Discussion
The c.2548C>T; p.(Gln850Ter) RB1 germline gene variant is registered in the COSMIC databases, but no relation to Rb was documented so far. Therefore, this was the first time to describe the variant in Rb. Germline loss of function RB1 gene mutations are known to be causative in Rb [6] and are associated with increased risk of osteosarcoma development [20]. The predisposition to sarcomas has been attributed to genetic susceptibility due to inactivation of the RB1  gene as well as to the genotoxic effect of radiotherapy applied to treat Rb. Bone and soft tissue sarcomas in hereditary Rb survivors occur most frequently within the radiation field in the facial bone (orbita), but they may also occur elsewhere.RB1 alterations also serve as unfavourable prognostic marker for the clinical classification and management of osteosarcoma patients [21].
In agreement with RB protein disordered structure, the C-terminal region has already been reported to enable protein-protein interactions. Structural studies revealed interactions of RB protein with heterodimer of E2F transcription factor 1 (E2F1) and transcription factor Dp-1 (DP1 (829-874) [18], with catalytic subunit of protein phosphatase 1 (PP1c) (870-882) [19], with complex of cyclin dependent protein kinase 2 (CDK2) and cyclin A (868-878) [20], and with mouse importin-α (858-877) [19] (numbers in parentheses show regions of retinoblastoma-associated protein that form interactions in the complexes) (Figure 3 ). Additionally, interactions between the RB protein and the complex of cyclin dependent protein kinase 9 (CDK9) and cyclin T2 were also reported and this mutagenesis study revealed that the interactions were mediated by the C-terminus of RB protein (835-928) [22]. Out of these proteins only E2F1 has exclusive nuclear localization based on Human Protein Atlas [23], the other relevant human proteins are localized in the cytoplasm. Consequently, deletion of the region 851-928 of RB protein most probably influences a series of functionally relevant protein-protein interactions, rather, than deletion-induced structural changes promoting directly the pathogenic phenotype.
The diversity of cancers in which MET mutations have been identified suggests that mutationally actived MET protein plays a significant role in the tumorigenic process in a wide range of cell types. The juxtamembran domain mutations were shown to attenuate MET receptor ubiquitination and degradation and prolong MET signaling [24]. There are no eukaryotic linear motifs in MET protein which include Thr1010 residues, but based on PhosphoSitePlus database [25] this residue is known to be phosphorylated. The functional characteristics of p.(Thr1010Ile) sequence variant has already been reported. However, transforming nature of this variant was described in a study. The investigation revealed that this variant was present in individuals with or without cancer, and no evidence was found regarding the transformative capacity of p.(Thr1010Ile) variant [26]. This finding may indicate that the structural integrity of MET is retained in the mutant. However, structural consequences of the p.(Thr1010Ile) variant were not investigated so far. In the COSMIC database, this mutation was described as gemline and somatic form as well. It proved to be more active than the wild-type MET in the athymic nude mice tumorigenesis assay, suggesting its potential effect on tumorigenesis [27].
In a novel large cohort study, patients with heritable Rb had a significantly increased risk for second tumor, while patients with nonheritable Rb did not. The overall mortality rate was 48% for heritable Rb and 23% for patients with nonheritable form. The cumulative mortality rate from second cancers at the age 60 years was 34% among those with heritable Rb and 12% among those with nonheritable Rb. Sarcoma was the most common histological type of malignancy in patients with heritable Rb, and carcinoma was the most common type in nonheritable Rb [28].
Loss of RB1 gene function has also been found to be associated with increased risk of osteosarcoma metastasis and a poor histological response to chemotherapy, compared with osteosarcoma patients with intact RB1 fuction [29]. The response of osteosarcoma patients to chemotherapy was assessed by the degree of histological necrosis of tumor tissues induced by chemotherapy. The majority of bone sarcomas occured within the radiation field in the head region, but up to 40% were diagnosed outside the treatment field, primarily in the lower extremities. Osteosarcoma is most frequently seen in the femur and osteoblastic osteosarcoma is the most common histological subtype making up 41-89% of this malignancy [21]. But both chondrosarcoma and Ewing sarcoma have been reported [30]. Sarcomas occuring in the radiation field were diagnosed with a lag time one year shorter than those diagnosed outside of the field [5]. This timing suggests that diverse biologic mechanism may be involved with bone sarcoma development including radiation damage and anatomical site. In our study, we demonstrated different somatic mutation profiles for sarcomas with chondroblastic and osteoblastic phenotypes by the use of a 67 gene solid tumor NGS panel. TP53 and other gene aberrations were limited to the osteoblastic form of the lower extremity, suggesting an independent evolution from the chondroblastic type originating at the site of the irradiation therapy.
The loss or inactivation of RB1 function results in a significant 1.62-fold increase in the mortality rates in patients with osteosarcoma compared with those in patients without this gene aberratios [21]. Osteosarcoma is characterised by its high potential to metastise to the lungs or other bones [31]. Mutations of the RB1 gene result in its dissociation from E2F and subsequent transcription of multiple genes involved in cell cycle progression lead to malignant transformation and the progression of osteosarcoma [32]. Metastatic osteosarcoma is typically difficult to control and known to indicate poor prognosis [33].
Because of the expanding number of registered Rb cases [28], the novel rare cases publication is very important to understand the molecular mechanism of this malignancy. To the best of our knowledge, this is the first report to detect this novel form of RB1mutation and synchronous MET gene mutation causing non-heritable bilateral retinoblastoma and consequential chondroblastic and osteoblastic osteosarcoma, the latter one developing pulmonary metastatses. Structural and functional prediction of the germline mutant proteins suggested an indirect parallel involvement in the pathogenesis of this uniqe series of retinoblastoma related neoplasias. The results of the 67 gene NGS panel clearly differentiated the histologically identified osteosarcoma types by two different sets of gene variants.