Molecular pathology and its diagnostic use in bone tumors

Cancer Genetics 205 (2012) 193e204

REVIEW

Molecular pathology and its diagnostic use in bone tumors Karoly Szuhai a, Anne-Marie Cleton-Jansen b, Pancras C.W. Hogendoorn b, e b,* Judith V.M.G. Bove a

Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands; b Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands Bone tumors are considered by most pathologists difficult to diagnose as they are rare, have overlapping morphology, need radiological correlation, and the usefulness of immunohistochemistry is limited, making conventional morphology the cornerstone of the diagnosis. Over the past decade, more and more has become known of the molecular background of bone tumors. Three groups of bone tumors are recognized, namely, tumors with specific translocations combined with a relatively simple karyotype involving chromosomal translocations (Ewing sarcoma, aneurysmal bone cyst), tumors with specific gene mutations or amplifications (chondrosarcoma, fibrous dysplasia, chordoma), and sarcomas with genetic instability and as a consequence complex karyotypes (osteosarcoma). Technical advancements will rapidly reveal new alterations in the more rare sarcoma subtypes for which the molecular background has remained enigmatic. Opening the archives and using new technologies, as well as refinement of existing technologies for decalcified paraffin-embedded tissue, may bring to light more specific genetic aberrations in bone tumors that can be applied in molecular diagnostics in the near future. Keywords Bone tumor, molecular pathology, molecular diagnostics ª 2012 Elsevier Inc. All rights reserved.

Bone tumors are rare, and pathologists often find their classification difficult. An accurate diagnosis is essential to predict biological behavior and for therapeutic decision making. Over the past two decades, considerable progress has been made in our understanding of the genetic background of cancer. Genetic changes, for instance chromosomal translocations or gene mutations, give the cells in which they arise a growth advantage, ultimately leading to tumor formation. For different bone and soft tissue tumors, more and more genetic data have become available. The detection of specific genetic changes has found its way into routine diagnostics at pathology laboratories. Unlike soft tissue tumors, where the classification is aided by immunohistochemistry to confirm the line of differentiation, immunohistochemistry in cases of primary bone tumors is less helpful in discriminating among the different histological types. Thus, molecular diagnosis is playing an increasingly important role as an additional approach to classification. Although traditional morphological evaluation remains at the Received March 1, 2012; received in revised form March 30, 2012; accepted April 4, 2012. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergen.2012.04.001

cornerstone of bone tumor diagnosis, molecular diagnostic assays can increase the accuracy of the diagnosis and assist in subtyping bone tumors. The types of changes found in sarcomas can be roughly divided into three groups: sarcomas with specific translocations combined with a relatively simple karyotype (Ewing sarcoma, aneurysmal bone cyst) (Table 1), tumors with specific gene mutations or amplifications (chondrosarcoma, fibrous dysplasia, chordoma), and sarcomas with genetic instability and, as a consequence, complex karyotypes (osteosarcoma). However, the distinction is slightly artificial, since tumors with specific translocations or gene mutations as a relatively early event obtain secondary genetic alterations such as TP53 mutations or CDKN2A/B deletions that may lead to complex karyotypes and are relevant for prognosis. Almost 100% of Ewing sarcomas and almost 70% of primary aneurysmal bone cysts carry a translocation involving the EWSR1 or the USP6 (TRE17) gene, respectively. The fusion genes resulting from the balanced translocations subsequently may act as aberrant transcription factors that induce expression of other genes that cause uncontrolled proliferation, as is the case for the EWSR1-FLI1 fusion gene in Ewing sarcoma. Alternatively, one of the genes involved in

194 Table 1

K. Szuhai et al. Specific genetic aberrations in bone tumors

Neoplasm Specific translocations Ewing sarcoma/PNET

Ewing sarcoma-like tumors without ETS involvement

Aneurysmal bone cyst

Mesenchymal chondrosarcoma Myoepithelial tumor of bone

Epithelioid hemangioendothelioma Bizarre parosteal osteochondromatous proliferation (Nora lesion) Subungual exostosis Specific amplifications/gains Chordoma Low grade (parosteal and intramedullary) osteosarcoma Specific gene mutations Fibrous dysplasia Enchondroma, central and periosteal chondrosarcoma Osteochondroma

Translocation

Involved gene(s)

t(11;22)(q24;q12) t(21;22)(q22;q12) t(7;22)(p22;q12) t(17;22)(q12;q12) t(2;22)(q33;q12) t(16;21)(p11;q22) t(2;16)(q35;p11) t(6;22)(p21;q12) inv(22)(q12) t(4;22)(q31;q12) t(2;22)(q31;q12) t(1;22)(p36.1;q12) Complex ring chromosome with amplification of the translocated segments t(16;17)(q22;p13) t(1;17)(p34.1e34.3;p13) t(3;17)(q21;p13) t(9;17)(q22;p13) t(17;17)(q12;p13) NA t(6;22)(p21;q12) t(19;22)(q13;q12) t(1;22)(q23;q12) t(1;3)(p36;q25) t(1;17)(q32;q21)

EWSR1-FLI1 EWSR1-ERG EWSR1-ETV1 EWSR1-ETV4 EWSR1-FEV FUS-ERG FUS-FEV EWSR1-POU5F1 EWSR1-ZSG (ZNF278) EWSR1-SMARCA EWSR1-SP3 EWS-ZNF278 EWSR1-NFATc2 CDH11-USP6 (Tre2) TRAP150-USP6 ZNF9-USP6 OMD-USP6 COL1A1-USP6 HEY1-NCOA2 EWSR1-POU5F1 EWSR1-ZNF444 EWSR1-PBX1 WWTR1-CAMTA1 RDC1

t(X;6)(q24-q26;q15-21)

COL12A1-COL4A5

6q27 12q14-15 (ring/marker chromosome)

T CDK4, MDM2, HMGA2, GLI, SAS

GNAS IDH1, IDH2 EXT1, EXT2

Abbreivation: NA, the cytogenetic alteration was not assigned.

the translocation is under control of the other gene involved in the translocation, which usually leads to overexpression, as is the case for USP6 overexpression in aneurysmal bone cysts. Genomic instability and the presence of a complex karyotype are the landmarks of osteosarcoma and high grade chondrosarcoma. It was always assumed that a high number of translocations present in carcinoma were not important for carcinoma pathogenesis and changes are secondary because of genomic instability. Recurrent translocations, due to the complexity of the chromosomal changes, could not be demonstrated cytogenetically. However, the identification of a high incidence (79%) of gene fusions in prostate cancer (1) suggested that also in bone tumors more of these cryptic chromosomal aberrations may be present. Recently, two novel recurrent fusion products were identified: the HEY1NCOA2 fusion in mesenchymal chondrosarcoma (2) and the WWTR1-CAMTA1 fusion resulting from the t(1;3)(p36;q25) translocation in epithelioid hemangioendothelioma of various sites, including bone (3e5).

In this review, we discuss the different techniques that can be used to detect genetic alterations in bone tumors, and we discuss some of the entities illustrating the different genetic subtypes of bone tumors.

Molecular techniques used to detect cytogenetic changes Genome-wide screening Cytogenetics (classical banding and multicolor fluorescence in situ hybridization based) Classical cytogenetic studies, using metaphases harvested from growing cells, were important tools in the discovery of recurrent chromosomal alterations. Karyotyping provides a genome-wide screen for structural and numerical alterations that are associated with tumorigenesis. These tests are well suited for the detection of unexpected alterations

Molecular pathology in bone tumors that in turn could lead to the identification of characteristic, tumor-associated chromosomal rearrangements. Cases with recurrent translocations have been used to identify breakpoint-spanning genes. The two major limitations of metaphase-based karyotyping are (i) the need for vital, dividing cells, and (ii) the limited resolution of the detection. In several clinical settings, fresh, vital specimens are not available for testing, precluding karyotyping. For most solid tumors, the complexity of the chromosomal changes and the lesser quality of the metaphase chromosomes hamper the identification of all involved chromosomal segments. In a recent study, it was shown that the use of a core-needle biopsy sample is superior to the use of a fine-needle biopsy sample when pretreatment resection specimens are not available for culture (6). The introduction of multicolor fluorescence in situ hybridization (M-FISH)ebased karyotyping, using specific whole-chromosome paint probe sets for each chromosome identified by a distinctly colored label has improved the detection sensitivity of chromosomal rearrangements, for example combined binary ratio labeling (COBRA)-FISHebased karyotyping (7). However, as these tests are expensive and require specific instruments, it has never become a widely introduced tool in the diagnostic setting. For diagnostics, however, for most of the identified translocation types, specific tests (FISH, or reverse transcriptionepolymerase chain reaction [RT-PCR], as discussed in a subsequent section) have been developed. These tests, next to the high sensitivity and specificity, can be applied on archival material. Alternatively, the highly selective nature of these tests (FISH, RT-PCR) precludes the detection of tumor-associated secondary changes that might be relevant for prognosis. Array comparative genomic hybridization During the last decade, array comparative genomic hybridization (array CGH) has become a powerful technique to detect genomic alterations as a result of genomic gain or loss. This technique has been successfully applied both in constitutional (germline) and acquired (tumor-associated) conditions. For routine application, parameters such as the resolution of an array test (i.e., the number of reporter elements present on a chip), the quality of a sample (degraded, fresh), and the purity of the sample (percentage of tumor cells) are important factors. The broadly used array platforms are designed for genome-wide scanning of alterations and therefore provide only limited information on alterations in individual genes at the exon-intron level. To overcome this limitation, custom-designed, oligonucleotidebased array CGH approaches were used. The detection and mapping of exon-sized genomic alterations of target genes both in acquired and constitutional genetic conditions was attainable (8,9). Through novel technical advances, the analysis of DNA extracted from decalcified, formalin-fixed, paraffin-embedded bone tissue became possible (10). We await the direct diagnostic application of the array CGH approach, but for the detection of copy number alterations, especially when multiple loci might be involved or the size of the alterations might be variable, the array CGH approach might be better suited than FISH-based approaches. It has been shown that homozygous deletion of the CDKN2A/2B region has negative prognostic value in various tumor types. The routinely used FISH probe sets showed contradictory

195 results in some instances. Microdeletion covering a fraction of the genomic regions covered by the FISH probes might be responsible for false negativity. However, as these deletions can be smaller (5e10 kb) than the actual probe size (90e150 kb), a microdeletion might remain undetected using this approach. Alternative techniques, such as array CGH or multiplex ligation-dependent probe amplification (MLPA), have been shown to readily detect microdeletions (11,12). The advance of such approaches has already been shown in constitutional genetic applications (common microdeletion and duplication syndrome genomic regions). Moreover, a balanced translocation also will not be detected using array CGH.

Targeted detection Translocation detection The detection of specific translocations not only is important for confirmation of the diagnosis as suggested by conventional morphology, but is especially important in those cases with unusual morphology or clinical presentation. FISH Specific translocations can be identified using FISH if the available probes overlap or flank the breakpoint. The presence of a translocation breakpoint then leads to the separation of the differentially labeled (typically a red/green combination) probe set (Figure 1A). Interphase FISH analysis is the primary choice if only formalin-fixed paraffin-embedded tissue is available. A huge advantage of FISH analysis is that the translocation can be ascertained in nuclei of non-dividing (interphase) cells, originating from fresh, frozen or formalinfixed paraffin embedded tumor material. Analysis using FISH is therefore a good choice when only formalin-fixed paraffin-embedded material is available. However, FISH is not always easy on paraffin material and can fail, especially if the material is decalcified in formic or acetic acid. FISH is considerably less sensitive to contamination than RT-PCR. A diagnostic test using a split-apart (breakapart) FISH probe set does not reveal the translocation partner. This might be of great relevance in tumor entities with the involvement of the EWSR1 gene. This locus is promiscuous and involved in a wide variety of bone and soft tissue tumors with unrelated clinical features. For the identification of the exact translocation partner auxiliary tests (co-localization probe set, or RT-PCR-based test as discussed in the next section) should be performed. The results of the FISH analysis should therefore always be interpreted within the histological and immunohistochemical context (Table 1, Figure 2). RT-PCR When fresh-frozen samples are available, reversetranscriptase PCR (RT-PCR) is often the primary choice. Most translocations result in the formation of a transcribed chimeric gene that can be detected using RT-PCR. For RTPCR, RNA is required, which can be isolated from frozen tumor samples. RNA isolation from formalin-fixed, paraffinembedded tissue in bone tumors can be severely hampered by decalcification procedures, and therefore sampling frozen tissue is advisable. The advantage of this technique is that it requires only small amounts of material, making it applicable

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Figure 1 Sarcoma-associated genomic changes can roughly be categorized into three major classes: (A) tumors with simple translocation, (B) tumors with point mutations (with or without secondary genetic changes), and (C) tumors with complex karyotypes without identified specific changes. (A) Typical histological appearance of Ewing sarcoma (small round blue cell tumor) using hematoxylineeosin (HE) staining (left panel). Ewing sarcoma is characterized by the presence of a balanced translocation between chromosome 11 and chromosome 22 (upper left part of the right panel) after COBRA-FISH karyotyping. The translocation leads to the disruption of the EWSR1 gene detected by a split-apart probe set bracketing the EWSR1 locus (red/green). Distantly located red-green signals indicate the presence a break within the EWSR1 locus in interphase cells (left bottom part of the right panel), or visible separation of the involved chromosomes in a metaphase. (B) Typical histological appearance of a central chondrosarcoma using HE staining (left panel). Partial sequence of IDH1 using Sanger sequencing detecting a R132C (c.394C>T) mutation, which is the most frequent hot spot mutation in chondrosarcoma. (C) Typical histological appearance of conventional osteosarcoma using HE staining with osteoid (dense, pink, amorphous intercellular material)(left panel). The image shows a COBRA-FISH karyotype of a conventional osteosarcoma case with a complex karyotype involving both numerical and structural alterations.

Molecular pathology in bone tumors

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Figure 2 An overview of translocation partnerships of the TET family of genes, EWSR1, FUS, TAF15, in various tumor entities. Translocation to various genes in the same gene family is believed to be pathognomonic to a given entity (e.g., EWSR1 translocation to members of the ETS family of transcription factors in Ewing sarcoma). Complementation by another TET member, FUS, has taken away this specificity and the presence of FUS/ERG translocation both in Ewing sarcoma and acute myeloid leukemia brings additional complications to molecular diagnostics. Similar shared translocation variants have been observed in clear cell sarcoma and angiomatoid fibrous histiocytoma. Arrows are pointing from the TET family translocation partners to translocation partners; members of a gene family are indicated with identical colors. Annotation on arrows indicates associated tumor entities. The asterisk ()) indicates myoepithelial tumors, which include soft tissue myoepithelioma and myoepithelial carcinoma.

to needle biopsies and aspirates. One should realize that fusion genes can be very heterogeneous; for instance, 18 different chimera transcripts have been described for EWSR1-FLI1 in Ewing sarcoma. This should be taken into

account when designing the primers. Sequencing of the detected product allows the identification of translocation variants at exon levels. New or rare transcripts can be missed with RT-PCR (Table 1, Figure 2).

198 Mutation detection Detection of nucleotide alterations in genes, and the methods for their detection, depends on whether they are recurrent and specific, or dispersed throughout the coding sequence. The most straightforward way to detect a sequence variation is the hotspot mutation approach, which is typically used to screen for single nucleotide substitutions in oncogenes. These mutations are limited in their variation, since they activate the gene in a dominant mechansim. The recently reported IDH1 hotspot mutation in chondrosarcoma is a typical example of a gene with a few unique nucleotide substitutions that are likely drivers of tumorigenesis (13,14). Specific nucleotide alterations can be detected with robust and extremely sensitive methods that address individual nucleotide changes even in patient material that is seriously contaminated with non-tumor tissue, such as stroma or inflammatory infiltrate. These methods include real-time PCR with hydrolysis probes (15) and pyrosequencing (16). Alternative approaches are mass spectrometry (www.sequenom. com) and KASPar genotyping (17). Each of these methods addresses a known nucleotide substitution, small deletion, or insertion. Screening for tumor suppressor gene inactivation often requires a much more elaborate procedure, including scanning of the entire sequence, since hot-spot mutations are less frequent. For some genes, hot-spot regions have been defined, such as for TP53. The so-called guardian of the genome is most commonly mutated in exons 5 to 8. The failure to detect mutations in the hot-spot exons necessitates screening of the remaining coding sequence. Pre-screening may be performed to identify the presence of sequence variations; high resolution melting curve analysis is the current method of choice (15). Once the presence of a variation has been identified, its exact nature can be determined with Sanger sequencing. The sensitivity of Sanger sequencing is lower than the methods discussed previously; therefore, it is important to make sure that the tumor tissue is not too heavily contaminated with non-neoplastic cells. Enrichment of tumor cells by micro- or macrodissection may be needed. Alternatively, next-generation sequencing can be applied, especially if many samples must be screened.

K. Szuhai et al. fusion to a member of the ETS family of transcription factors has been well established. In about 95% of cases, the gene fusion is EWSR1-FLI1 involving the N-terminal segment of the EWSR1 gene and the C-terminal segment of the FLI1 gene containing the ETS DNA-binding domain. In less than 5% of Ewing sarcoma cases, the fusion involves the ERG gene located on chromosome 21q22. Variant translocations t(2;22)(q33;q12), t(7;22)(p22;q12), and t(17;22)(q12;q12) with fusion of the EWSR1 gene to FEV, ETV1, or E1AF, respectively, have been reported (18). For EWSR1-FLI1 in Ewing sarcoma, 18 different chimera transcripts have been described, and it has been proposed that type I (EWSR1 exon 7/FLI1 exon 6) versus type II (EWSR1 exon 7/FLI1 exon 5) might be prognostically relevant (19). However, recent prospective studies have shown that fusion subtypes have no prognostic significance (20,21). Diagnostic RT-PCR tests have been designed to detect the two most frequent fusion types (EWSR1/FLI1 and EWSR1/ERG) leading to a falsenegative result in cases of rare translocation variants. The split-apart FISH probe approach permits the detection of all translocation variants involving the EWSR1 locus. Since its primary description, several other tumor entities, both soft tissue and bone related tumors, have been found to carry EWSR1 fusions with various partner genes. Moreover, Ewing sarcomas with “unusual” histomorphological characteristics, or atypical Ewing sarcomas, have been described with the involvement of fusion genes that are not members of the ETS family of transcription factors (Table 1, Figure 2). This finding emphasizes that conclusions cannot always be based on EWSR1 split-apart FISH tests, and FISH and RT-PCR negative cases should be tested for alternative translocation variants. Another example of this is the high prevalence of CIC fusion to DUX4 in small blue round cell tumor as a result of either a t(4;19) or t(10;19) (22,23). FISH results with atypical patterns, such as the amplification of the proximal EWSR1 region, may indicate the presence of translocation variants, as have been shown for Ewing sarcomaelike tumors for the EWSR1/NFATC2 translocation. So far, this Ewing sarcomalike tumor is the only entity involving the EWSR1 locus with a high level of amplification of the fusion gene (24). These recent developments further complicate molecular diagnostics of Ewing sarcoma.

Tumor entities Aneurysmal bone cyst Ewing sarcoma The diagnosis of small blue round cell tumors (SBRCT) can be a daunting task. The differential diagnosis is broad and includes Ewing sarcoma, small cell osteosarcoma, mesenchymal chondrosarcoma, lymphoma, metastases of alveolar rhabdomyosarcoma, poorly differentiated synovial sarcoma, myxoid/round cell liposarcoma, neuroblastoma, melanoma, or small cell carcinoma. The histological pattern in combination with immunohistochemistry may result in a conclusive diagnosis in several of the aforementioned entities. However, an atypical presentation regarding primary location or patient’s age, or atypical histology, or an unusual immunohistochemical profile may lead to a diagnostic challenge. In these instances, the use of an auxiliary molecular test for the identification of a tumor-specific translocation will help to establish the diagnosis. For Ewing sarcoma, the presence of an EWSR1 gene

Primary aneurysmal bone cyst (ABC) is a cystic lesion of bone composed of blood-filled spaces separated by connective tissue septae containing fibroblasts, osteoclasttype giant cells and reactive woven bone. ABC can occur as primary tumors, which are usually meta-epiphyseal in patients in their first two decades, or as secondary tumors to another bone tumor such as osteoblastoma or chondroblastoma. In 1999, a recurrent translocation, t(16;17), was shown in several cases of primary ABC (25). The t(16;17) translocation was later shown to relocate the promoter of CDH11, a gene that is strongly expressed in bone, in front of the USP6 gene (TRE2, TRE17) (26). Over the past few years, many different translocations has been described in ABC (Table 1) (26,27), all resulting in oncogenic activation of the USP6 gene on chromosome 17p13. Thus, the pathogenesis of most primary ABCs involves upregulation of USP6

Molecular pathology in bone tumors transcription driven by other, highly active promoters (28). The USP6 gene product is involved in actin remodeling (29). USP6 induces expression of matrix metalloproteinases (30), alters bone morphogenic protein (BMP) signaling, and inhibits the differentiation of preosteoblasts (31). Rearrangements in USP6 were shown to be restricted to the spindle cells, and were absent in the multinucleated giant cells, inflammatory cells, endothelial cells, and osteoblasts (32). When evaluating FISH results for molecular diagnostics, one should realize that the translocation is found in a variable number of cells, ranging from 7% to 82% (32). USP6 rearrangements are absent in its morphological mimics in bone, including giant cell tumor, telangiectatic osteosarcoma, cherubism, and brown tumor of hyperparathyroidism (33), making it a valuable diagnostic tool when the morphology or clinical presentation is not typical. Interestingly, USP6 rearrangements were also reported in 2 of 12 cases of ABC-like myositis ossificans, and in 44 of 48 cases of nodular fasciitis. In the latter, USP6 has different fusion partners, 65% of which include the MYH9 gene (34).

Fibrous dysplasia Fibrous dysplasia is a benign fibro-osseous lesion in the medulla of bone, involving one or more bones. The monostotic form is six times more common than polyostotic fibrous dysplasia. The latter can be associated with endocrine  au lait pigmentation in non-hereditary abnormalities and cafe McCuneeAlbright syndrome. Fibrous dysplasia has long been regarded a non-neoplastic process, a dysplasia of bone as its name implies. However, the finding of recurrent chromosomal abnormalities (35) indicates its neoplastic nature. Fibrous dysplasia is characterized by activating mutations in the Gs alpha (GNAS1) gene localized on chromosome 20q13.32, which can be detected in up to 93% of cases (36). It encodes the alpha subunit of the stimulatory guanine nucleotide-binding protein (G-protein) (37). G-proteins couple extracellular receptors to intracellular effector enzymes and ion channels, mediating the cellular response to an external stimulus. In McCuneeAlbright syndrome, patients carry a postzygotic somatic activating GNAS1 mutation. Therefore, fibrous dysplasia, McCuneeAlbright syndrome, and nonskeletal isolated endocrine lesions are all associated with GNAS1 mutations and represent a spectrum of phenotypic expressions of the same basic disorder, probably reflecting different patterns of somatic mosaicism (37). GNAS1 mutations are also found in (intramuscular and cellular) myxomas (38e40). The co-occurrence of fibrous dysplasia and myxomas is known as Mazabraud syndrome (41). In the diagnostic setting, the demonstration of a GNAS1 mutation can help. Mutation hot spots include R201H (57%), R201C (38%), and Q227L (5%) (36). Fibroblastic (or fibrous dysplasia-like) low grade osteosarcoma is the most important differential diagnosis of fibrous dysplasia because of its low grade malignant behavior. There seems to be a low prevalence of GNAS1 mutations in fibroblastic low grade osteosarcoma (one of five cases) (42). In the long bones, especially the tibia and fibula, osteofibrous dysplasia should also be considered in the differential diagnosis of fibrous dysplasia. GNAS1 mutations are however absent in osteofibrous dysplasia (36,43). In the jaw, cemento-ossifying

199 fibroma and cemento-osseous dysplasia are in the differential diagnosis in which GNAS mutations are not found (44,45).

Chondrogenic tumors Osteochondroma and secondary peripheral chondrosarcoma Multiple osteochondroma (MO) (previously known as hereditary multiple exostosis) is a skeletal disorder characterized by the presence of bony outgrowths at the epiphyseal region of long bone and is caused by mutations of the EXT1 or EXT2 genes (46e49). Similarly, mutations of the EXT1 gene were detected in the cartilage cap of sporadic osteochondroma cases (50). Loss of the wild-type allele in hereditary cases (51) and homozygous loss of both alleles in sporadic cases (52) indicates that inactivation of both EXT alleles is required for osteochondroma formation, which was confirmed in mouse models (53). Probably, loss of heparan sulfate due to EXT inactivation leads to disturbance of hedgehog signaling and loss of polarization of chondrocytes (54e56). The complexity of osteochondroma formation, however, is not yet completely clarified by the bi-allelic inactivation, as mosaic distribution of cells with retained chromosome 8 regions (presumably normal cells) was observed in the cartilage cap of both human osteochondromas and animal model systems (53,57). Interestingly, in peripheral chondrosarcoma arising secondarily within the cartilaginous cap of osteochondroma EXT was shown to be wild type in most cases, suggesting that malignant transformation predominantly occurs in the wild-type cells (57). Most hereditary cases (70e75%) are caused by point mutations resulting in truncated proteins, whereas deletions involving single or multiple exons were found in about 10% of all hereditary cases using MLPA. However, in the remaining 10e15% of multiple-osteochondroma cases, genomic alterations cannot be detected implying the potential role of other alterations such as inversions, translocations, or somatic mosaicism. With the use of a custom-designed, oligucleotide-based array CGH, the detection and mapping of exonsized mosaic genomic alterations of the EXT1/2 genes were shown (8). Therefore, it is likely that instead of a putative EXT3 gene, the presence of low level mosaic mutation or other copy number neutral genome alterations cause EXT1and EXT2-negative multiple osteochondromas cases. Enchondroma and central chondrosarcoma Whereas the initial event for central cartilaginous tumors has been unknown for a long time, somatic heterozygous isocitrate dehydrogenase 1 (IDH1) hot spot (R132C and R132H) or IDH2 (R172S) mutations have recently been identified (13). Ollier disease and Maffucci syndrome are rare non-hereditary skeletal disorders characterized by multiple enchondromas (Ollier disease) combined with spindle cell hemangiomas (Maffucci syndrome). In total, 65 of 75 patients with Ollier disease (87%) and 17 of 21 patients with Maffucci syndrome (81%) were shown to carry mutations in their tumors (14,58). Mutations mostly affect the Arg132 position in exon 4 of IDH1 and include R132C (w65%), R132H (w15%), R132 G (w5%) or other very rare variants affecting the R132 position. Mutations in IDH2 (R172S)

200 are rare. Both IDH1 and IDH2 catalyze the oxidative decarboxylation of isocitrate into a-ketoglutarate in the Krebs tricarboxylic acid cycle. IDH1 and IDH2 catalyze the identical biochemical reaction but differ in their subcellular localization: IDH1 is located in the cytoplasm and peroxisomes, whereas IDH2 is located in the mitochondria. Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 are also found in primary (38e70%) and secondary (86%) central chondrosarcomas as well as in periosteal chondrosarcomas (4 of 4, 100%) and are absent in other cartilaginous tumors or chondroblastic osteosarcomas (13,14,59,60). The high mutation frequency in enchondromas and the fact that they are early events suggest a causal rather than a bystander role for IDH1 or IDH2 mutations in tumorigenesis, and future studies should reveal the exact mechanism by which these mitochondrial deficiencies lead to tumor development. The genetic or epigenetic alterations underlying malignant progression toward central chondrosarcoma are so far unknown. For central chondrosarcoma, several active signaling pathways were identified, including pRB (61,62); IHH/PTHLH/Bcl-2 (63e66); Src, Akt, and PDGFR with no effect of imatinib (67,68); IGF (69); and estrogen signaling (70,71) as well as hypoxic (72) and glycolytic pathways (73). An important role for overexpression of Bcl-2 family members in the chemoresistance of chondrosarcoma was recently shown (74).

Chordoma Chordoma is a rare, malignant, locally destructive tumor with a characteristic morphology and immunohistochemical profile (cytokeratin 19 and T [brachyury] positive) (75e77). Chordoma is typically located at the clivus, vertebral, or sacral bones. Genome-wide studies using array CGH showed a high frequency of homozygous deletion of the chromosome 9p21 locus containing the CDKN2A/2B genes in 70% of the analyzed cases and a gain of the chromosome 6q27 locus containing the T gene (78). The rare occurrence of familial chordoma has been linked to T duplication in members of four of seven families with a history of familial chordoma (79). This observation led to detailed investigation of the involvement of both the T locus and the expression of the T gene at both the RNA and protein level. In about half of the investigated samples, a gain of the T region either regionally or by means of a whole chromosome gain was observed. However, similar expression levels of the T gene were detected both at the RNA and protein levels irrespective of the copy number status (with or without gain). The expression of T protein, with the exception of hemangioblastoma, is exclusive to chordoma and is a specific and sensitive marker for chordoma. The mechanism responsible for the elevated expression of the T gene in cases without copy number alterations still remains unclear. Efforts to show involvement of promoter or gene mutation or the involvement of up- or downstream regulatory elements were unsuccessful (80).

Osteosarcoma Osteosarcoma is an extremely genetically instable tumor type with many translocations, amplifications, and deletions that obscure the detection of driver genes that were selected

K. Szuhai et al. for tumorigenic activity (81). Recently, a possible mechanism for this instability has been proposed: chromothripsis, a cataclysmic event in which chromosomes are fragmented and subsequently aberrantly assembled (82). When CGH reports are compared, there is a general consensus about gain of chromosome arms 6 p, 8q, and 17 p (83e89). Two well-known tumor suppressor genes have been shown to be altered in osteosarcoma, TP53 on chromosome arm 17p and RB1 at 13q. Either somatic or hereditary gene mutations are seen with loss of heterozygosity of the other allele and most times also a large part of the remaining chromosome arm. RB1 is an important gene in controlling cell cycle signaling, as well as CDKN2A. This latter gene, located at 9p, has been shown to be mutated in osteosarcomas that have no RB1 mutations (90). A third member of the RB pathway, CDK4, negatively affects RB1 function and has been shown to be amplified in osteosarcoma (91). The p53 pathway is compromised in osteosarcoma. The TP53 gene is mutated in 22% of high grade central osteosarcoma (92), but the presence of a mutation is not associated with clinical outcome, like in other tumor types. It was reported that TP53 mutations correlate with an increased genomic instability in osteosarcoma (93). Amplification of MDM2 targets the same pathway as p53. MDM2 encodes an E3 ligase specific for p53 protein, which targets it for ubiquitin-mediated proteasomal degradation. The frequency of MDM2 amplification in central high grade osteosarcoma is determined to be w6% but is mutually exclusive of TP53 mutations (93). Checkpoint kinase 2 (CHK2) is required for blocking the cell cycle in response to DNA damage and other stresses and is activated by phosphorylation mediated by ATM. CHK2 was shown to be mutated in 4 of 57 osteosarcomas (94). As can be appreciated from this non-exhaustive list of genes that are targeted in osteosarcoma, most of these are genes involved in cell cycle checkpoint regulation, which complies with the complex karyotypes of these tumors and reflects their chromosomal instability. None of the genetic changes are recurrent and specific, so their use in diagnostics is limited. The deletion of the CDKN2A locus has prognostic value, since survival of patients with this alteration is very poor (11,95). Telomeres are repeated DNA sequences that protect chromosomes from erosion. Cancer cells usually have a mechanism to maintain their telomeres, either by activation of the enzyme telomerase, encoded by telomerase reverse transcriptase (TERT ) or by a mechanism, designated alternative lengthening of telomeres (ALT), which is poorly defined. In osteosarcoma, ALT is more frequent than in other tumor types and found in two-thirds of cases (96,97). It has been suggested that patients with TERT messenger RNA expression have a worse prognosis than those with ALTmediated telomere maintenance (97), although another study reports that the presence of both mechanisms predicts poor outcome (96). A third study even claims that telomerase activity is associated with good response to chemotherapy, especially in high grade central osteosarcomas (98). These contradicting conclusions may be attributed to different detection methods and small tumor series. Genome-wide expression studies have shed more light on genetic alterations in osteosarcoma. The significance of these studies may be hampered by small sample size, but

Molecular pathology in bone tumors a recent study was successful in the identification of a very significant pathway involving macrophage function and associated with better prognosis through suppression of metastases, which has been be confirmed in an independent tumor series (99).

Conclusions Over the past decade, much more has become known regarding the molecular etiology of bone tumors. The identification of tumor specific, recurrent alterations may be used as ancillary tools in establishing histopathological diagnosis. The wide variety of different histomorphological bone tumor entities and the lack of immunohistochemical support, initiated an intense quest for novel alterations. Technical advancements, especially results coming from next-generation sequencing technology-based reports will reveal new alterations in the remaining rare sarcoma subtypes for which the molecular background has remained enigmatic. With the recent identification of IDH1/2 mutations in central chondrosarcoma, an entity originally regarded as a tumor with a complex karyotype, current concepts will change. Other approaches, such as the computational identification of gene expression differences occurring at exon levels for expressed fusion genes using exon-level expression array data sets, have been shown to work successfully. This approach resulted in the identification of a TMPRSS2/ERG fusion in prostate cancer (1). More recently, the HEY1-NCOA2 fusion product was identified in mesenchymal chondrosarcoma (2). In both cases, complex intrachromosomal alterations led to the formation of the fusion gene without detectable chromosomal alterations using classical karyotyping approaches. Opening the archives using new technologies as well as refining existing technologies for decalcified paraffin-embedded tissue (10) may bring more knowledge about other rare sarcoma subtypes in the near future. Although the use of immunohistochemistry is widespread and very helpful in soft tissue tumors, for bone tumors conventional morphology is the cornerstone of the diagnosis. The identification of specific genetic alterations in bone tumors enables the development of molecular diagnostic tests for this group of tumors that can be used in everyday pathology practice.

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