The 8th AJCC has incorporated "H" or hereditary into the TNM staging of RB, thus envisaging the role of genetics in management - H1 carry the RB1 cancer-predisposing gene; H0 are tested and proven to have normal Rb alleles and HX carry unknown risk(55). In the current era of personalized medicine and cancer-care accessibility, availability and reliability - NGS/MLPA techniques have revolutionised the genetic diagnostic scenario of RB-care globally and also selectively in India [22-26]. Incorporating genetic testing as part of RB-care has significant advantages - these opportunities and challenges are highlighted in the current study. For example, our four ULRB cases who would have otherwise not been monitored closely post treatment completion with the mutation were switched to 3-6 monthly surveillance, like any other BLRB patient with an RB1 mutation in our study cohort. With the advent of ophthalmic artery chemotherapy, intra vitreal and intracameral chemotherapy, globe salvage in RB has reached a paradigm shift (56),(57), thus availability of tumour tissue for pathology studies now is a rarity and in such situations an objective test like NGS/MLPA serves as a useful clinical marker tool. Although, ophthalmic artery chemotherapy involves targeted chemotherapy to just the affected eye and obviates need of systemic chemotherapy, this modality is best reserved for non germline cases. Hence, before using this supra-selective modality in a unilateral disease, the clinical team needs to be sure that the other eye is not at-risk due to an inherited mutation(58). Genetic test as a prognostic marker has been applied in medulloblastoma, paediatric gliomas (59, 60) and breast cancer(61). However, in comparison, clinical adoption of RB genetic diagnostics is poor amongst the clinicians in India and other developing countries.
The mutation detection rates across countries in BLRB varied from 100 to 16.6% and in ULRB from 56.3 to 9.5% (Table 5), the wide variation could be due various reasons inclusive of the fact that the studies were performed prior to highly sensitive NGS/MLPA tests era. Price et al., in United Kingdom studied 403 unrelated patients, 209 blood and 194 tumour samples and identified 533 variations, including RB1 gene mutations and loss of heterozygosity (LOH)(39). In another Netherlands large cohort study, 529 RB patients were screened with a 92% detection rate in BLRB and 10 % in ULRB, the latter being the lowest in the mutation spectrum(27). In the largest mutation meta-analysis of 932 RB patients, it was found that globally the most frequent mutations reported were R320X (nearly 50 times), R579X (nearly 40 times) and R251X (nearly 30 times)(62). All the studies uniformly found deletions, duplications, missense, nonsense, splice and frameshift mutations, once again establishing that RB1 gene has no hotspot (10-13, 16, 17, 20-24, 26, 30-32, 34-36, 63-66). In our study, we found 20 RB1 gene variations in 29 probands (79%), inclusive of three novel mutations, 16 were previously reported mutations, four heterozygous deletions of the whole RB1 gene. We had one case each of frameshift and commonly reported R251X and R320X and it is to be noted that those with the arginine/termination mutations have a risk to develop SPT (15). In our study, we identified mutations in 86% of BLRB patients and 19% in ULRB – which is comparable to other global studies, however we could not find any mutation in 4 BLRB patients and this could be because of various reasons including mosaicism and MYCN gene mutations, which we did not study. Mosaicism is a tricky issue in RB diagnostics and prenatal genetic counselling, hence may go unnoticed suggests Rushlow et al(63, 67).
RB1 mutations have shown variable genotype-phenotype correlations - Taylor et al (68), observed complete penetrance in nonsense, frameshift mutations and large rearrangements, whereas missense mutations showed heterogenous phenotypes and low penetrance, which was contradicted by Zhang et al (69). Lidderman et al, suggested that frameshift, nonsense and aberrant splice mutations result in hereditary RB, whereas missense, inframe and promoter mutations result in variable phenotype with reduced penetrance, (70) similar to Lohman and Gallie and Harbour (71, 72). RB1 gross alterations were found in 15% of 433 BLRB and 6.5% of 262 ULRB patients – these patients developed fewer tumours compared to those with null mutations and interestingly, those with cytogenetic or sub-microscopic whole gene deletions often had ULRB, however all those with gross deletions with one breakpoint inside the RB1 gene had BLRB (73)Notably, in our cohort all cases of ULRB, irrespective of their mutation type, had optic nerve invasion and were severe enough to warrant enucleation. Prior knowledge of mutation may influence enucleation decisions in the subset of ULRB patients, who all had the mutation, the other eye is also ‘at risk’ and must be treated potentially as a ‘bilateral’ case. In the four c.1333C>T (p.R445Ter) BLRB patients, three had disease progression despite treatment, in one bilateral globe salvage was successful by using plaque brachytherapy, two needed unilateral enucleation and one case needed bilateral enucleation due to progressive disease unresponsive to multimodality treatment (Fig. 2). The variable clinical phenotype and response to treatment despite the same mutation, could be due to epigenetic molecular events in the tumor (74). In pineal cyst, a pre-malignant form of pinealoblastoma, BLRB is more common than ULRB where germline mutations are invariably identified (75) and we had a patient with pinealoma, trilateral RB who had the pW78X mutation.
The clinical and pathological factors that are considered as predictors of poor outcome, are the presence of subretinal seeds for recurrence anterior segment, choroidal, optic nerve involvement (76) and glaucoma (77). Pathological high-risk factors have been known to be strong predictors of metastasis in RB and in our study the mutation group had statistically significant progression, recurrence and higher percentage of optic nerve invasion, subretinal seeds and high-risk pathological factors but lower percentage of enucleation compared to the non-mutation group. Those with germline mutations have an 8% lifetime risk of developing osteosarcomas, apart from small cell sarcomas of the lung, and urinary bladder cancers. Radiotherapy is contraindicated in patients with germline mutations and this valuable information could help the clinician to modify treatment options. There are studies describing ill effects of radiation on RB, which however do not have the mutation data (78). Interestingly, analysis of radiation effects based on genetic susceptibility and age clearly shows that radiation induced side effects occur in patients who are genetically predisposed (79). Rarely the hereditary retinoblastoma could be a part of chromosome 13q deletion syndrome, such children additionally suffer from developmental delay, mental retardation and craniofacial abnormalities, thus warranting active surveillance and rehabilitation from the beginning (80), With the advent of preimplantation genetic diagnosis for retinoblastoma, the avenues for families of hereditary retinoblastoma have tremendously improved (81). This further envisages the importance of genetic analysis in comprehensive retinoblastoma care.
Testing the RB1 gene for mutation is a challenging task, owing to its size, heterogeneity of mutations (with 200 reported), lack of hotspot and the variable intronic lengths (82). In our study, about three patients were exclusively referred for mutation analysis from outside our organisation and they continued receiving clinical care for the RB elsewhere. This envisages the fact that clinical management of RB is well addressed across the country, however the same level of care doesn’t exist for genetic testing, even though CAP certified private companies provide NGS/MLPA RB testing at a cost. Moreover, established RB guidelines specifies the role of genetic testing in RB care (8). Centres for RB care without a genetic support, must be aware of this need and should sensitize the family on the role and usefulness of genetic testing and also inform them of the additional cost of care to the family which is not covered by insurance (83). The conclusions made in the study was based on small size and the techniques failed to detect mutations in all the BLRB patients.