Molecular Diagnostics of Bone and Soft Tissue Tumours

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1 Molecular Diagnostics of Bone and Soft Tissue Tumours Judith V.M.G. Bovée Pathologist / Associate professor, Department of Pathology, Leiden University Medical Center, The Netherlands ABSTRACT Bone and soft tissue tumours are relatively rare and their classification is often found to be difficult by the pathologist. An accurate diagnosis is essential to predict biological behaviour and for therapeutic decision making. Over the last decades our knowledge on the genetic background of the different mesenchymal tumours has evolved, which has led to the 2002 WHO classification integrating pathology and genetics. Approximately 15-20% of bone- and soft tissue tumours has a specific translocation which can be used for diagnosis. The detection of specific translocations is not only important to confirm the diagnosis as was suggested by classical morphology and immunohistochemistry, but is especially useful in those cases with an unusual morphology, immunohistochemical profile or clinical presentation. Translocations can be demonstrated using RT-PCR on RNA isolated from frozen tumour tissue, or FISH on non-decalcified paraffin embedded material. In addition, a small subset of bone and soft tissue tumours is characterized by specific somatic gene mutations, e.g. KIT and PDGFRA mutations in gastrointestinal stromal tumours (GIST), and GNAS1 mutations in fibrous dysplasia and myxoma. Mutation analysis can be useful for diagnosis, and in case of GIST for predicting the response to the tyrosine kinase inhibitor Imatinib (Glivec). INTRODUCTION Bone- and soft tissue tumours are relatively rare and constitute <1% of all malignancies in adults. 1 These tumours are generally regarded difficult by surgical pathologists, since they constitute a very heterogeneous, relatively uncommon group of tumours containing > 40 entities. Moreover, there is considerable morphological overlap between the different diagnostic entities. 2 Distinction is however essential since these entities differ widely in treatment and outcome. Keywords: Molecular diagnostics, pathology, sarcomas, bone tumours, soft tissue tumours Over the past 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 Address for communication: Dr. Judith V.M.G. Bovée, Pathologist / Associate professor, Department of Pathology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands Tel: , Fax: , j.v.m.g.bovee@lumc.nl 151

2 Judith V.M.G. Bovée advantage, ultimately leading to cancer. Also for the different bone- and soft tissue tumours more and more genetic data become available. Molecular diagnostic possibilities arose during the last decade, detecting tumorspecific translocations and mutations. 3 The latter development has led to the 2002 WHO classification of bone and soft tissue tumours integrating morphology and genetics. 2 This classification is at present widely accepted and used. Histological grading schemes have been developed for soft tissue sarcomas as a group. The updated French grading system has been internationally accepted. 4, 5 This is a valuable predictor of patient survival for many types of soft tissue sarcomas, although the exclusion of its use for certain tumor types is now acknowledged. 6-8 Accurate histological subtyping is therefore essential. In classifying soft tissue tumours the pathologist can be assisted by immunohistochemistry and molecular diagnostics. Immunohistochemistry is used to confirm the line of differentiation. In addition, molecular diagnostics is playing an increasingly important role as additional technique. Although the tradional morphological and immunohistochemical evaluation remain at the cornerstone of bone and soft tissue tumour diagnosis, molecular diagnostics can increase the accuracy of the diagnosis and can assist in subtyping bone and soft tissue tumours. CHROMOSOMAL TRANSLOCATIONS Translocations can be found in approximately 20% of all malignancies. 9 They are associated with certain tumour types. The occurrence of the translocation is considered a very early step in tumourigenesis. 9 In contrast to what is known about haematopoietic malignancies, data on cytogenetic changes in solid tumours is limited. In sarcomas, translocations are exclusively found in certain tumour types (Table 1). 3, 9 Almost 100% of the Ewing sarcomas / PNETs, myxoid liposarcomas and synoviosarcomas carry a translocation involving the EWSR1-, the DDIT3-, and one of the SSX-genes, respectively. In contrast, translocations are absent in the more frequently occurring osteosarcomas, chondrosarcomas and leiomyosarcomas. 9 It is estimated that approximately 15-20% of bone and soft tissue tumours carry a translocation. 9 It was always assumed that translocations were not important for carcinoma pathogenesis, since these could not be demonstrated cytogenetically. However, it was recently shown that the high expression of two genes at cdna microarray analysis of prostate cancer was due to a gene fusion, that could be demonstrated in 79% of the tumours. 10 It can therefore not be excluded that also in Table 1. Specific translocations in bone and soft tissue tumours. Alveolar rhabdomyosarcoma t(2;13)(q35;q14) PAX3-FKHR (FOXO1A) t(1;13)(p36;q14) PAX7-FKHR t(2;2)(q35;p23) PAX3-NCOA1 Alveolar soft part sarcoma der(17)t(x;17)(p11.2;q25) ASPL-TFE3 Aneurysmal bone cyst t(16;17)(q22;p13) CDH11-USP6 (TRE 2) t(1;17)(p ;p13) TRAP150-USP6 t(3;17)(q21;p13) ZNF9-USP6 t(9;17)(q22;p13) OMD-USP6 t(17;17)(q12;p13) COL1A1-USP6 Angiomatoid fibrous histiocytoma t(12;16)(q13;p11) FUS-ATF1 t(12;22)(q13;q12) EWSR1-ATF1 Bizarre parosteal osteochondromatous t(1;17)(q32;q21) RDC1? proliferation (Nora lesion) Clear cell sarcoma t(12;22)(q13;q12) ATF1-EWSR1 t(2;22)(q32;q12) EWSR1-CREB1 Congenital fibrosarcoma and t(12;15)(p13;q25) ETV6-NTRK3 Mesoblastic nephroma 152

3 Molecular Diagnostics of Bone and Soft Tissue Tumours Dermatofibrosarcoma protuberans der(22)t(17;22)(q22;q13) COL1A1-PDGFB (and giant cell fibroblastoma) Desmoplastic small round cell tumour t(11;22)(p13;q12) WT1-EWSR1 Ewing sarcoma / peripheral primitive neuroectodermal tumour (PNET) t(11;22)(q24;q12) EWSR1-FLI1 t(21;22)(q22;q12) EWSR1-ERG t(7;22)(p22;q12) EWSR1-ETV1 t(17;22)(q12;q12) EWSR1-ETV4 t(2;22)(q33;q12) EWSR1-FEV t(16;21)(p11;q22) FUS-ERG Endometrial stromal sarcoma t(7;17)(p15;q11) JAZF1-JJAZ1 (SUZ12) t(6;7)(p?;p15) JAZF1-PHF1 Inflammatory myofibroblastic tumor t(2;19)(p23;p13.1) ALK-TPM4 t(1;2)(q22-23;p23) TPM3-ALK t(2;17)(p23;q23) CLTC-ALK Extraskeletal myxoid chondrosarcoma t(9;22)(q22;q12) EWSR1- NR4A3 (NOR1, CHN) t(9;17)(q22;q11.) RBP56-NR4A3 t(9;15)(q22;q21) TCF12-NR4A3 Lipoblastoma different rearrangements chromosome 8 HAS2-PLAG1 Lipoma t(3;12)(q27-28;q14-15) HMGA2 (HMGIC)-LPP Many other translocations involving 12q14 (HMGA2) Low-grade fibromyxoid sarcoma t(7;16)(q33;p11) FUS-CREB3L2 FUS-CREB3L1 Myxoid / roundcell liposarcoma t(12;16)(q13;p11) TLS (FUS) - GADD153 (CHOP, DDIT3) t(12;22)(q13;q12) EWSR1- GADD153 Mesenchymal chondrosarcoma der(13;21)(q10;q10) Pulmonary chondroid hamartoma t(6;14)(p21;q24) HMGA1-RAD51L1 t(3;12)(q27-28;q14-15) HMGA2 (HMGIC)-LPP Pericytoma t(7;12)(p22;q13) ACTB-GLI1 Synoviosarcoma t(x;18)(p11;q11) SYT (SS18) -SSX1 SYT-SSX2 SYT-SSX4 Subungual exostosis t(x;6)(q24-q26;q15-21) COL12A1-COL4A5 Giant cell tumour of tendon sheath t(1;2)(p13;q35) COL6A3-CSF1 (localized type) 153

4 Judith V.M.G. Bovée sarcomas more of these cryptic chromosomal aberrations may be found in the future. In many of the translocations in sarcomas the EWSR1 or the FUS gene is involved (Table 2). These promiscuous genes are strongly homologous and encode RNA binding proteins. Most probably, EWSR1 and FUS have a similar effect when they are involved in a chromosomal translocation. The type of DNA binding domain originating from the fusion partner probably determines the tumor type that is induced by the translocation (Table 2). Ewing sarcoma / PNET was one of the first solid tumours in which a characteristic translocation was found. 11 All chromosomal translocations identified so far in Ewing sarcoma / PNET lead to fusion of an ETS binding domain to a highly potent EWS (or, rarely, FUS) transactivation domain. 12 These oncoproteins subsequently act as aberrant transcription factors dysregulating gene expression patterns initiating Ewing tumor formation. Target genes were shown to stimulate cell proliferation (upregulation of PDGF-C, Table 2. Different fusion partners of EWSR1 and FUS in different tumour types Fusion gene Tumor EWSR1 FLI1 ERG ETV1 ETV4 FEV WT1 Desmoplastic small round cell tumour NR4A3 Extraskeletal myxoid chondrosarcoma ATF1 Clear cell sarcoma Angiomatoid fibrous histiocytoma CREB1 Clear cell sarcoma GADD153 Myxoid / round cell liposarcoma FUS ATF1 Angiomatoid fibrous histiocytoma ERG Acute myeloid leukemia CREB3L1 Low-grade fibromyxoid sarcoma CREB3L2 Low-grade fibromyxoid sarcoma GADD153 Myxoid / round cell liposarcoma TRANSLOCATIONS AND PATHOGENESIS Balanced translocations lead to the formation of fusion genes. These hybrid genes subsequently induce expression of other genes that induce uncontrolled proliferation, as is the case for the EWSR1-FLI1 fusion gene in Ewing sarcoma/ PNET. Alternatively, one of the genes involved in 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 cyst. Approximately half of the genes involved in translocations encode transcription factors and tyrosine kinases. CCDN1, c-myc), evade growth inhibition (downregulation of cyclin-dependent kinase inhibitors and TGF-beta receptor type II), escape from senescence (upregulation of htert), escape from apoptosis (repression of IGFBP-3 promoter), induce angiogenesis (VEGF), invasion and metastases (MMPs). 12 Aneurysmal bone cyst 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. It has been for long regarded a reactive process, in which a local circulatory 154

5 Molecular Diagnostics of Bone and Soft Tissue Tumours abnormality would have led to increased venous pressure and resultant dilation of the local vascular network. Moreover, ABC-like areas can be seen in association with other bone tumors such as giant cell tumor or chondroblastoma (secondary ABC). However, in 1999 a recurrent translocation t(16, 17) was shown in several cases of primary ABC. 13 The t(16, 17) translocation was later shown to relocate the promotor of CDH11, a gene that is strongly expressed in bone, in front of the USP6-gene (TRE2, TRE17). 14 Over the past few years a series of different translocations has been decribed in ABC (Table 1), 14, 15 all resulting in oncogenic activation of the USP6 gene on chromosome 17p13. Thus, the pathogenesis of most primary ABCs involves upregulation of USP6 transcription driven by other, highly active promoters. 16 The mechanism by which USP6 upregulation causes ABC formation has been unclear so far. The USP6 gene product is involved in actin remodeling. 17 USP6 rearrangements were shown to be restricted to the spindle cells, and were absent in the multinucleated giant cells, inflammatory cells, endothelial cells and osteoblasts. 18 This suggests that the neoplastic spindle cells induce a vigourous, reactive host response mimicking young granulation tissue, explaining why for a long time the lesions were regarded reactive. 18 Remarkably, in 17 secondary ABCs no USP6 or CDH11 rearrangements could be demonstrated. 18 This suggests that despite its morphological similarity with primary ABC, secondary ABC represents a nonspecific morphologic pattern occurring in other bone tumors. synoviosarcoma, rhabdomyosarcoma, small cell osteosarcoma and mesenchymal chondrosarcoma. The differential diagnosis of these tumouors is difficult due to the absence of distinguishing morphological features. However, using immunohistochemistry and conventional histochemical stainings many of these entities can be distinguished. Although the transmembrane glycoprotein MIC2/CD99 is highly expressed in, CD99 immunoreactivity is also found in other small blue round cell tumours. On small bone biopsies the distinction between Ewing sarcoma/pnet, mesenchymal chondrosarcoma and small cell osteosarcoma can be very difficult since the osteoid in case of small cell osteosarcoma and the cartilaginous matrix in case of mesenchymal chondrosarcoma may be lacking. Moreover, all three can demonstrate CD99 positivity. 19 Real- Time PCR on RNA isolated from frozen tumor tissue can then be used to detect the t(11;22)(q24;q12) or the t(21;22)(q22;q12) 20 characteristic of Ewing sarcoma. If only paraffin embedded material is available FISH can be used to demonstrate that the EWSR1 gene, on chromosome 22, is involved in a translocation (Figure 1). A disadvantage of the latter technique is that the fusion partner of the EWSR1 normal t(11;22) der 11 der USEFULNESS OF SPECIFIC TRANSLOCATIONS IN MOLECULAR DIAGNOSTICS The detection of specific translocations is not only important for confirmation of the diagnosis as suggested by conventional morphology and immunohistochemistry, but is especially important in those cases with unusual morphology, immunohistochemistry or clinical presentation. Small blue round cell tumours De term small blue round cell tumor encompasses a heterogeneous group of tumours that have in common that they are histologically characterized by the presence of undifferentiated small round cells with scant cytoplasm. The differential diagnosis of a small blue round cell tumour includes, neuroblastoma, non Hodgkin lymphoma, poorly differentiated (round cell) Figure 1. Basic principle of translocation detection using FISH analysis for t(11;22) in Ewing sarcoma / PNET. Two individual adjacent DNA probes located at the EWSR1 gene on chromosome 22 are labelled with different fluorescent dyes (usually green and red). In the normal situation these probes will hybridize closely together when FISH is performed using interphase nuclei (lower panel). In case of a translocation the individual probes will split apart and will be detected as two separate signals. 155

6 Judith V.M.G. Bovée gene remains unknown. This can be difficult since EWSR1 is promiscuous and is also involved in translocations in other tumours (Table 2). In case of localization in soft tissue the distinction between poorly differentiated synoviosarcoma and Ewing sarcoma / PNET can be problematic, since the morphology and the immunohistochemical profile can be identical (CD99 positive, EMA positive). The demonstration of an EWSR1 or SYT rearrangement can be essential to come to a correct diagnosis and therapy. Translocation detection is not only used for classification purposes, but is also used for detection of minimal residual disease (MRD) of Ewing sarcoma in bone marrow specimens. While on H&E it is sometimes impossible to distinguish haematopoiesis (especially erythropoiesis) from tumor cells, using the highly sensitive PCR technique the presence or absence of a known translocation type can be demonstrated. SOMATIC GENE MUTATIONS In addition to the 15-20% of bone and soft tissue tumours that are characterized by specific translocations, there is a small subgroup in which specific gene mutations can be found. These somatic mutations can often be found in diagnostics, but they will become increasingly important for targeted therapy. GASTROINTESTINAL STROMAL CELL TUMOUR (GIST) AND KIT Gastrointestinal stromal cell tumour (GIST) is the most frequent mesenchymal tumour of the GI tract and is mainly located in the stomach and the duodenum. The histology is characteristic and can demonstrate both a spindle cell phenotype (~70%), an epithelioid phenotype (~20%) and a mixture of both. The clinical behaviour of GIST is difficult to predict. The most important parameters were found to be tumour size and mitotic rate and therefore these two parameters are used to estimate the risk of aggressive behaviour of GIST. 21 The majority of GISTs (85-90%) carry an activating mutation in the KIT proto-oncogene, localized at chromosome 4q The gene encodes a type III receptor tyrosine kinase protein. Mutations in the KIT gene are mainly found in the juxtamembrane domain (exon 11), and less frequently in exons 9, 13 en 17 (Table 3). These mutations induce ligand independent signal transduction leading to cell proliferation. GIST was one of the first solid tumours in which therapy specifically targeted at a molecular alteration Table 3. Molecular classification of GIST (based on (22-24)) Domain Frequency Type mutation Response to Imatinib KIT Exon 9 Extracellular ~10% AY duplication Intermediate response / insertion Exon 11 Juxtamembrane ~80% Insertions and deletions; Best response pointmutations are rare Exon 13 Kinase I % K642E (V654 A in Sensitive in vitro, also secondary resistance) clinical reponse Exon 17 Activation loop 0.6% N822K and N822H Sensitive in vitro, also clinical reponse PDGFRA Exon 12 Juxtamembrane Together 4.7-7% Point mutations Sensitive in vitro, also clinical reponse Exon 18 Activation loop Point mutations D842 (62%) poor response, other mutations sensitive wildtype - ~5% No mutations Poor response 156

7 Molecular Diagnostics of Bone and Soft Tissue Tumours turned out to be effective. The activity of KIT can be inhibited by Imatinib (STI 571, Glivec), a small molecule kinase inhibitor. The overexpression of KIT due to an activating mutation can be demonstrated using CD117 immunohistochemistry. Approximately 95% of GISTs is positive for CD117, if according to the international consensus agreement no antigen retrieval is used. Of the KIT mutation negative GISTs, 35% harbour a mutation in the platelet derived growth factor receptor type A (PDGFRA), a tyrosine kinase receptor within the same family as KIT. Immunohistochemistry for CD117 is often weak or even negative in this specific subgroup. In addition, these cases more often have an epithelioid morphology. A subset of GISTs with PDGFRA mutations respond well to Imatinib 22, 23 (table 3). In addition to immunohistochemistry for CD117, mutation analysis is used in the diagnosis of GIST. This is indicated when a CD117 negative GIST is suspected, since a subset of these can be sensitive to Imatinib. Mutation analysis can be performed by direct sequence analysis using DNA isolated from paraffin embedded tumor tissue. The type and location of the mutation can predict the response to Imatinib. Approximately 80% of the mutations is located in exon 11, and these are associated with a good response to Imatinib. Approximately 10% of the mutations are found in exon 9. These are often more aggressive tumours, mainly located in the small bowel. 24 GISTs carrying an exon 9 mutation have a worse response to Imatinib than GISTs with exon 11 mutations. Mutations in exon 13 and exon 17 are rare and are mostly specific point mutations. Mutation analysis is also important for secondary resistance. A subset of tumours that primarily were sensitive to imatinib become resistant to the drug. This is caused by additional point mutations, especially in the kinase domains of KIT (exon 13 and exon 17) or PDGFRA (D842V), by amplification of KIT or PDGFRA, or by loss of KIT expression (KIT independent resistance). 25 These tumours can subsequently be treated with Sutent (Sunitinib). 26 At present alternative strategies are being developed that interfere elsewhere in the KIT cascade. Expression of CD117 is also reported for other soft tissue tumours. However, the numbers are small when according to the consensus no antigen retrieval is used. 27 Moreover, this was not associated with KIT mutations, nor with response to Imatinib. There is however a role for Imatinib in the treatment of dermatofibrosarcoma protuberans (DFSP). This is a superficially located low-grade sarcoma with an often longstanding clinical course. DFSP has a specific chromosomal aberration, most often a ring chromosome, resulting in amplification of PDGFB (Table 1). Its activity can be inhibited using Imatinib. FIBROUS DYSPLASIA, MYXOMAS AND GNAS1 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 abnormalities and cafe au lait pigmentation in non-hereditary Mc Cune Albright Syndrome (MIM ). Fibrous dysplasia has for long been regarded a nonneoplastic process, a dysplasia of bone as its name implies. However, the finding of recurrent chromosomal abnormalities 28 indicate its neoplastic nature. Fibrous dysplasia is characterized by activating mutations in the Gs alpha (GNAS1) gene localized on chromosome 20q12-q13.3. It encodes the alpha subunit of the stimulatory G-protein (guanine nucleotide binding protein). 29 G- proteins couple extracellular receptors to intracellular effector enzymes and ion channels, mediating the cellular response to an external stimulus. In Mc Cune Albright syndrome patients carry a postzygotic somatic activating GNAS1 mutation. Therefore, fibrous dysplasia, Mc Cune Albright syndrome and non-skeletal 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. 29 GNAS1 mutations are also found in (intramuscular and cellular) myxomas. 30 The co-occurrence of fibrous dysplasia and myxomas is known as Mazabraud syndrome. 31 In the diagnostical setting the demonstration of a GNAS1 mutation can be helpful. Fibroblastic (or fibrous dysplasialike) low grade osteosarcoma is the most important differential diagnosis of fibrous dysplasia because of its low grade malignant behaviour. While the R201 mutation is found in nearly all cases of fibrous dysplasia, there seems to be a low prevalence of GNAS1 mutations in fibroblastic low grade osteosarcoma (1 out of 5 cases 32 ). 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. 33 CONCLUSION During last decade the molecular genetic background has become increasingly important for the classification of 157

8 Judith V.M.G. Bovée bone and soft tissue tumours. To come to an accurate diagnosis additional molecular diagnostics are frequently used in referral centers where many sarcomas are treated. The demonstration of specific translocations or gene mutations is not only helpfull in confirming the diagnosis as was already suspected based on conventional morphology and immunohistochemistry. It is especially important in cases with an unusual morphology, immunohistochemical profile or clinical presentation. Moreover, it has become clear over the past decade that mutation analysis can be essential to estimate the usefulness of certain targeted therapy. The prototype is tyrosine kinase inhibition by Imatinib (Glivec) in GIST, in which the type of mutation can predict the reponse. The ongoing elucidation of the molecular genetic changes in the different mesenchymal tumour types will contribute to a better classification of these tumours, in which morphology and genetics can be integrated. Moreover, in the future it may also lead to the identification of possible targets for therapy, and contribute to tailored therapy. ACKNOWLEDGEMENTS The author would like to thank Dr. A.M. Cleton-Jansen, Prof. Dr. P.C.W. Hogendoorn, I. Briaire-de Bruijn, P. Wijers- Koster, K. Kleiverda, R. van Eijk, S. Tol-Rensen, Dr. J.J. Baelde and Prof. dr. J. Morreau, Department of Pathology LUMC for their involvement in the development of molecular diagnostics of bone and soft tissue tumours. This paper is based on a previous paper in Dutch Moleculaire diagnostiek van bot- en wekedelentumoren Nederlands Tijdschrift voor Oncologie 2007; 4(6): , for which permission was obtained. REFERENCES 1 Hogendoorn PCW. Pathology of soft tissue sarcomas with emphasis on molecular diagnostic techniques. Eur.J.Cancer X, S205-S World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Soft Tissue and Bone. Lyon: IARC Press; Lazar A, Abruzzo LV, Pollock RE, Lee S, Czerniak B. 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