The spectrum of germline mutations in retinoblastoma patients and their implications on clinical outcomes

Retinoblastoma is the most common intraocular malignancy of childhood. According to the "two-hit" hypothesis first proposed by Knudson, the disease occurs when both alleles of the RB1 gene are mutated. One of these mutations may be inherited from the family, or both mutations may arise somatically in retinal cells. It is now known that the patients with germline mutations tend to present at an earlier age and with bilateral involvement. The purpose of this study is to evaluate whether the clinical outcome of the patients who have germline mutations differ from those without mutations. The clinical and genetic data from a total of 51 cases was evaluated. Germline mutations were analyzed via Sanger sequencing and Multiplex Ligand-Associated Probe Amplification methods. Germline mutations were detected in 30 patients (58.8%), 8 of which were novel mutations. It was found that the patients with germline mutations presented with an earlier age of diagnosis (13.1 ± 15.3 vs. 30.4 ± 31.7 months respectively; p˂0.05), a higher ratio of bilaterality (56.7% vs. 23.8% respectively; p˂0.05), and a higher ratio of multifocality (70.7% vs. 40.9% respectively; p˂0.05) when compared to the patients without mutations. Germline mutations were also detected in 44.8% of unilateral cases. Among the eyes of patients with and without mutations, the percentage distribution of groups defined according to the International Classification of Retinoblastoma also varied. It was found that this difference was caused by statistically different percentages in Group A and Group D eyes (17.4% vs 0% respectively for Group A, 40.0% vs 26.9% respectively for Group D; p˂0.05). A higher need for intravenous chemotherapy (5.6 ± 3.8 vs. 2.9 ± 3.6 cycles, respectively; p˂0.05) and local therapies (2.6 ± 3.8 vs 1.6 ± 3.8 sessions, respectively; p˂0.05) were recorded in patients with germline mutations compared to those without mutations. In addition, we also investigated whether the clinical outcome changes with respect to different types of mutations. Among the detected mutations, nonsense mutations were the most frequent ones by a ratio of 33.3%, followed by missense (23.3%), frameshift (20.0%) and splice site (16.7%) mutations. Nonsense and frameshift mutations were associated with an earlier age at diagnosis (6.7 ± 4.8 and 7.5 ± 7.9 months, respectively), compared to missense and splice site mutations (21.0 ± 22.5 and 21.2 ± 21.5 months, respectively) (p˂0,05). We also found that the laterality of tumor differed among patients with different mutation types; all the patients who had missense mutations (%100) had unilateral disease, whereas the other types of tumors mostly presented with bilateral involvement (p˂0,05). Furthermore, the enucleation rate was observed to be higher for the eyes of patients who had frameshift and splice site mutations (54.5% and 57.1%, respectively), while it was lower for the eyes of patients with nonsense and missense mutations (16.7% and 14.3%, respectively) (p˂0,05). In our study, it was concluded that the existence of germline mutations and types of mutations in retinoblastoma patients might affect the clinical outcome and the need for therapy. In addition to earlier age at diagnosis and higher rates of bilaterlity and multifocality, a higher need for intravenous chemotherapy and local therapy was also detected in patients with germline mutations. It was also worth mentioning that a germline mutation was detected nearly in half of the unilateral patients. Furthermore, we found that different mutation types might be a factor influencing the age at diagnosis and laterality, as in nonsense and frameshift mutations were associated with an earlier age at diagnosis and patients with missense mutations tended to present with unilateral involvement. Moreover, higher enucleation rates were recorded for the patients with frameshift and splice site mutations. In order to construct an appropriate follow-up algorithm for each patient, as well as to provide an effective service for family planning and screening for secondary malignancies, genetic testing and counselling should be a part of the routine care for every retinoblastoma patient and their families.