Introduction
Retinoblastoma (Rb, OMIM#180200) is a malignant tumor of the developing retina that affects children before the age of five years with an estimated incidence between 1 in 16,000 and 1 in 18,000 live births [1]. Rb occurs in both heritable (25-30%) and non-heritable (70-75%) forms. A heritable form is defined by the presence of a germline heterozygotic mutation in the RB1gene (Genbank accession number L11910.1; NCBI RefSeq NM_000321.2), wich is followed by a second somatic hit in the developing retina. As a result, tumors affecting either one (unilateral) or both (bilateral) eyes may develop. In the non-heritable form both mutations occur in somatic cells, usually leading to unilateral malignancy [2]. In addition to the highly malignant early onset Rb, the risk of developing second cancers, e.g. osteosarcomas and other soft-tissue sarcomas, rarely melanomas is increased. Molecular diagnostics is required to clear heredity status and to deliver the best options for the management of the disease [3, 4]. Due to the genetic predisposition, second primary malignancies may arise spontaneously or following radiotherapy, which have become the leading cause of death in Rb survivors. Osteosarcomas in retinoblastoma patients occurred 1.2 years earlier and the latency period between radiotherapy and osteosarcoma onset was 1.3 years shorter inside than outside of the radiation field [5].
The RB1 gene shows a wide spectrum of mutations, including single nucleotide variants (SNVs), small insertions/deletions (indels), and large deletions/duplications. These mutations are distributed throughout the entire length of the gene, spanning over 27 exons, and no hotspots have been reported [6]. New advances in molecular genetic testing, and especially next-generation sequencing (NGS), allow the comprehensive demonstration of all SNVs and large aberrations throughout the full length of the gene. Pathogenic mutations in both alleles of theRB1 gene are related to the development of this neoplasm in the large majority of the cases. Alternatively, complex mutation patterns missing the RB1 gene aberration were identified in rare cases of manifest Rb, indicating to oncogenic interactions between different signal transduction pathways [7].
The aim of our study was (i) to identify germline genetic aberrations in a patient with bilateral Rb of non-parental origin, (ii) to exclude the parental carrier status, (iii) to characterize histological and genetic features of samples originating from the two Rb and the four anatomically distinct osteosarcoma tumors (iv) to genetically differentiate between the irradiation-related orbitofacial and the non-related de novo osteosarcoma of the lower extremity, (v) structural and functional prediction of the germline mutant proteins reconstructed after DNA sequencing, and, finally (vi) identify potential interaction between defected proteins using prediction analysis. For this purpose, histology, including immunohistochemistry (IHC) and NGS solid tumor gene panel (Illumina MiSeq platform) analysis were performed using samples from both enucleated eyes and from the four available osteosarcoma tissues. Autopsy sample of the skin was taken as normal non-neoplastic control tissue. In addition, in silico prediction methods were applied to analyse the secondary structure and functionality of detected germline variants and to predict protein-protein interaction. To exclude potential parental origin conventional Sanger sequencing was used.