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.