IDH1 and IDH2 Mutations

Mutations in isocitrate dehydrogenase 1 and 2 ( IDH1 and IDH2 ), first identified in glioblastoma multiforme are found in 87% of enchondromas, 38%–70% of primary central chondrosarcomas, 54% of dedifferentiated chondrosarcomas, and 15% of periosteal chondrosarcomas. The occurrence of IDH1 and IDH2 mutations in enchondroma indicates that this is an early genetic event in the development of central chondrosarcoma (see Fig. 4.1 ). Also, in intracranial chondrosarcomas, but not in chordomas, IDH1 and IDH2 mutations have been reported, making it a good diagnostic marker in the differential diagnosis between the two different entities.

COL2A1 Mutations

Collagen type-II alpha1 ( COL2A1 ) mutations have been identified in 37% and 19.3% of central chondrosarcomas and 31.7% of enchondromas. Type-II collagen is the major extracellular component of cartilage and is important for the strength and flexibility of the tissue. Mutations in the COL2A1 gene can lead to abnormalities in collagen formation. Immunohistochemical analysis has revealed that only a small part of chondrosarcomas have complete loss of the COL2A1 protein, while most others retain focal staining. Mutations in COL2A1 are associated with several developmental disorders called type-II collagenopathies, but the incidence of enchondromas or chondrosarcomas is not increased in these disorders. Also, in mice expressing a mutant COL2A1 , increased chondrocyte proliferation was not observed but the structure of the collagen network was altered and the growth plate was disorganized. Downregulation of COL2A1 in cultured mouse chondrocytes resulted in upregulation of collagen I. Also Hedgehog signaling was affected by knock-down of COL2A1 , which disrupted chondrocyte differentiation. Whether mutations in COL2A1 are drivers of chondrosarcoma formation is still to be determined.

Dysregulation of the Hedgehog Pathway

The Indian hedgehog (IHH) pathway is important for bone development. Chondrocytes of the growth plate undergo differentiation, resulting in longitudinal growth of the bone. This process is regulated by Indian hedgehog and parathyroid hormone–like hormone (PTHLH) in a negative feedback loop. This feedback loop inhibits terminal differentiation of chondrocytes. Chondrocytes are differentiating toward hypertrophic chondrocytes which will be replaced by osteoblasts. Prehypertrophic chondrocytes produce IHH, stimulating proliferation of chondrocytes and stimulating the cells of the perichondrium to produce PTHLH. PTHLH inhibits IHH as well as terminal differentiation of chondrocytes. Constitutive activation of the hedgehog pathway results in failure to induce terminal differentiation of growth plate chondrocytes, which can result in the development of enchondromas.

Expression studies show that the hedgehog pathway members are highly expressed in enchondromas and in central chondrosarcomas, maintaining tumor cells in a less differentiated, but proliferative state. Levels of expression are variable, but there is no correlation with histological grade. Blocking of the hedgehog pathway with triparanol or IPI-926 reduces tumor volume in chondrosarcoma xenografts, while cyclopamine does not decrease proliferation of chondrosarcoma cell lines suggesting that the tumor microenvironment affects this pathway. Also the potency of the different compounds used in these studies can differ.

Patients with enchondromatosis show mutations in one of the genes involved in the IHH pathway in 8% of cases. Also, mice expressing a mutant parathyroid hormone–like hormone receptor specifically in chondrocytes have a deregulated growth plate and form cartilaginous lesions.

Hedgehog signaling is activated by binding of hedgehog ligand to the patched receptor, releasing the smoothened receptor and freeing Gli transcription factors to travel to the nucleus to activate transcription. Similar to the mutant PTHLH receptor expressing mice, mice constitutively expressing Gli2 in their chondrocytes also develop cartilaginous lesions.

Primary cilia have been shown to be involved in IHH signaling and to regulate its activity. Primary cilia are also important for the columnar organization of the growth plate. Chondrosarcoma cells express fewer cilia than do normal chondrocytes, and fewer cilia correlates with deregulated hedgehog signaling. Inhibition of the hedgehog pathway in enchondromas and central chondrosarcomas is a potential therapeutic strategy for patients with inoperable or metastatic disease. A phase-II clinical trial investigating the efficacy of GDC-0449 smoothened inhibitor in 45 patients with progressive advanced chondrosarcoma found only modest activity, with stable disease in only 10 patients with grade-I tumors after 6 months.

Recently, mutations in IHH genes have been identified in 18% of chondrosarcomas using whole exome sequencing. If these tumors respond better to hedgehog pathway inhibitors, this mutation may be a marker for selecting patients for such treatment.

P53 and pRb Pathway Alterations in the Progression From Low to High-Grade Chondrosarcoma

Mutations in the TP53 gene are the most frequently observed mutations in cancer and are often associated with a more aggressive phenotype and worse prognosis. Mutations in TP53 can lead to either loss or gain of function. Wild-type p53 has important functions in controlling cell proliferation and apoptosis, and loss of function mutations lead to cancer, as shown in a TP53 knock-out mouse model. However, point mutations in TP53 will lead to pathogenic accumulation of p53 in the nucleus, and mutant TP53 knock-in mice develop highly invasive tumors.

In chondrosarcoma, TP53 mutations are observed in 20% of cases and are associated with more malignant tumors and a higher histological grade. Also, increased nuclear p53 expression is correlated with a higher histological grade and worse survival. Mice constitutively expressing Gli, crossed with p53 knock-out mice, developed low-grade chondrosarcomas, indicating that deregulation of p53 is important in the progression of enchondromas to chondrosarcoma.

The retinoblastoma (pRb) pathway is important in the control of the cell cycle. It governs the restriction point transition (R-point), determining whether a cell progresses to S-phase or retreats to G0. RB gene mutations have been found in several cancer types but not in chondrosarcoma. Also, pRb expression was normal in chondrosarcomas.

Amplification of 12q13 occurs consistently in central chondrosarcoma. Both cyclin-dependent kinase 4 ( CDK4 ) and mouse double minute 2 homolog ( MDM2 ) are located in this region; they have important roles in the pRb and p53 pathways. CDK4 forms a complex with cyclin D1 and together they control the transition through the G1 restriction point by inactivating pRb. Consistent with the 12q13 amplification observed in a subset of chondrosarcomas, CDK4 and Cyclin D1 expression is upregulated in 62%–73% of high-grade chondrosarcomas. Furthermore inhibition of CDK4 decreases cell proliferation in chondrosarcoma cell lines. CDKN2A/p16 is a negative regulator of the CDK4-cyclin D1 complex and is downregulated in 20% of chondrosarcomas, which correlates with histological grade. Moreover, in 28% of chondrosarcomas mutations in CDKN2A are observed. However, no loss of CDKN2A/p16 has been found in enchondromas. Decreased cell growth, but not apoptosis, was observed in cells in which CDKN2A/p16 was reexpressed. MDM2 is a negative regulator of p53, targeting it for proteosomal degradation. MDM2 is overexpressed in 34% of high-grade chondrosarcomas, indicating that p53 is degraded at a higher rate in these tumors.

The combined incidence of mutations in either the p53 or the pRb pathway in high-grade chondrosarcomas is 96%. This suggests that these pathways are important in the progression of chondrosarcoma toward a more malignant histological phenotype.

Apoptosis and Prosurvival Pathways Implicated in Chondrosarcoma

A downstream target in the hedgehog pathway is Bcl-2, which is upregulated in the growth plate and inhibits differentiation of proliferating chondrocytes after PTHrP binds to its receptor. In most conventional chondrosarcomas, and rarely in other chondrosarcoma subtypes, Bcl-2 is highly expressed.

By upregulating antiapoptotic proteins of the Bcl-2 family, chondrosarcoma cells acquire resistance to chemotherapy, a major problem in the treatment of these tumors. This has been shown in a cell model where chondrosarcoma cells could be sensitized to chemotherapy by a Bcl-2 family inhibitor (ABT-737).

Survivin is also highly expressed in chondrosarcoma. Survivin plays important roles in the cell cycle and the apoptotic pathways. As it is expressed only at very low levels in normal tissue, it is an ideal therapeutic target. In preclinical studies a small molecule survivin inhibitor (YM155) decreased cell viability in chondrosarcoma cell lines, a response dependent on TP53 mutation status. Also downregulation of survivin using siRNAs decreased cell viability and increased cell sensitivity to chemotherapeutics.

Several studies have shown the importance of the Akt-Pi3K-mTOR pathway in chondrosarcoma. In 69% of conventional chondrosarcomas and 44% of dedifferentiated chondrosarcoms, S6 was phosphorylated, indicating that the PI3K/mTOR was active. Also treatment with a dual PI3K/mTOR inhibitor (Bez235) reduced cell viability in chondrosarcoma cell lines and inhibited tumor growth in a mouse xenograft model. In a rat chondrosarcoma model, treatment with everolimus inhibited tumor growth.

EXT1 and EXT2 Mutations in Osteochondroma and Peripheral Chondrosarcoma

Multiple osteochondromas (MO) is an autosomal dominant condition in which mutations in the tumor suppressor genes, exostosin glycosyltransferase 1 or exostosin glycosyltransferase 2 ( EXT1 and EXT2 ), have been identified. Patients with this disease develop osteochondromas at the surfaces of their bones and show heterozygous germline EXT1 or EXT2 mutations, with loss of the wild-type allele in the osteochondroma cartilaginous cap. Also, homozygous deletions in EXT1 have been identified in a subset of sporadic osteochondromas. Both hereditary and sporadic osteochondromas can progress toward peripheral chondrosarcoma (see Fig. 4.1 ). This occurs in ∼1% of sporadic and up to 5% of hereditary osteochondromas.

Exostosin-1 and exostosin-2 are both type-II glycosyltransferases, which participate in the biosynthesis of the heparan sulfate chains of proteoglycans. Proteoglycans regulate the signaling of several growth factors in many different ways. They can act as receptors or coreceptors in signaling pathways, regulate receptor trafficking by endo- and exocytosis, facilitate ligand secretion, function as a signal to other cells, or present a ligand to a receptor. In addition, they can regulate the structure of the extracellular matrix by providing a signaling gradient to ligands. In the growth plate, heparan sulfate is important for the establishment of a gradient of IHH (as discussed above). When the biosynthesis of heparan sulfate is deregulated, the diffusion of IHH signaling is disturbed. This disrupts the polar organization of growth plate chondrocytes and the formation of the bony collar, leading to the formation of an osteochondroma.

In a conditionally inducible mouse model, where both Ext1 alleles were inactivated specifically in chondrocytes, osteochondromas developed, mimicking the human situation. The osteochondromas consisted of a mixture of wild-type and mutated cells, indicating that cells need normal heparan sulfate synthesis to survive. In another mouse model with heterozygous loss of both Ext1 and Ext2 , heparan sulfate was decreased and more exostoses appeared in comparison to mice with heterozygous loss of Ext1 or Ext2 . Zebra fish with homozygous loss of Ext2 in their chondrocytes show osteochondroma-like lesions and altered mesenchymal stem-cell differentiation.

Osteochondromas can progress to peripheral chondrosarcomas in a small percentage of cases. Most peripheral chondrosarcomas have at least one wild-type allele for EXT1 and EXT2 , indicating that functional EXT is required for cells to survive and to undergo malignant progression. Most likely, additional genetic alterations are needed for progression to peripheral chondrosarcoma and they probably arise in the EXT1 and EXT2 wild-type cells.

p53 and pRb Pathway Alterations

In peripheral chondrosarcoma, pRb and p53 pathway alterations occur in most cases, especially in tumors with higher histological grade. A mouse model with conditionally inducible Ext1 inactivation specifically in chondrocytes combined with conditional loss of Trp53 or Ink4a/Arf developed peripheral chondrosarcomas from Ext -null and Ext -mutated cells, mimicking the human situation. The tumors lacked both primary cilia and polar organization. This indicates that in addition to the EXT mutation, an alteration in one of the pathways regulating the cell cycle is needed for the development of peripheral chondrosarcoma from an osteochondroma.