visible tumors that are round, white retinal lesions that obscure the underlying choroidal pattern; invisible definitive tumors can be detected even earlier by optical coherence tomography [Fig.
1].46,47 Centrifugal growth results in small tumors being round; more extensive growth produces lobular growth, likely related to genomic changes in single [clonal] cells that provide a proliferative advantage.48 Next, tumor seeds spread out of the main tumor into the subretinal space or vitreous cavity. Vitreous seeding is associated with loss of chromosome 16q,
including the cadherin 13 gene, a genomic change that may induce poor cohesive forces between tumor cells.49,50 Advanced vitreous tumor seeds can migrate to the anterior chamber producing a pseudohypopyon. Enlarging tumor can push the iris lens diaphragm forward causing angle closure glaucoma. Advanced tumors may induce iris neovascularization. Rapid necrosis of tumor can cause an aseptic orbital inflammatory reaction
resembling orbital cellulitis, sometimes showing central retinal artery occlusion on pathology.48,51 Untreated, retinoblastoma spreads into the optic nerve and brain, or hematogenous spread occurs through choroid, particularly to grow in bone marrow. Tumor extension through sclera can present as orbital extension and proptosis. Depending on the exact RB1 mutation, most, but not all, carriers of an RB1 mutation will develop retinoblastoma and other cancers throughout life. Each
offspring of a person carrying an RB1 mutated gene has 50% risk to inherit the RB1 mutated gene [Fig. 2]. A measure of expressivity of a mutant retinoblastoma allele is the diseaseeye ratio [DER] [number of eyes affected with tumor divided by the number of carriers of the
RB1 mutation].52 Nonsense and frame-shift germline mutations that lead to absent or truncated dysfunctional pRB result in 90% bilateral retinoblastoma [nearly complete penetrance, DER = 2]. Partially functional RB1 mutated alleles show variable penetrance and expressivity
[Fig. 2C] with fewer tumors and later onset,47 and some carriers never develop retinoblastoma [DER = 1-1.5]. Reduced penetrance mutated RB1 alleles include [i] mutations in exons 1 and 2,53 [ii] mutations near the 3’ end of the gene in exons 24 to 27,54,55 [iii] splice and intronic mutations,56 and [iv] missense mutations.57 Counterintuitively, large deletions encompassing the RB1 gene and MED4 gene also cause reduced expressivity/penetrance because
RB1-/- cells cannot survive in the absence of MED458 and the most common M2 event in retinoblastoma tumors is loss of the normal allele and reduplication of the mutated allele. In comparison, patients with large deletions with 1 breakpoint inside the RB1 gene typically
present with bilateral disease.59 There are at least 2 specific RB1 mutant alleles showing a parent-of-origin effect: c.607+1G>T substitution60,61 [Fig. 2D] and c.1981C>T [p.Arg661Trp] missense
mutation.62 Both may be explained by differential methylation of intron 2 CpG85, which skews RB1 expression in favor of the maternal allele.63 When the mutated allele is maternally inherited, there is sufficient tumor suppressor activity to limit retinoblastoma development to 10% of
carriers. However, when the mutated RB1 allele is paternally transmitted, very little pRB is expressed, leading to retinoblastoma in 68% of carriers.WHAT IS RETINOBLASTOMA?
HOW CAN CANCER OCCUR AT SUCH A YOUNG
AGE?
WHAT CAUSES RETINOBLASTOMA?
WHAT CAUSES RETINOBLASTOMA TO BE UNILATERAL VERSUS BILATERAL?
WHAT CAUSED THESE MUTATIONS? DID I CAUSE THEM?
DOES ONLYRB1MUTATION CAUSE RETINOBLASTOMA?
COULD WE HAVE DISCOVERED RETINOBLASTOMA
EARLIER?
DO ALL AFFECTED
INDIVIDUALS WITHRB1MUTATIONS DEVELOP RETINOBLASTOMA?
WHAT FACTORS AFFECT RETINOBLASTOMA CANCER STAGING?
Treatment and prognosis of retinoblastoma depend on the stage of disease at initial presentation. Factors predictive of outcomes include size, location of tumor origin, extent of subretinal fluid, presence of tumor seeds, and the presence of high-risk features on pathology.5 Multiple staging systems have predicted likelihood to salvage an eye without using radiation therapy, but published evidence is confusing because significantly different staging versions have been used.1,5 The 2017 TNMH classification is based on an international consensus and evidence from an international survey of 1728 eyes and separates well initial clinical and pathological features predictive of outcomes to save the eye, in retrospective comparison with 5 previous eye staging systems.5
DOES GERMLINE STATUS AFFECT RETINOBLASTOMA STAGING?
Retinoblastoma is the first cancer in which staging recognizes the impact of genetic status on outcomes in the 2017 TNMH staging [Fig. 4]: presence of a positive family history, bilateral or trilateral disease, or high sensitivity positive RB1 mutation testing is stage H1; without these features before testing blood, HX; and H0 for those relatives shown to not carry the proband’s specific RB1 mutation [Fig. 2].5 We propose H0* for patients tested and having neither M1 nor M2 RB1 mutated alleles of the tumor detectable in blood and parents showing no evidence of the M1 allele of their offspring, but with remaining low risk [T/p.Arg661Trp mutation of RB1. PLoS Genet. 2016;12:e1005888.
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Keywords:
retinoblastoma; RB1 gene; bilateral; unilateral; DNA sequencing; genetic counseling; prenatal screening