Regular paper

Osteosarcoma’s genetic landscape painted by genes’ mutations

Wiktoria Urban1#, Dagmara Krzystańska1#, Michał Piekarz2#, Jerzy Nazar2#
and Anna Jankowska1

1Department of Cell Biology, Poznan University of Medical Sciences, Poznań, Poland; 2Department of Orthopaedics and Traumatology of Locomotor System, Heliodor Swiecicki Clinical Hospital in Poznan, Poznan University of Medical Sciences, Poznań, Poland

Purpose: Osteosarcoma (OS) is one of the most common primary bone tumors. Direct pathogenesis remains unknown, however, genes’ mutations are proven to participate in the process. This study aimed to examine the most frequently mutated genes in OS to appoint candidates for the cancer markers. Methods: Using the COSMIC Catalogue twenty the most frequently mutated genes were selected leading to an up-to-date genetic OS landscape summary. The genes can be classified into four categories: suppressor genes (TP53, RB1, NCOR1, SMAD2, NF1, TSC2, KMT2C), proto-oncogenes (GNAS, BRAF, MLLT3), epigenetic and post-translational modification-related genes (SMARCA4, ARID1A, ATRX, BCOR, H3F3A) and cell growth and survival regulating genes (EGFR, CAMTA1, LRP1B, PDE4DIP, MED12). Results and conclusions: Their role in cancerogenesis was confirmed by the analysis of available articles published previously. The results of the study indicate that examination of selected genes’ mutations might help to identify patients’ predisposition to OS development, as well as monitor the disease progression, and establish prognosis. However, to fully understand the pathogenesis of OS further studies are required.

Keywords: osteosarcoma, genes’mutations, COSMIC catalogue, biomarkers

Received: 02 June, 2023; revised: 14 August, 2023; accepted:
14 August, 2023; available on-line: 17 September, 2023

e-mail: ajanko@ump.edu.pl

#These authors equally contributed to the work.

Abbreviations: ALP, alkaline phosphatase; ANPEP, alanyl aminopeptidase; AP-1, activator protein 1; ARID1A, AT-rich interactive domain-containing protein 1A; ATRX, alpha thalassemia/mental retardation syndrome X-linked; BCOR, BCL6 Corepressor; BRAF, serine/threonine kinase; c-Myc, cellular myelocytomatosis oncogene; CAMTA1, calmodulin binding transcription activator 1; CD133, CD133 antigen; COSMIC, Catalogue Of Somatic Mutations In Cancer; CRP, C-reactive protein; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; FLNA, filamin A; FUCA1, alpha-L-fucosidase 1; GNAS, guanine nucleotide binding protein alpha stimulating; H3F3A, H3 histone family 3A; HER-4, human epidermal growth factor receptor 4; ICB, immune checkpoint blockade; ICIs, immune checkpoint inhibitors; KTM2C, lysine methyltransferase 2C; LAMA3, laminin subunit alpha 3; LGALS1, galectin 1; LRP1B gene, low density lipoprotein receptor-related protein 1B; MATN3, matrilin 3; MED12, mediator complex subunit 12; miRNA , microRNA; MLLT3, MLLT3 super elongation complex subunit 3; MMGL, myomegalin-like; MSCs, mesenchymal stem cells; NANOG, homeobox protein NANOG; NCOR1, nuclear receptor corepressor 1; NF1, neurofibromin 1; OS, Osteosarcoma; PDE4DIP, phospodiestrase 4D-interacting protein; Rb, retinoblastoma protein; RB1, RB transcriptional corepressor 1; SEC, super elongation complex; SGCG, sarcoglycan gamma; SMAD2, SMAD family member 2; SMARCA4, SWI/SNF-related, matrix-associated, actin-dependant regulator of chromatin, subfamily a, member 4; SOX4, SRY-box transcription factor 4; TCGA, The Cancer Genome Atlas; TICs, tumor-initiating cells; TP53, tumor protein p53; TSC, tuberous sclerosis complex; TSC2, TSC complex subunit 2; TWIST, Twist Family BHLH Transcription Factor; VEGFA,Vascular endothelial growth factor A; WNT5A, Wnt Family Member 5A

Introduction

Osteosarcoma (OS) is one of the most common primary bone tumors. It occurs intraosseous, mainly in the metaphyseal region of the long bones. OS is inflicting an accelerated osteoid matrix production, which is connected to being developed at sites where bone grows expeditiously. Current data prove that OS has two main peaks of incidence: in childhood and adolescence. OS is the third most common type of tumor affecting young people, with the highest incidents in the early twenties (Wu & Livingston, 2020; Sun et al., 2020; Misaghi et al., 2018; Czarnecka et al., 2020).

Osteosarcoma is divided into several subtypes (Fig. 1). It can be categorized depending on the region of bone it affects (the surface and central part of the bone, and within the medulla). The staging scheme divides OS into two classes depending on the grade of the tumor (low or high), as well as the localization of the compartment (extra- or intra-compartmental). Additionally, it is classified considering the metastatic level of the tumor. The most common classification groups OS as osteoblastic, chondroblastic, fibroblastic, and small cell subtypes. Among mention above types of ostesarcoma, the most often diagnosed one is a high-grade tumor, occurring extra-cortically (Misaghi et al., 2018).

Even though the exact mechanisms responsible for OS pathogenesis are not known, it is confirmed that genetics play a role in the tumor origin. The most common characteristic of OS includes genome disorganization, together with alterations of tumor suppressor and DNA repair, as well as changes in cell cycle aneuploids with chromosomal alterations (de Azevedo et al., 2020).

It was proved that OS can be developed as a consequence of alteration in tumor suppressor genes, including TP53 and Rb1, during hereditary disorders such as Li-Fraumeni cancer family syndrome and retinoblastoma (Porter et al., 1992; Fletcher & Unni, 2002; Misaghi et al., 2018; Sun et al., 2020). However, also other genes, such as transcription factors, and tumor suppressor genes, including CAMTA1, or KMT2C might participate in osteosarcoma development (He et al., 2021; Chen et al., 2016; Chiappetta et al., 2019).

Currently, there are no laboratory tests or specific biomarkers to diagnose osteosarcoma (Misaghi et al., 2018). Even though there are some molecules expressed in OS that are proposed as their potential markers. The most promising one appears to be alkaline phosphatase (ALP), which increased serum level and seems to correlate positively with tumor volume (Limmahakhun et al., 2011). Additionally, C-reactive protein (CRP), Cathepsin D, osteocalcin, SATB2, and aspartic endoprotease can help distinguish OS (Misaghi et al., 2018; Czarnecka et al., 2020; Agustina et al., 2018; Machado et al., 2016; Tallegas et al., 2022).

In addition to proteins, microRNA expression also seems to be valuable in the diagnosis of osteosarcomas. Overexpression of miR-421 and miR-191 proves to be linked with proliferation, migration, and malignant character of the tumors. Their levels in the serum of OS patients were higher compared to samples from healthy volunteers (Czarnecka et al., 2020).

Moreover, the presence of tumor-initiating cells (TICs) may help in OS diagnostics. TICs expressing markers of stem cell phenotype, such as NANOG or SOX4 were observed in both primary and metastatic tumor tissue (Yan et al., 2016). These proteins, together with CD133+, which is a distinctive marker of TICs observed in osteosarcoma cell lines (Czarnecka et al., 2020). It has been proved that the expression of CD133 in OS patients is related to distant metastasis and poor prognosis. making this a tumor marker (Xie et al., 2018).

Finally, the prevalence of mesenchymal stem cells (MSCs) connected with OS progression is proposed to be the tumor marker. Changes in the expression of c-Myc, Rb, AP-1 or TWIST were shown to contribute to the transformation of MSCs into osteosarcoma tumor cells (Yang et al., 2020). Most sarcomas characterize a permanent mesenchymal state. However, their phenotype can change by factors regulating epithelial-mesenchymal transition – EMT. These factors have been shown to be highly correlated with the invasiveness and higher risk of metastasis in malignant OS (Wu et al., 2019; Yu, Yustein & Xu, 2021). An EMT-related genes’ panel (including LAMA3, LGALS1, SGCG, VEGFA, WNT5A, MATN3, ANPEP, FUCA1, and FLNA) was used as a predictive marker in a multi-cohort study of OS. The panel proved to be a reliable tool for estimating the overall survival of OS patients. Moreover, it was proposed as a selection method for patients with metastases for personalized treatment (Yiqi et al., 2020).

Even though numerous studies describe molecules, whose expression may characterize osteosarcomas, all pointed proteins and/or miRNA are not specific to OS. Thus, further studies are needed to establish biomarkers helping to diagnose and treat OS.

The present study aims to provide a comprehensive, up-to-date study of genes the most frequently mutated in osteosarcoma using the COSMIC Catalogue. Such genes would become candidates for OS markers.

COSMIC, the Catalogue Of Somatic Mutations In Cancer (https://cancer.sanger.ac.uk) is a database providing tools to explore somatic mutations and their correlation with human cancers. The catalogue is the collection of somatic mutations. The data used to build this catalogue derives strictly from the scientific literature. COSMIC additionally provides information about patients’ genetic predispositions and environmental factors participating in the process of cancerogenesis (Tate et al., 2019). 

Materials and methods

To identify the most common somatic mutations occurring in osteosarcoma patients, the COSMIC Cancer Browser tool (https://cancer.sanger.ac.uk/cosmic/browse/tissue) was used. The catalogue provides information about 282 osteosarcoma samples (classified into 9 histological subtypes). The biological relevance of the 20 most frequently mutated genes pointed out by the COSMIC Cancer Browser was investigated by the analysis of publications available on PubMed (https://pubmed.ncbi.nlm.nih.gov/). To search for the publications, specific keywords, such as: selected genes’ name, osteosarcoma, cancer, and mutation were used. For each gene research was divided into two keyword panels – first: selected genes’ name, cancer, mutation; second: selected genes’ name, osteosarcoma, mutation.

Results and discussion

Genes the most frequently mutated in OS

The analysis of the COSMIC Catalogue provided a list of genes in which somatic mutation may participate in osteosarcoma development. Out of 18,309 entries of genes, the top 20 genes, based on the frequency of their mutation were selected. They are shown in Table 1 and shown in Fig. 2.

All these genes are directly or indirectly involved in oncogenesis (Table 2). Seven out of these 20 genes – TP53, RB1, NCOR1, SMAD2, NF1, TSC2, KMT2C are known to be tumor suppressor genes. Thus, their mutations lead to the expression of malfunctioning protein products and strictly correlate with tumourigenesis. The following three genes: GNAS, BRAF, and MLLT3 are protooncogenes. Other genes – SMARCA4, ARID1A, ATRX, BCOR, and H3F3A are related to the epigenetic status of gene expression or posttranslational histone modifications. Finally, EGFR, CAMTA1, LRP1B, PDE4DIP, and MED12 are associated with cell growth, metabolism, and survival.

Molecular basis of OS

Genome destabilization, aneuploids with chromosomal changes, dysregulation of cell cycle and tumor suppressor genes, as well as a lack of DNA repair are some of the most prevalent characteristics of OS (de Azevedo et al., 2020). Epigenetic changes are also recognized as risk factors for OS (Sharma, Kelly & Jones, 2009). Still, the pathogenesis of OS is complex and poorly understood. Thus, the identification of additional genes involved in oncogenesis may lead to advances in understanding OS biology, as well as shortening the time needed to establish diagnosis and introduce proper treatment.

The results of the COSMIC Catalogue analysis allowed distinguishing 20 genes the most frequently mutated in cancer. All recognized genes (Table 2) are known to be key regulators of vital cellular processes such as DNA repair, cell proliferation and differentiation. Thus, their mutations might lead to cancer development.

The most frequently mutated genes and their possible role in OS biology

Among the genes examined during the analysis of the COSMIC Catalogue, the most frequently mutated gene in OS was TP53. Its mutation characterized 25% of samples available in the database. TP53 encodes the P53 protein, which is an essential transcription factor with a tumor suppressor function. It stimulates several processes that result in cell cycle arrest, DNA repair, alterations in metabolism in response to cellular stress, apoptosis, and cell senescence. Moreover, it plays an important role in ontogenesis, myogenesis, and angiogenesis (Synoradzki et al., 2021). Thus, mutated TP53 appears to be essential for cancer development and its progression. Mutations in TP53 are known to increase protein instability, which affects clinical characteristics of osteosarcoma, such as metastatic potential, tumor type, and grade as well as its aggressiveness. According to Chen et al. TP53 mutations are a reliable predictive indicator for osteosarcoma patient survival (Chen et al., 2016).

Another gene commonly mutated in OS is Retinoblastoma transcriptional corepressor 1 gene – RB1. Its mutations were observed in 8% of samples available in the COSMIC catalogue. Between 50% and 70% of osteosarcoma cases exhibit mutations in RB1 (Wu & Livingston, 2020). RB1 encodes retinoblastoma protein controlling the transition from the G1 to S phase. This makes it a crucial regulator of cell cycle progression. It is suggested that during OS oncogenesis, alterations of RB1 may correlate with TP53 inactivation (Wu & Livingston, 2020; Li et al., 2022). Indeed, using mice models it was proved that deletion of RB1 leads to osteosarcomas development. Osteoblast development and mineralization as well as an increase in the expression of osteogenic markers were positively impacted by the gene deletion (Li et al., 2022).

Yet another gene mutated in 8% of OS is ATRX – alpha thalassemia/mental retardation syndrome X-linked. The gene encodes the protein that plays a role in chromatin remodeling and DNA repair. Loss of ATRX function has been linked to the accumulation of repetitive DNA sequences and alterations in the epigenetic regulation of the gene expression, which may contribute to the development and progression of tumor (He et al., 2015). Mutations in ATRX have been observed in a variety of cancer types (Haase et al., 2018). In osteosarcoma, ATRX dysfunction was linked with chromosomal instability, which contributes the tumor development. The gene’s mutations were also associated with poor prognosis and resistance to chemotherapy (Masliah-Planchon et al., 2018). Thus, ATRX is pointed out as a promising biomarker of osteosarcoma.

6% of OS samples in the COSMIC catalogue manifested mutation of the KTM2C gene. KTM2C has been shown to act as a tumor suppressor. Its expression in OS has been shown to be downregulated, like in a variety of other human cancers, including breast and prostate cancer (Gala et al., 2018; Lian et al., 2022; Liu et al., 2021). Loss of KTM2C expression is associated with poor prognosis and resistance to chemotherapy (Lian et al., 2022; Liu et al., 2021). What is more, KTM2C has been shown to regulate the other tumor suppressor genes, such as TP53 and p16INK4a. By removing methyl groups from histones, KTM2C also activates the expression of oncogenes, such as MYC (Liu et al., 2021; Lian et al., 2022; Gala et al., 2018). All these data demonstrate that the KTM2C gene can play an important role in OS development.

6% of available OS samples of the COSMIC catalogue were mutated in the LRP1B gene – Low Density Lipoprotein Receptor-Related Protein 1B that encodes cell surface receptor for LDL. LRP1B has been previously shown to play a role in tumor growth and progression. Additionally, it regulates angiogenesis in some of the cancer types, such as non-small cell lung cancer, renal cell cancer, and neuroglioma (Wang & Xiong, 2021). The gene overexpression observed in various cancers is linked to poor prognosis and decreased patient survival (Brown et al., 2021; Príncipe et al., 2021; Wang & Xiong, 2021). LRP1B is associated with the occurrence of osteosarcoma and its overexpression correlates with poor prognosis and decreased patient survival. Therefore the gene might be one relevant factor helping in OS diagnosis (Xu et al., 2021).

A significant role in tumorigenesis and tumor progression is also played by the CAMTA1 gene encoding a transcription factor – Calmodulin Binding Transcription Activator 1 (He et al., 2021). The gene down-expression correlates with chemoresistance (Pan et al., 2022). It has been shown that the expression of long noncoding RNA CAMTA1 (lncCAMTA1) in breast cancer affects cells viability and promotes their migratory state. Knock-down of lncCAMTA1 leads to cancer cell apoptosis (Lu et al., 2018). CAMTA1 mutations were observed in 5% of OS COSMIC samples. Even though there is a lack of information about the gene significance for osteosarcoma, still in Epithelioid hemangioendothelioma, that can arise from soft tissues and bones, CAMTA1 was proposed to be an immunohistochemical marker (Anderson & Jo, 2021).

Yet another gene shown to play a role in tumor development and its progression is NCOR1 (Nuclear Receptor Corepressor 1) gene. COSMIC catalogue analysis showed that as many as 4% of OS samples showed NCOR1 mutation. The gene encodes a corepressor involved in the regulation of gene expression (Tang et al., 2020). NCOR1 was observed to be frequently upregulated in breast cancer, and its high expression is associated with good prognosis and better patient survival (Noblejas-López et al., 2018). Interestingly, in bladder cancer, NCOR1 strongly correlated with immunogenicity, and its mutations were shown to cause activation of anti-tumor immunity, as well as overexpression of immune-related genes (Lin et al., 2021). According to Luo et al. NCOR is widely amplificated in osteosarcoma, as it might participate in tumor growth as a regulator of other gene transcription (Luo et al., 2019).

Four percentage of OS samples of the COSMIC catalogue showed mutations of the MLLT3 gene. MLLT3 protein is part of a super elongation complex (SEC), which is needed to increase the catalytic function of RNA polymerase II (Calvanese et al., 2019). The knock-out of the MLLT3 gene can lead to decreased proliferation of osteosarcoma cell lines, associated with the JNK signaling pathway. Thus, MLLT3 can be an oncogene candidate involved in osteosarcomas development (Sun et al., 2017).

As much as 4% of samples deposited in the COSMIC catalogue were characterized by PDE4DIP gene mutations. Even though there is no information about the gene significance in osteosarcomas, still, PDE4DIP protein, also known as MMGL (myomegalin-like) protein is involved in several intracellular metabolic pathways, which may control OS biology (Soejima et al., 2001; Lehnart et al., 2005).

Three percentage of OS samples carried a mutation of GNAS (Guanine Nucleotide Binding Protein, Alpha Stimulating). The gene encodes a signaling molecule involved in many cellular processes involving cAMP signaling. It has been shown to play a role in tumorigenesis (Nomura et al., 2014; Zauber, Marotta & Sabbath-Solitare, 2016; Patra et al., 2018; Afolabi et al., 2022). GNAS mutations were observed in various cancers, such as colon, pancreas, and gastrin adenocarcinoma as well as in fibrous dysplasia, which is known to have the potential to form malignancies such as osteosarcoma (Sugiura et al., 2018).

Three percentage of samples deposited in the COSMIC catalogue manifested mutations of the SMARCA4 gene. SMARCA4 encodes the catalytic subunit of SWI/SNF complexes, which is associated with chromatin remodeling and making genes accessible for transcriptional factors. Unfortunately, there is a lack of information about the significance of gene alterations in osteosarcomas.

Also, the ARID1A gene product (AT-rich interactive domain-containing protein 1A) is a part of the SWI/SFI complex, associated with chromatin remodeling (Xu et al., 2019). ARID1A mutations were observed in many cancers before (Jones et al., 2010). Recent studies show that in osteosarcomas the expression of ARID1A is down-regulated compared to non-tumor tissues (Xu et al., 2019). This can be correlated with the gene mutations measured in 3% of OS samples of the COSMIC catalogue.

Yet another mutated gene affecting the structure of chromatin and contributing to tumorigenesis is H3F3A (Sturm et al., 2012; Park et al., 2016). H3F3A overexpression has been shown to correlate with aggressive phenotype and chemoresistance leading to lower survival rates and relapse in lung cancer patients (Park et al., 2016). COSMIC catalogue analysis showed that 2% of OS samples are mutated in H3F3A. This data finds approval in Koelsche et al. study showing hotspot mutations in H3F3A in six osteosarcoma cases analysed by the authors. Observed alternations were correlated with age and increased the incidence of osteosarcoma in patients over the age of 30 (Koelsche et al., 2017).

Two percentage of the samples of the COSMIC catalogue were carrying mutations in the BCOR gene. BCOR protein is involved in the process of deacetylation of histones and when over-expressed inhibits BCL-6 tumor suppressor ­(Huynh et al., 2000). Mutations in BCOR are associated with many types of cancer, such as clear cell sarcoma of the kidney, endometrial stromal sarcoma, or small round blue cell sarcomas (Astolfi et al., 2019; Ueno-Yokohata et al., 2015; Marinõ-Enriquez et al., 2018; Sbaraglia et al., 2020).

Among samples deposited in the COSMIC catalogue, approximately 2% carry mutations in the MED12 gene. MED12 is part of a larger, multiprotein complex that mediates polymerase II RNA during the process of transcription (Ding et al., 2008). MED12 is associated with 70% of uterine leiomyomas and fibroepithelial tumors of the breast (Croce & Chibon, 2015; Lerwill et al., 2022). As for now, only two clinical case reports described MED12 mutations in osteosarcomas. Le and others (Le et al., 2021) showed that the patient with MED12-mutated high-grade uterine sarcoma developed osteosarcoma with the same L36R missense mutation. Another team described MED12 mutation in osteosarcoma which further led to the development of breast malignant phyllodes tumor (Tokoyoda et al., 2018). More study is needed to assess the link between MED12 mutations and the occurrence of osteosarcoma, especially since according to The Cancer Genome Atlas (TCGA) data, mutations in MED12 can be biomarkers helping to select patients for the therapy with immune checkpoint inhibitors (ICIs) (Zhou et al., 2022).

Two percentage of OS samples from the COSMIC catalogue displayed changes in the EGFR gene encoding receptor for the epidermal growth factor (EGFR). The receptor is known for its crucial role in the signaling pathway responsible for cell proliferation, differentiation, and/or survival (Wang et al., 2004). Mutations of EGFR in a variety of malignancies are well documented. Most frequent is the gene’s amplification leading to EGFR overexpression increasing cellular signaling (Sigismund, Avanzato & Lanzetti, 2018; Cheng et al., 2021). The gene importance for OS has been already documented. Sheng-Lin and others (Sheng-Lin et. al., ????) reported that high expression of EGFR together with HER-4 (human epidermal growth factor receptor 4) was associated with distant metastasis and the level of EGFR expression was proposed to be a potential prognostic biomarker of OS (Wang et al., 2018).

Among OS samples deposited in the COSMIC catalogue, 2% manifested mutations of the SMAD2 gene. This low number of samples can indicate that the gene mutations are not osteosarcoma specific. However, is worth mentioning that SMAD2, as a part of the TGF-beta superfamily, plays an important role in cell signaling, proliferation, and differentiation (Piek et al., 1999) and may act as an oncosuppressor (Piek et al., 1999; Pasche, 2001). Moreover, it has been shown that there are statistically significant changes in SMAD2 expression in OS compared to healthy tissue. The correlation between alterations of Smad signaling, the high proliferation rate, and the invasive phenotype of osteosarcoma cells was observed in OS with Serine/threonine kinase 39 (STK39) knockdown (Won et al., 2010; Yang et al., 2013; Huang et al., 2017).

Another gene selected during our study was the NF1 gene. According to the data from the COSMIC catalogue 2% of OS samples were carrying the gene’s mutations. The product of NF1 is neurofibromin 1 – a cytoplasmic protein expressed in neurons, Schwann cells, and leukocytes. The protein is a part of the RAS/MAPK signaling pathway controlling cell proliferation (Huang et al., 2017). It is suggested that NF1 can act also as an oncosuppressor. Although the NF1 gene is associated mostly with neural malignancies, its mutations are observed in many cancers, e.g. lung cancers or melanomas (Trovó-Marqui & Tajara, 2006). Further studies are needed to prove NF1 role in OS development.

An additional gene involved in the inhibition of cell proliferation is the tumor-suppressor gene TSC2 (Inoki et al., 2002). The gene’s mutations were described in tuberous sclerosis complex (TSC) and non-small cell lung cancer. It was proved that patients with TSC1/TSC2 loss can benefit from target therapy including treatment with immune checkpoint blockade (ICB) (Huang et al., 2022). Kuroda et al. reported a clinical case of a young patient with tuberous sclerosis complex (TSC) who developed osteosarcoma. Analysis of the tumor sample showed additional mutations in TP53 as well as TSC2 genes (Kuroda et al., 2021). This observation is in agreement with COSMIC catalogue data showing that approximately 2% of OS samples were characterized by mutated TSC2.

Two percentage of OS samples were also characterized by mutated proto-oncogene BRAF. BRAF activates signaling pathways involved in the cell cycle, including proliferation, differentiation, and cell death. The gene mutations may result in malignancies development. In fact, dysregulation of gene expression was observed in several cancers, including melanoma, colorectal cancer, and non-small cell lung cancer (Śmiech et al., 2020). Pignochino et al. reported that in 4 of 30 osteosarcoma patients analysed in their study, BRAF gene mutation was confirmed (Pignochino et al., 2009).

Conclusions

COSMIC catalogue analysis allows to identify genes that are the most frequently mutated in osteosarcoma. Since 20 of the selected genes have previously been shown to be involved in cancerogenesis, thus their mutation might also play a role in OS development. However, to prove their clinical significance and the possibility of using them as OS biomarkers further studies are needed. The results of such a study would help to identify patient predisposition to OS development, follow cancer progression, and establish patients’ treatment and prognosis.

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