👤 J V Bovée

Periosteal chondrosarcoma is a rare, malignant cartilage-forming neoplasm originating from the periosteal surface of bone. We collected 38 cases from the archives of the Netherlands Committee on Bone Tumours, with the aim of studying histological features and evaluating the involvement of isocitrate dehydrogenase 1 (IDH1), EXT, Wnt/β-catenin, the pRB pathway (CDK4 and p16), and the TP53 pathway (p53 and MDM2). Histology showed a moderately cellular matrix with mucoid-myxoid changes and, in 42% of cases, formation of a neocortex. Occasional intramedullary extension (26%) and subsequent host bone entrapment (40%) were seen. Histological grading revealed grade 1 (53%) and grade 2 (45%). The EXT1 protein was normally expressed, and mutations in IDH1 were observed in only 15% of cases. pRb signalling was deregulated by loss of p16 expression in 50% of cases, and Wnt signalling was lost in 89%. No alterations were found in CDK4, p53, or MDM2. We report the first large histological and molecular study on periosteal chondrosarcoma showing that histopathological examination and molecular aberrations do not predict prognosis. Although the mutation frequency of IDH1 was low, we confirm the supposed relationship with central chondrosarcoma. Moreover, we identify loss of canonical Wnt signalling and deregulation of pRb signalling as possible events contributing to its histogenesis. Show less

Peripheral chondrosarcoma (PCS) develops as malignant transformation of an osteochondroma, a benign cartilaginous outgrowth at the bone surface. Its invasive, lobular growth despite low-grade histology suggests a loss of chondrocyte polarity. The known genetics of osteochondromagenesis include mosaic loss of EXT1 or EXT2 in both hereditary and non-hereditary cases. The most frequent genetic aberrations in human PCS also include disruptions of CDKN2A or TP53. In order to test the sufficiency of either of these to drive progression of an osteochondroma to PCS, we added conditional loss of Trp53 or Ink4a/Arf in an Ext1-driven mouse model of osteochondromagenesis. Each additional tumour suppressor silencing efficiently drove the development of growths that mimic human PCS. As in humans, lobules developed from both Ext1-null and Ext1-functional clones within osteochondromas. Assessment of their orientation revealed an absence of primary cilia in the majority of mouse PCS chondrocytes, which was corroborated in human PCSs. Loss of primary cilia may be responsible for the lost polarity phenotype ascribed to PCS. Cilia deficiency blocks proliferation in physeal chondrocytes, but cell cycle deregulation is sufficient to rescue chondrocyte proliferation following deciliation. This provides a basis of selective pressure for the frequent cell-cycle regulator silencing observed in peripheral chondrosarcomagenesis. Mosaic loss of Ext1 combined with loss of cell cycle regulators promotes peripheral chondrosarcomagenesis in the mouse and reveals deficient ciliogenesis in both the model and the human disease, explaining biological behaviour including lobular and invasive growth. Show less

Multiple osteochondromas (MO) is a syndrome in which benign cartilage-capped neoplasms develop at the surface of the long bones. Most cases are caused by exonic changes in EXT1 or EXT2, but 15% are negative for these changes. Here we report for the first time a family of MO patients with germline genomic alterations at the EXT1 locus without detectable mutations or copy number alterations of EXT exonic sequences. Array-CGH showed an 80.7 kb deletion of Intron 1 of EXT1 and a 68.9 kb duplication proximal of EXT1. We identified a breakpoint between the distal end of the duplicated region and a sequence distal of the deleted region in the first intron. This breakpoint was absent in non-affected family members. The configuration of the breakpoint indicates a direct insertion of the duplicated region into the deletion. However, no other breakpoint was found, which suggests a more complex genomic rearrangement has occurred within the duplicated region. Our results reveal intronic deletion and duplication as a new causative mechanism for MO not detected by conventional diagnostic methods. Show less

Endochondral bone formation requires a cartilage template, known as the growth plate, and vascular invasion, bringing osteoblasts and osteoclasts. Endochondral chondrocytes undergo sequences of cell division, matrix secretion, cell hypertrophy, apoptosis, and matrix calcification/mineralisation. In this study, two critical steps of endochondral bone formation, the deposition of collagen X-rich matrix and blood vessel attraction/invasion, were investigated by immunohistochemistry. Fourteen multiple osteochondromas and six secondary peripheral chondrosarcomas occurring in patients with multiple osteochondromas were studied and compared to epiphyseal growth plate samples. Mutation analysis showed all studied patients (expect one) to harbour a germ-line mutations in either EXT1 or EXT2. Here, we described that homozygous mutations in EXT1/EXT2, which are causative for osteochondroma formation, are likely to affect terminal chondrocyte differentiation and vascularisation in the osteocartilaginous interface. Contrastingly, terminal chondrocyte differentiation and vascularisation seem to be unaffected in secondary peripheral chondrosarcoma. In addition, osteochondromas with high vascular density displayed a higher proliferation rate. A similar apoptotic rate was observed in osteochondromas and secondary peripheral chondrosarcomas. Recently, it has been shown that cells with functional EXT1 and EXT2 are outnumbering EXT1/EXT2 mutated cells in secondary peripheral chondrosarcomas. This might explain the increased type X collagen production and blood vessel attraction in these malignant tumours. Show less

Secondary peripheral chondrosarcoma is the result of malignant transformation of a pre-existing osteochondroma, the most common benign bone tumor. Osteochondromas are caused by genetic abnormalities in EXT1 or EXT2: homozygous deletion of EXT1 characterizes sporadic osteochondromas (non-familial/solitary), and germline mutations in EXT1 or EXT2 combined with loss of heterozygosity define hereditary multiple osteochondromas. While cells with homozygous inactivation of EXT and wild-type cells shape osteochondromas, the cellular composition of secondary peripheral chondrosarcomas and the role of EXT in their formation have remained unclear. We report using a targeted-tiling-resolution oligo-array-CGH (array comparative genomic hybridization) that homozygous deletions of EXT1 or EXT2 are much less frequently detected (2/17, 12%) in sporadic secondary peripheral chondrosarcomas than expected based on the assumption that they originate in sporadic osteochondromas, in which homozygous inactivation of EXT1 is found in ~80% of our cases. FISH with an EXT1 probe confirmed that, unlike sporadic osteochondromas, cells from sporadic secondary peripheral chondrosarcomas predominantly retained one (hemizygous deleted loci) or both copies (wild-type) of the EXT1 locus. By immunohistochemistry, we confirm the presence of cells with dysfunctional EXT1 in sporadic osteochondromas and show cells with functional EXT1 in sporadic secondary peripheral chondrosarcomas. These immuno results were verified in osteochondromas and secondary peripheral chondrosarcomas in the setting of hereditary multiple osteochondromas. Our data therefore point to a model of oncogenesis in which the osteochondroma creates a niche in which wild-type cells with functional EXT are predisposed to acquire other mutations giving rise to secondary peripheral chondrosarcoma, indicating that EXT-independent mechanisms are involved in the pathogenesis of secondary peripheral chondrosarcoma. Show less

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a "second hit," that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome. Show less

Multiple osteochondromas (MO) is a hereditary skeletal disorder characterized by the presence of cartilage capped bony outgrowths at bone surface. Causative mutations in EXT1 or EXT2 genes have been described in 85-90 % of MO cases. However, in about 10-15 % of the MO cases, genomic alterations can not be detected, implying the potential role of other alterations. We have designed a custom-made Agilent oligonucleotide-based microarray, containing 44,000 probes, with tiling coverage of EXT1/2 genes and addition of 68 genes involved in heparan sulfate biosynthesis and other related pathways. Out of the 17 patient samples with previously undetected mutations, a low level of deletion of the EXT1 gene in about 10-15% of the blood cells was detected in two patients and mosaic deletion of the EXT2 was detected in one patient. Here we show that for the first time somatic mosaicism with large genomic deletions as the underlying mechanism in MO formation was identified. We propose that the existence of mosaic mutations and not alterations of other heparan sulfate biosynthesis related genes play a significant role in the development of MO in patients who are tested negative for mutations in Exostosins. Show less

Multiple osteochondromas (MO) is an autosomal dominant disorder caused by germline mutations in EXT1 and/or EXT2. In contrast, solitary osteochondroma (SO) is nonhereditary. Products of the EXT gene are involved in heparan sulfate (HS) biosynthesis. In this study, we investigated whether osteochondromas arise via either loss of heterozygosity (2 hits) or haploinsufficiency. An in vitro three-dimensional chondrogenic pellet model was used to compare heterozygous bone marrow-derived mesenchymal stem cells (MSCs EXT(wt/-)) of MO patients with normal MSCs and the corresponding tumor specimens (presumed EXT(-/-)). We demonstrated a second hit in EXT in five of eight osteochondromas. HS chain length and structure, in vitro chondrogenesis, and EXT expression levels were identical in both EXT(wt/-) and normal MSCs. Immunohistochemistry for HS, HS proteoglycans, and HS-dependent signaling pathways (eg, TGF-β/BMP, Wnt, and PTHLH) also showed no differences. The cartilaginous cap of osteochondroma contained a mixture of HS-positive and HS-negative cells. Because a heterozygous EXT mutation does not affect chondrogenesis, EXT, HS, or downstream signaling pathways in MSCs, our results refute the haploinsufficiency theory. We found a second hit in 63% of analyzed osteochondromas, supporting the hypothesis that osteochondromas arise via loss of heterozygosity. The detection of the second hit may depend on the ratio of HS-positive (normal) versus HS-negative (mutated) cells in the cartilaginous cap of the osteochondroma. Show less

Primary cilia are specialized cell surface projections found on most cell types. Involved in several signaling pathways, primary cilia have been reported to modulate cell and tissue organization. Although they have been implicated in regulating cartilage and bone growth, little is known about the organization of primary cilia in the growth plate cartilage and osteochondroma. Osteochondromas are bone tumors formed along the growth plate, and they are caused by mutations in EXT1 or EXT2 genes. In this study, we show the organization of primary cilia within and between the zones of the growth plate and osteochondroma. Using confocal and electron microscopy, we found that in both tissues, primary cilia have a similar formation but a distinct organization. The shortest ciliary length is associated with the proliferative state of the cells, as confirmed by Ki-67 immunostaining. Primary cilia organization in the growth plate showed that non-polarized chondrocytes (resting zone) are becoming polarized (proliferating and hypertrophic zones), orienting the primary cilia parallel to the longitudinal axis of the bone. The alignment of primary cilia forms one virtual axis that crosses the center of the columns of chondrocytes reflecting the polarity axis of the growth plate. We also show that primary cilia in osteochondromas are found randomly located on the cell surface. Strikingly, the growth plate-like polarity was retained in sub-populations of osteochondroma cells that were organized into small columns. Based on this, we propose the existence of a mixture ('mosaic') of normal lining (EXT(+/-) or EXT(wt/wt)) and EXT(-/-) cells in the cartilaginous cap of osteochondromas. Show less

Histologic grade is currently the best predictor of clinical course in chondrosarcoma patients. Grading suffers, however, from extensive interobserver variability and new objective markers are needed. Hence, we have investigated DNA copy numbers in chondrosarcomas with the purpose of identifying markers useful for prognosis and subclassification. The overall pattern of genomic imbalances was assessed in a series of 67 chondrosarcomas using array comparative genomic hybridization. Statistical analyses were applied to evaluate the significance of alterations detected in subgroups based on clinical data, morphology, grade, tumor size, and karyotypic features. Also, the global gene expression profiles were obtained in a subset of the tumors. Genomic imbalances, in most tumors affecting large regions of the genome, were found in 90% of the cases. Several apparently distinctive aberrations affecting conventional central and peripheral tumors, respectively, were identified. Although rare, recurrent amplifications were found at 8q24.21-q24.22 and 11q22.1-q22.3, and homozygous deletions of loci previously implicated in chondrosarcoma development affected the CDKN2A, EXT1, and EXT2 genes. The chromosomal imbalances in two distinct groups of predominantly near-haploid and near-triploid tumors, respectively, support the notion that polyploidization of an initially hyperhaploid/hypodiploid cell population is a common mechanism of chondrosarcoma progression. Increasing patient age as well as tumor grade were associated with adverse outcome, but no copy number imbalance affected metastasis development or tumor-associated death. Despite similarities in the overall genomic patterns, the present findings suggest that some regions are specifically altered in conventional central and peripheral tumors, respectively. Show less

The tumor suppressor genes EXT1 and EXT2 are involved in the formation of multiple osteochondromas, which can progress to become secondary peripheral chondrosarcomas. The most common chondrosarcoma subtype is primary central chondrosarcoma, which occurs in the medullar cavity of bone. The EXT1/EXT2 protein complex is involved in heparan sulfate proteoglycan (HSPG) biosynthesis, which is important for signal transduction of Indian hedgehog (IHH), WNT, and transforming growth factor (TGF)-beta. The role of EXT and its downstream targets in central chondrosarcomas is currently unknown. EXT1 and EXT2 were therefore evaluated in central chondrosarcomas at both the DNA and mRNA levels. Immunohistochemistry was used to assess HSPG (CD44v3 and SDC2), WNT (beta-catenin), and TGF-beta (PAI-1 and phosphorylated Smad2) signaling, whereas IHH signaling was studied both by quantitative polymerase chain reaction and in vitro. mRNA levels of both EXT1 and EXT2 were normal in central chondrosarcomas; genomic alterations were absent in these regions and in 30 other HSPG-related genes. Although HSPGs were aberrantly located (CD44v3 in the Golgi and SDC2 in cytoplasm and nucleus), this was not caused by mutation. WNT signaling negatively correlated with increasing histological grade, whereas TGF-beta positively correlated with increasing histological grade. IHH signaling was active, and inhibition decreased cell viability in one of six cell lines. Our data suggest that, despite normal EXT in central chondrosarcomas, HSPGs and HSPG-dependent signaling are affected in both central and peripheral chondrosarcomas. Show less

Multiple osteochondromas (MO) is characterised by development of two or more cartilage capped bony outgrowths (osteochondromas) of the long bones. The prevalence is estimated at 1:50,000, and it seems to be higher in males (male-to-female ratio 1.5:1). Osteochondromas develop and increase in size in the first decade of life, ceasing to grow when the growth plates close at puberty. They are pedunculated or sessile (broad base) and can vary widely in size. The number of osteochondromas may vary significantly within and between families, the mean number of locations is 15-18. The majority are asymptomatic and located in bones that develop from cartilage, especially the long bones of the extremities, predominantly around the knee. The facial bones are not affected. Osteochondromas may cause pain, functional problems and deformities, especially of the forearm, that may be reason for surgical removal. The most important complication is malignant transformation of osteochondroma towards secondary peripheral chondrosarcoma, which is estimated to occur in 0.5-5%. MO is an autosomal dominant disorder and is genetically heterogeneous. In almost 90% of MO patients germline mutations in the tumour suppressor genes EXT1 or EXT2 are found. The EXT genes encode glycosyltransferases, catalyzing heparan sulphate polymerization. The diagnosis is based on radiological and clinical documentation, supplemented with, if available, histological evaluation of osteochondromas. If the exact mutation is known antenatal diagnosis is technically possible. MO should be distinguished from metachondromatosis, dysplasia epiphysealis hemimelica and Ollier disease. Osteochondromas are benign lesions and do not affect life expectancy. Management includes removal of osteochondromas when they give complaints. Removed osteochondromas should be examined for malignant transformation towards secondary peripheral chondrosarcoma. Patients should be well instructed and regular follow-up for early detection of malignancy seems justified. For secondary peripheral chondrosarcoma, en-bloc resection of the lesion and its pseudocapsule with tumour-free margins, preferably in a bone tumour referral centre, should be performed. Show less

Multiple osteochondromas is a hereditary syndrome that is characterized by the formation of cartilage-capped bony neoplasms (osteochondromas), for which exostosis (multiple)-1 (EXT1) has been identified as a causative gene. However, 85% of all osteochondromas present as solitary (nonhereditary) lesions in which somatic mutations in EXT1 are extremely rare, but loss of heterozygosity and clonal rearrangement of 8q24 (the chromosomal locus of EXT1) are common. We examined whether EXT1 might act as a classical tumor suppressor gene for nonhereditary osteochondromas. Eight nonhereditary osteochondromas were subjected to high-resolution array-based comparative genomic hybridization (array-CGH) analysis for chromosome 8q. The array-CGH results were validated by subjecting tumor DNA to multiple ligation-dependent probe amplification (MLPA) analysis for EXT1. EXT1 locus-specific fluorescent in situ hybridization (FISH) was performed on nuclei isolated from the three tissue components of osteochondroma (cartilage cap, perichondrium, bony stalk) to examine which parts of the tumor are of clonal origin. Array-CGH analysis of tumor DNA revealed that all eight osteochondromas had a large deletion of 8q; five tumors had an additional small deletion of the other allele of 8q that contained the EXT1 gene. MLPA analysis of tumor DNA confirmed these findings and identified two additional deletions that were smaller than the limit of resolution of array-CGH. FISH analysis of the cartilage cap, perichondrium, and bony stalk showed that these homozygous EXT1 deletions were present only in the cartilage cap of osteochondroma. EXT1 functions as a classical tumor suppressor gene in the cartilage cap of nonhereditary osteochondromas. Show less

Mutational inactivation of EXT1 or EXT2 is the cause of hereditary multiple osteochondromas. These genes function in heparan sulphate proteoglycan (HSPG) biosynthesis in the Golgi apparatus. Loss of heterozygosity of the EXT1 locus at 8q24 is frequently found in solitary osteochondromas, whereas somatic mutations are rarely found. We investigated the expression of EXT1 and EXT2 (quantitative RT-PCR) and of different HSPGs (immunohistochemistry) in solitary and hereditary osteochondromas and in cases with malignant progression to secondary peripheral chondrosarcoma, in relation to possible mutations and promoter methylation. The mutation status of patients with multiple osteochondromas correlated with decreased EXT1 or EXT2 expression found in their resected tumours. We could not show somatic point mutations or promoter hypermethylation in 17 solitary tumours; however, EXT1 expression was decreased in 15 cases, whereas EXT2 was not. Intracellular accumulation of syndecan-2 and heparan sulphate-bearing isoforms of CD44 (CD44v3) was found in most tumours, which concentrated in the Golgi apparatus as shown by confocal microscopy. This contrasted with the extracellular expression found in normal growth plates. In conclusion, mutational inactivation of either EXT1 or EXT2 leads to loss of mRNA expression of the corresponding gene. We hypothesize that loss of EXT expression disrupts the function of the EXT1/2 complex in HSPG biosynthesis, resulting in the intracellular accumulation of HSPG core proteins that we found in these tumours. Show less

Multiple Osteochondromas is an autosomal dominant disorder characterised by the presence of multiple osteochondromas and a variety of orthopaedic deformities. Two genes causative of Multiple Osteochondromas, Exostosin-1 (EXT1) and Exostosin-2 (EXT2), have been identified, which act as tumour suppressor genes. Osteochondroma can progress towards its malignant counterpart, secondary peripheral chondrosarcoma and therefore adequate follow-up of Multiple Osteochondroma patients is important in order to detect malignant transformation early.This review summarizes the considerable recent basic scientific and clinical understanding resulting in a multi-step genetic model for peripheral cartilaginous tumorigenesis. This enabled us to suggest guidelines for clinical management of Multiple Osteochondroma patients. When a patient is suspected to have Multiple Osteochondroma, the radiologic documentation, histology and patient history have to be carefully reviewed, preferably by experts and if indicated for Multiple Osteochondromas, peripheral blood of the patient can be screened for germline mutations in either EXT1 or EXT2. After the Multiple Osteochondroma diagnosis is established and all tumours are identified, a regular follow-up including plain radiographs and base-line bone scan are recommended. Show less

A 40 year old man with hereditary multiple exostoses (HME), affecting predominantly his left proximal tibia, distal femur, and proximal femur, underwent resection of an osteochondroma near the trochanter major of his left proximal femur because of malignant transformation of the cartilaginous cap towards secondary peripheral chondrosarcoma. The patient had a history of a papillary thyroid carcinoma four years previously. At examination of the resected specimen, a third malignant tumour, an intermediate grade osteosarcoma (grade II/IV), was found in the osseous stalk of the osteochondroma. Although no mutations were found in the EXT1 and EXT2 genes, the genes involved in HME, or in exons 5-8 of the p53 gene, the development of three malignancies before the age of 40 suggests that this patient is genetically prone to malignant transformation. Show less

Hereditary multiple exostoses is an autosomal dominant disorder characterised by the presence of multiple osteochondromas, resulting in a variety of skeletal deformities. It is a genetically heterogeneous condition for which two genes, EXT1 and EXT2, have been isolated. The EXT1 gene, located at 8q24, has been shown to harbour mutations in 44-66% of the hereditary multiple exostoses-families. Mutations in the EXT2 gene, located at 11p11-p12, are detected in about 30% of the families. Additional linkage to chromosome 19p suggests the existence of an EXT3 gene. EXT1 has been shown to act as a tumour suppressor gene in hereditary multiple exostoses, resulting in osteochondroma formation when both copies of EXT1 are lost. Diagnostic germ-line mutation analysis is operative in the Clinical Genetic Center Leiden, the Netherlands. Show less

Chondrosarcomas are malignant cartilage-forming tumors arising centrally in bone (central chondrosarcoma) or within the cartilaginous cap of osteochondroma (peripheral chondrosarcoma). For hereditary multiple osteochondromas, two responsible genes, EXT1 and EXT2, have been cloned. Their recently elucidated role in heparan sulfate biosynthesis and Hedgehog diffusion leads to the hypothesis that EXT inactivation affects fibroblast growth factor (FGF) and Indian Hedgehog (IHh)/parathyroid hormone-related peptide (PTHrP) signaling, two important pathways in chondrocyte proliferation and differentiation. The expression of PTHrP, PTHrP-receptor, Bcl-2, FGF2, FGFR1, FGFR3, and p21 is investigated by immunohistochemistry in osteochondromas (n = 24) and peripheral (n = 29) and central (n = 20) chondrosarcomas. IHh/PTHrP and FGF signaling molecules are mostly absent in osteochondromas. Although no somatic EXT mutations were found in sporadic osteochondromas, the putative EXT downstream targets are affected similarly in sporadic and hereditary tumors. In chondrosarcomas, re-expression of FGF2, FGFR1, PTHrP, Bcl-2, and p21 is found. Expression levels increase with increasing histological grade. Up-regulation of PTHrP and Bcl-2 characterizes malignant transformation of osteochondroma because PTHrP and Bcl-2 expression is significantly higher in borderline and grade I peripheral chondrosarcomas compared with osteochondromas. In contrast, up-regulation of PTHrP and Bcl-2 seems to be a late event in central cartilaginous tumorigenesis because expression is mainly restricted to high-grade central tumors. Show less

Osteochondromas occur as sporadic solitary lesions or as multiple lesions, characterizing the hereditary multiple exostoses syndrome (EXT). Approximately 15% of all chondrosarcomas arise within the cartilaginous cap of an osteochondroma. EXT is genetically heterogeneous, and two genes, EXT1 and EXT2, located on 8q24 and 11p11-p12, respectively, have been cloned. It is still unclear whether osteochondroma is a developmental disorder or a true neoplasm. Furthermore, it is unclear whether inactivation of both alleles of an EXT gene, according to the tumor-suppressor model, is required for osteochondroma development, or whether a single EXT germline mutation acts in a dominant negative way. We therefore studied loss of heterozygosity and DNA ploidy in eight sporadic and six hereditary osteochondromas. EXT1- and EXT2-mutation analysis was performed in a total of 34 sporadic and hereditary osteochondromas and secondary peripheral chondrosarcomas. We demonstrated osteochondroma to be a true neoplasm, since aneuploidy was found in 4 of 10 osteochondromas. Furthermore, LOH was almost exclusively found at the EXT1 locus in 5 of 14 osteochondromas. Four novel constitutional cDNA alterations were detected in exon 1 of EXT1. Two patients with multiple osteochondromas demonstrated a germline mutation combined with loss of the remaining wild-type allele in three osteochondromas, indicating that, in cartilaginous cells of the growth plate, inactivation of both copies of the EXT1 gene is required for osteochondroma formation in hereditary cases. In contrast, no somatic EXT1 cDNA alterations were found in sporadic osteochondromas. No mutations were found in the EXT2 gene. Show less