Liquid biopsy in bone sarcomas and identification of new biomarkers

Bone sarcoma malignancies: origin, treatment, and diagnosis

Bone sarcomas are rare, highly heterogeneous oncologic entities that originate in mesenchymal tissues from bone. Primary bone cancers account for less than 0.2% of all cancers. The high morphological heterogeneity of cancer cells and tumor tissues leads to multiple biological behaviors that require specific and complex therapeutic strategies. Osteosarcoma, Ewing sarcoma (ES), and chondrosarcoma are the most prevalent bone sarcomas [ ]. In 2021, the American Cancer Society estimates around 3610 new diagnoses of bone sarcoma cases, and 2060 estimated deaths. In adults, over 40% of primary bone cancers are chondrosarcomas, while in children/teenagers and young adults, osteosarcoma and Ewing's tumors account for 56% and 34%, respectively [ ]. Osteosarcoma and chondrosarcoma are considered the result of a disturbed differentiation program for mesenchymal stem cells (MSCs), and ES is characterized by the expression of a fusion protein resulting from a chromosomal translocation between the EWS gene on chromosome 2 and a gene in the ETS family in undifferentiated MSC [ ].

The main areas affected by osteosarcoma are the epiphyses of long bones. Genetic analyses confirmed the high heterogeneity of osteosarcoma [ ]. Around 10%–20% of patients show clinically detectable metastases at time of diagnosis, mostly located in the lungs. ES develops in flat bones and mostly affects the metaphysis of long bones. Overall survival is also associated with the metastatic status of patients. For localized tumors, the overall survival is 50%–60% at 5 years, which drops to only around 20% when metastasis is detected. At time of diagnosis, 20%–25% of patients show clinically detectable metastases [ ]. Chondrosarcoma is the third bone sarcoma in terms of incidence. Chondrosarcoma is mainly localized in the pelvic bone, scapula, and long bones. While high-grade chondrosarcomas can be associated with metastases, these tumors are characterized by a high rate of local recurrence and consequently by high morbidity [ ].

The conventional therapeutic approach to osteosarcoma and ES combines surgery and chemotherapy [ , ]. The conventional cocktail of chemotherapeutic agents used in osteosarcoma is doxorubicin, cisplatin, and methotrexate, and ifosfamide can be also added. In ES, chemotherapy includes vincristine, ifosfamide, doxorubicin, and etoposide. Multiple new approaches are currently being assessed in osteosarcoma and ES: new formulations of chemotherapy agents [ ], tyrosine kinase inhibitors [ , ], bone targeting [ ], DNA repair targeting [ ], immunotherapies [ , ], fusion protein targeting, or cyclin-dependent kinase inhibitors. Chondrosarcomas are chemo- and radioresistant tumors, thus the treatment chosen for local and advanced chondrosarcoma patients is surgery with proper margin resection (see chapter 48 ). Unfortunately, adequate margins can only be achieved in 45%–75% of patients, which is related to a high risk of local recurrence. Currently, several clinical trials using new molecules such as tyrosine kinase inhibitors or mTOR inhibitors are being assessed in the treatment of chondrosarcoma patients [ ]. However, the high heterogeneity of the tumor microenvironment of bone sarcomas points to the development of personalized medicine as the most effective therapeutic strategy.

Current and potential bone sarcoma biomarkers

Current sarcoma diagnosis, prognosis, and treatment decisions include a combination of imaging-based CT, MRI scans, and biopsies [ ]. Biomarkers have many potential applications in oncology, including differential diagnosis, determination of prognosis, prediction of response to treatment, and monitoring of progression of disease. Because of the critical role that biomarkers play at all stages of the disease, their rigorous evaluation is mandatory, and includes analytical validation, clinical validation, and assessment of clinical utility, prior to incorporation into routine clinical care. Several authors have assessed the potential role of different molecules such as biomarkers for bone sarcomas. Here we will summarize certain current and potential biomarkers for osteosarcoma, ES, and chondrosarcoma ( Table 35.1 ).

Many studies show that several biomarkers undergo critical changes as osteosarcoma progresses. Luo et al. observed decreased expression of ALP, Runx2, OSX, and OPN, involved in osteogenic differentiation. The authors suggested that defects in this pathway may be involved in tumorigenesis, and thus these four genes may serve as biomarkers [ ]. Similarly, Shimizu et al. found FGF-2 and LIF were able to reduce the osteogenic differentiation of osteosarcoma. In addition, FGF-2 promoted the proliferation and migration of neoplastic cells and altered the response of cells to drug therapy. Blocking this factor may be an interesting strategy for modulating the progression of osteosarcoma [ ]. Another molecule involved in osteoblast differentiation that seemed to be involved in osteosarcoma prognosis is CCN3. The assessment for CCN3 expression levels at diagnosis may be useful for classifying patients with different prognoses [ ]. In another study, Sandhu et al. observed that serum sialic acid levels increased significantly in osteosarcoma patients [ ]. Yu et al. found an increase in the mitotic arrest defective protein 2 (MAD2) in osteosarcoma patients with metastasis and poor survival [ ]. Babkina et al. found that levels of PGF-2, PIGF, endostatin, and FGF-1 were increased in osteosarcoma patients compared to the control group [ ]. Reduced expression of RECK was found as an independent prognosis marker for osteosarcoma patients that played a part in determining the best therapy for individual patients [ ]. IGF-1R was described as an independent prognostic marker for osteosarcoma patients; moreover, increased levels of this molecule were related to metastasis [ ]. CRIP1 was found preferentially expressed in patients with longer survival without metastases [ ]. Lu et al. observed that the coexpression of WNT-5a and ROR2 markers was present in advanced stages of osteosarcoma and promoted the diffusion of the tumor [ ]. One study performed by Jin et al. confirmed decreased levels of gelsolin in the serum of osteosarcoma patients [ ]. Snail2 was found to be correlated with the severity of osteosarcoma and the risk of metastases and may be useful for the prognosis of bone sarcoma [ ]. Wei et al. analyzed the c-kit gene; they concluded that gene alteration is a prognostic marker for osteosarcoma patients [ ]. The genetic analysis of TGFBR1∗6A, a dominant polymorphism of the transforming growth factor β receptor 1 (TGFBR1), concluded that this variant was associated with increased susceptibility and metastasis diffusion in osteosarcoma [ ]. Similarly, Wang et al. demonstrated that the C 49G/A polymorphism of cytotoxic T-lymphocyte antigen-4 (CTLA-4) promoted the development of osteosarcoma, as this molecule decreased the immune response mediated by T cells [ ]. The overexpression of the cortactin (CTTN) gene, involved in osteosarcoma carcinogenesis, has been consolidated as a valid prognosis marker in pediatric osteosarcoma patients [ ]. Won et al. analyzed the role of the protein Runx2 in osteosarcoma. The authors confirmed that an increase in its expression resulted in a higher risk of diffusion of the osteosarcoma, and therefore considered the protein as a valid prognostic marker [ ]. Genetic alterations in the glutathione S-transferase M supergene family (GSTM) play a role in the body's defense mechanisms to carcinogens, increasing the risk of cancer and drug resistance. Salinas-Souza et al. analyzed the relationship between the GSTM1, GSTM2, and GSTM3 polymorphisms and the clinical outcome of osteosarcoma patients. They concluded that GST polymorphisms may allow osteosarcoma to progress [ ].

ES family tumors are characterized by translocations of the EWS gene on chromosome 22q12 with an ETS gene family member. These rearrangements provide a valuable tool for their accurate and unequivocal diagnosis. They also represent ideal targets for the development of tumor-specific therapies [ ]. Interestingly, some studies have demonstrated the correlation of their expression and EWS/FLI1 translocations. Aryee et al. found transcription factor “hypoxia-inducible factor-1” (HIF-1) that makes it possible to control EWS-FLI1 expression, resulting in variability in the clinical and prognostic features in tumors [ ]. In another study, CD99 expression was significantly related to EWS/FLI1 translocations in ES, suggesting a role as a diagnosis and prognosis marker [ ]. FLI1 has been described as a useful marker for the differential diagnosis of ES and other bone sarcomas [ ]. Besides the biomarkers associated with the EWS gene, other molecules have been suggested as biomarkers in ES. Bennani-Baiti et al. affirmed that CXCR4 expression increases the risk of tumor metastases, and CXCR7 expression is associated with shorter survival [ ]. The 1QG and CDT2 genes were found to have prognostic significance and can be used to select therapy [ ].

Several molecules have recently been reported as biomarkers in the diagnosis, prognosis, and treatment of chondrosarcoma. Hallor et al. found a similar pattern of genomic imbalances in the vast majority of chondrosarcoma cases studied. They found deletions of loci at the level of the CDKN2A, EXT1, and EXT2 genes, related to the progression of the disease. These three molecules were defined as good candidates for genetic biomarkers in the diagnosis and prognosis of chondrosarcomas [ ]. The ESMO/EUROBONET Working Group suggests several chondrosarcoma biomarkers: p53 was a late event involved in tumor progression; amplification of 12q13 and loss of 9p21 were genetic aberrations found in conventional chondrosarcomas; and a loss of INK4A/p16 expression in high-grade chondrosarcomas was also detected [ ]. One study performed by Rozeman et al. showed that higher expression of PTHR1 and Bcl-2 was associated with increasing histological grade in chondrosarcoma [ ]. Later, they found that PAI-1 might also be of interest as a prognostic marker in dedifferentiated peripheral chondrosarcomas [ ]. Liang et al. found that expression of Aurora Kinase A and B was significantly higher in patients with recurrence and metastasis than in the control group Moreover, expression of the marker was lower in low-grade tumors compared to medium- and high-grade tumors [ ]. Two studies confirmed the association between COX-2 and CD34 with patient survival [ , ].