Deep Exome Sequencing to Identify the Gene Causing Solitary |
An ESUN Article
Background
Chondrosarcoma is the second most frequent malignant bone tumour that forms cartilage. Adults in the third to sixth decade are affected (1). Chondrosarcoma is unique amongst mesenchymal tumours since it has benign precursor lesions, may progress from low- to high grade malignant and may occur in a syndrome. Our previous studies have shown that cartilage tumours arising at the surface of bone should be regarded separately from those arising in the medulla (2) (Table 1), although their histology is very similar. Central chondrosarcoma is most common (1;3) and may be secondary to an enchondroma (4). Enchondromas usually present in the long bones in the third or fourth decade (4). Malignant transformation towards central chondrosarcoma occurs in <1% of solitary enchondromas.
| Central | Peripheral | |
|---|---|---|
| Location | Medulla of the bone | Surface of the bone |
| Benign precursor | Enchondroma | Osteochondroma |
| Malignant form | Conventional central chondrosarcoma | Secondary peripheral chondrosarcoma |
| Frequency | 85% | 15% |
| Multiple tumors | Enchondromatosis (Ollier disease, Maffucci syndrome) | Multiple Osteochondromas |
| Risk of malignant transformation in case of multiple tumours | Up to 35% | 1-5% |
| Mode of inheritance | Non-hereditary | Autosomal dominant |
| Causative gene | Unknown | EXT1, EXT2 |
While most enchondromas are solitary, some patients demonstrate multiple enchondromas with a marked unilateral predominance particularly affecting the limbs (5;6), known as Ollier disease or Maffucci syndrome, the latter of which combines multiple enchondromas with soft tissue vascular lesions. The risk of malignant transformation is increased (10-35%) (5). In 2002 a mutation was reported in the Parathyroid Hormone Receptor type I (PTHR1) gene in 2 of 6 patients with Ollier disease (7). However, in a large multi-institutional series of 31 patients we failed to detect any mutations (8). Moreover, three additional mutations were found in PTHR1 in tumors from 3 of 28 Ollier patients reducing the function of PTHR1 to ~70% (9). Therefore, PTHR1 may contribute to Ollier disease in 8% of the cases, but is probably not causative.
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Cartilage Tumors
Enchondroma: benign tumor in the center of the bone consisting of cartilage
Central chondrosarcoma: malignant tumor in the center of the bone that still resembles cartilage
Ollier disease: rare congenital disorder in which patients develop multiple enchondromas usually during childhood, leading to deformity. The disease usually affects one side of the body more severe than the other side. There is a 35% risk that the enchondromas turn into malignant chondrosarcomas.
Approximately 15% (3) of chondrosarcomas (secondary (peripheral) chondrosarcoma) are located at the surface of bone and result from malignant transformation of osteochondroma (1). Osteochondroma is a mostly asymptomatic cartilage capped bony outgrowth representing ~35% of all primary benign bone tumours (10). Malignant transformation towards peripheral chondrosarcoma is low (<1% of cases). Although peripheral chondrosarcoma is far less common, many of the active signalling pathways in chondrosarcoma have been identified after the initial genetic event was elucidated. Osteochondromas can occur in multiple osteochondromas (MO) syndrome, an autosomal dominant disorder characterized by mutations in EXT1 or EXT2. The EXT gene products are involved in heparan sulphate biosynthesis, essential for the diffusion of hedgehog proteins. Both enchondromas and osteochondromas were shown to have active hedgehog signalling (11;12). However, EXT is not involved in central chondrosarcoma (13). We expect to further unravel the initiating event for enchondroma and central chondrosarcoma by studying Ollier disease as a model, similar to Multiple Osteochondromas elucidating many of the pathways involved in peripheral chondrosarcoma (table 1).
Previous Studies by our Group
In the past, we could not detect any significant differences between solitary and Ollier disease related chondrosarcomas at the molecular level in a pilot study using cDNA microarray and high-resolution array-CGH (14;15). Molecules involved in IHH/PTHLH signalling are expressed at similar levels (8;16). Therefore, solitary and Ollier disease-related chondrosarcomas seem similar justifying the study of Ollier disease as a model to understand central chondrosarcoma development. To study the genetic background of Ollier disease we performed SNP analysis using Affymetrix SNP6.0 on 15 enchondromas and 24 chondrosarcomas of different grades from 30 Ollier patients and normal DNA from 12 Ollier patients for paired comparison collected within the EuroBoNeT Network of Excellence (17). We studied tumour tissue since we hypothesized that Ollier disease is a mosaic condition, since it affects multiple bones with an often unilateral predominance. All samples were divided into three groups: normals, enchondromas and chondrosarcomas. Non-recurrent EC specific copy number alterations were found at FAM86D, PRKG1and ANKS1B. LOH with copy number loss of chromosome 6 was found in two ECs from two unrelated Ollier patients. One of these patients also had LOH at chromosome 3. However, no common genomic alterations were found for all ECs. Using an integration approach of SNP and expression array we identified loss as well as down regulation of POU5F1 and gain as well as up regulation of NIPBL. None of these candidate regions were affected in more than two Ollier patients suggesting these changes to be random secondary events in EC development. An increased number of genetic alterations and LOH were found in Ollier CS which mainly involves chromosomes 9p, 6q, 5q and 3p. In summary the absence of common copy number variations or loss of heterozygosity suggests that instead point mutations or epigenetic mechanisms seem to play a role in the origin of Ollier disease (17). Mutation analysis revealed absence of the reported G121E, A122T, R150C and R255H variations in PTHR1 in our series.
Hypotheses
1. Ollier disease is a mosaic condition.
The fact that in most patients only certain parts of the body are affected and that the disease is not inherited from the parents suggest that the as far unknown etiological factor affects the limb buds during early foetal life. An early postzygotic mutation resulting in asymmetric involvement of skeletal structures can be expected, as was shown for polyostotic fibrous dysplasia (18). This putative mosaicism indicates that the cause of Ollier disease is to be found by studying tumour tissue.
2. Ollier disease is caused by a point mutation.
Our preliminary results show absence of loss of heterozygosity in enchondromas from Ollier disease which suggests that the classical tumour suppressor model as found in Multiple Osteochondromas (19) does not apply to Ollier disease. Therefore, similar to fibrous dysplasia a single somatic point mutation probably causes the disease.
3. The same gene(s) is / are responsible for Ollier disease-related and solitary cartilage tumours.
We showed Ollier disease-related and solitary tumours to be similar at the DNA (20) and expression level (8;14;16). We therefore expect that the same gene is involved, in which the mutation in solitary chondrosarcomas occurs somatically, later in life. Thus, genes involved in central chondrosarcomagenesis are expected to be found by studying Ollier disease.
Purpose
The aim of the proposed studies is to identify the gene(s) causing solitary as well as Ollier disease related enchondroma and chondrosarcoma.
Research Plan and Experimental Design
We will start by deep exome sequencing of at least 3 Ollier related hand enchondromas and patient matched normal tissue, followed by three more pairs (see below). Normal DNA was isolated from blood or saliva, obtained after informed consent. Samples were coded and all procedures were performed according to the ethical guidelines “Code for Proper Secondary Use of Human Tissue in The Netherlands” (Dutch Federation of medical Scientific Societies). Patients are very well characterized (see preliminary results). We choose to start with hand enchondromas to have a homogeneous group, since our preliminary data show a difference in clinical behaviour in addition to minor differences in copy number alterations between small – and long bones enchondromas. Since we expect Ollier disease to be a mosaic condition, comparable to polyostotic fibrous dysplasia, an early somatic mutation is expected with mutated cells spread throughout the body. Tumor tissue however, is expected to be enriched for the cells containing the mutation. A coverage of 30-50 deep is needed to find variants in the mosaic condition (21). This is why exome sequencing is favored over whole genome sequencing.
This technique requires 1 microgram of DNA which is available for most of the tumours previously analyzed by SNP-array (see preliminary results). It is estimated that within a tumor several hundred changes occur in the DNA (22). Results of the tumour will therefore be compared to germline DNA from the same patient. In addition, it is necessary to combine the results of a number of patients or tumors to identify the driver changes and filter out bystander changes. Therefore, after the initial bio-informatical analysis of the 3 tumor-normal pairs an evaluation is made. If the candidate list is already very short, a set of 3 additional long bone enchondromas will be analyzed to find whether the difference in biological behavior can be attributed to genetic differences. If the candidate list is still long, 3 more hand tumors will be analyzed in order to strengthen the evidence. Since we hypothesize the disease to be germline mosaic, the inclusion of surrounding normal tissue will in this case be considered, if available.
The experiments will be performed at the Leiden Genome Technology Centre (LGTC) using the Illumina Genome Analyzer II or HiSeq 2000. Candidate genes will be verified using DNA of the larger cohort that is available and well documented ((frozen tissue n=~40, paraffin n=~70) see preliminary results), as well as of our series of solitary central chondrosarcomas (13;23). In addition, expression of the (candidate) gene(s) can be studied using available tissue microarrays (TMA) for Ollier tumours. A tissue microarray was previously constructed containing 66 tumours from 44 patients with Ollier disease, 24 solitary tumours, and 12 tumours from 7 patients with Maffucci syndrome. In addition, recently constructed TMAs containing 100 solitary conventional central chondrosarcomas can be used, in case antibodies are available for immunohistochemistry. This will be the starting point for additional functional studies.
Deep exome sequencing is a form of second generation sequencing. The second generation of sequencers can determine the base-order of millions of fragments of DNA, with lengths between 50 and 250 bases per fragment. A single run on an Illumia Genome Analyzer II generates 2 to 3 billion bases of sequence. This is in contrast with the first generation of sequencers, such as used for the human genome project, that can determine the base order of a single fragment of DNA with a length of a few hundred bases. The exome is the part of the DNA that actually codes for proteins. In humans this is about 2% of the DNA. In exome sequencing the exome DNA is separated from the rest of the DNA from a sample and then tested in the analyzer. This allows the 50 fold sequencing of each piece of coding DNA, and the identification of changes that are present in only a subset of the cells.
Impact and Clinical Relevance
Identification of the gene causing Ollier disease is expected to also play a role in the origin of the most common chondrosarcoma subtype, conventional central chondrosarcoma. By elucidating the initial causative event we will better understand central chondrosarcoma development, similar to recent advances for EXT and its role in peripheral cartilaginous tumours (24). In addition, identification of the exact pathways causing the disease may provide new targets for preventive (in case of Ollier disease) or therapeutic (in case of conventional chondrosarcoma) strategies. Since chondrosarcomas are resistant to conventional chemo- and radiotherapy there is nothing curative to offer patients with irresectable or metastatic disease, and new treatment strategies are therefore urgently required.
Conclusion
Using a whole exome sequencing approach of enchondromas in patients affected by Ollier disease, we intend to elucidate the underlying genetic event causing the disease which we expect to be mosaic. Since Ollier related and solitary chondrosarcomas share many similarities, it is expected that the same gene underlies the development of solitary central chondrosarcoma. By elucidating the initiating genetic event driving chondrosarcoma development the pathways crucial for tumour growth may be identified and may serve as target for preventive or therapeutic strategies against chondrosarcoma.
Editor's Note: This study is funded by a $50,000 grant from the Liddy Shriver Sarcoma Initiative.
References
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