1. 1 CD56 Expression in Bone and Osteoblastic Neoplasia: A Novel Osteoblast Marker? D.E. Hughes, R. Deb, D.C.Mangham, V.P. Sumathi and R.J. Grimer Royal Orthopaedic Hospital, Birmingham, U.K. D.E. Hughes, R. Deb, D.C.Mangham, V.P. Sumathi and R.J. Grimer Royal Orthopaedic Hospital, Birmingham, U.K.

2. 2 CD56/NCAMCD56/NCAM Cell adhesion molecule of the immunoglobulin superfamily Cell adhesion molecule of the immunoglobulin superfamily Involved in homotypic cell-cell interactions Involved in homotypic cell-cell interactions Possible role in neural morphogenesis Possible role in neural morphogenesis Restricted tissue distribution Restricted tissue distribution Cell adhesion molecule of the immunoglobulin superfamily Cell adhesion molecule of the immunoglobulin superfamily Involved in homotypic cell-cell interactions Involved in homotypic cell-cell interactions Possible role in neural morphogenesis Possible role in neural morphogenesis Restricted tissue distribution Restricted tissue distribution

3. 3 CD56 expression in normal tissues Neurons, gangliocytes Neurons, gangliocytes Plasma cells Plasma cells Natural killer cells Natural killer cells Neurons, gangliocytes Neurons, gangliocytes Plasma cells Plasma cells Natural killer cells Natural killer cells

4. 4 CD56 expression in tumours Myeloma Myeloma Neuroendocrine carcinomas Neuroendocrine carcinomas Neuroblastoma Neuroblastoma Phaeochromocytoma Phaeochromocytoma Some glial tumours Some glial tumours NK-type LGL NK-type LGL Myeloma Myeloma Neuroendocrine carcinomas Neuroendocrine carcinomas Neuroblastoma Neuroblastoma Phaeochromocytoma Phaeochromocytoma Some glial tumours Some glial tumours NK-type LGL NK-type LGL

5. 5 Previous evidence of CD56 expression in osteoblast lineage Expressed by calvarial cells in vitro Expressed by calvarial cells in vitro Described in association with CD56 expression in myeloma in osteoblasts Described in association with CD56 expression in myeloma in osteoblasts Reported in osteosarcomas Reported in osteosarcomas Not expressed in Ewing’s sarcoma Not expressed in Ewing’s sarcoma Not expressed in chondrosarcoma Not expressed in chondrosarcoma Expressed by calvarial cells in vitro Expressed by calvarial cells in vitro Described in association with CD56 expression in myeloma in osteoblasts Described in association with CD56 expression in myeloma in osteoblasts Reported in osteosarcomas Reported in osteosarcomas Not expressed in Ewing’s sarcoma Not expressed in Ewing’s sarcoma Not expressed in chondrosarcoma Not expressed in chondrosarcoma

6. 6 Aim of study To assess CD56 expression in normal cells of the osteoblast lineage To assess CD56 expression in normal cells of the osteoblast lineage To assess CD56 expression in tumour-like lesions in which bone is formed To assess CD56 expression in tumour-like lesions in which bone is formed To assess CD56 expression in osteoblastic neoplasms To assess CD56 expression in osteoblastic neoplasms To assess CD56 expression in normal cells of the osteoblast lineage To assess CD56 expression in normal cells of the osteoblast lineage To assess CD56 expression in tumour-like lesions in which bone is formed To assess CD56 expression in tumour-like lesions in which bone is formed To assess CD56 expression in osteoblastic neoplasms To assess CD56 expression in osteoblastic neoplasms

7. 7 MethodsMethods Cases selected from Royal Orthopaedic Hospital paraffin archive to represent a broad range of normal bone, reactive conditions, tumour-like conditions and osteoblastic tumours Cases selected from Royal Orthopaedic Hospital paraffin archive to represent a broad range of normal bone, reactive conditions, tumour-like conditions and osteoblastic tumours CD56 expression assessed by a standard immunohistochemical approach CD56 expression assessed by a standard immunohistochemical approach Cases selected from Royal Orthopaedic Hospital paraffin archive to represent a broad range of normal bone, reactive conditions, tumour-like conditions and osteoblastic tumours Cases selected from Royal Orthopaedic Hospital paraffin archive to represent a broad range of normal bone, reactive conditions, tumour-like conditions and osteoblastic tumours CD56 expression assessed by a standard immunohistochemical approach CD56 expression assessed by a standard immunohistochemical approach

8. 8 ImmunohistochemistryImmunohistochemistry Novacastra anti- CD56 clone 1B6 at 1:50 Novacastra anti- CD56 clone 1B6 at 1:50 High temperature antigen retrieval in citrate/EDTA High temperature antigen retrieval in citrate/EDTA Detection by Enivision system Detection by Enivision system Novacastra anti- CD56 clone 1B6 at 1:50 Novacastra anti- CD56 clone 1B6 at 1:50 High temperature antigen retrieval in citrate/EDTA High temperature antigen retrieval in citrate/EDTA Detection by Enivision system Detection by Enivision system Appendix CD56 staining of neural cells

9. 9 Case selection – benign Normal/reactive bone (31 cases) Normal/reactive bone (31 cases) Quiescent bone Quiescent bone Callus, periosteal reaction Callus, periosteal reaction Benign bone tumours and tumour-like conditions (24 cases) Benign bone tumours and tumour-like conditions (24 cases) Aneurysmal bone cyst Aneurysmal bone cyst Fibrous dysplasia Fibrous dysplasia Osteofibrous dysplasia Osteofibrous dysplasia Non-ossifying fibroma Non-ossifying fibroma Desmoplastic fibroma Desmoplastic fibroma Osteoid osteoma Osteoid osteoma Osteoblastoma Osteoblastoma Giant cell tumour Giant cell tumour Normal/reactive bone (31 cases) Normal/reactive bone (31 cases) Quiescent bone Quiescent bone Callus, periosteal reaction Callus, periosteal reaction Benign bone tumours and tumour-like conditions (24 cases) Benign bone tumours and tumour-like conditions (24 cases) Aneurysmal bone cyst Aneurysmal bone cyst Fibrous dysplasia Fibrous dysplasia Osteofibrous dysplasia Osteofibrous dysplasia Non-ossifying fibroma Non-ossifying fibroma Desmoplastic fibroma Desmoplastic fibroma Osteoid osteoma Osteoid osteoma Osteoblastoma Osteoblastoma Giant cell tumour Giant cell tumour

10. 10 Case selection – malignant Malignant osteogenic tumours (23 cases) Malignant osteogenic tumours (23 cases) High grade intramedullary osteosarcoma High grade intramedullary osteosarcoma Telangiectatic osteosarcoma Telangiectatic osteosarcoma Intermediate grade chondroblastic osteosarcoma Intermediate grade chondroblastic osteosarcoma Parosteal osteosarcoma Parosteal osteosarcoma Low grade central osteosarcoma Low grade central osteosarcoma Undifferentiated recurrences of known osteosarcomas Undifferentiated recurrences of known osteosarcomas Non bone-forming intra-osseous sarcomas Non bone-forming intra-osseous sarcomas Malignant osteogenic tumours (23 cases) Malignant osteogenic tumours (23 cases) High grade intramedullary osteosarcoma High grade intramedullary osteosarcoma Telangiectatic osteosarcoma Telangiectatic osteosarcoma Intermediate grade chondroblastic osteosarcoma Intermediate grade chondroblastic osteosarcoma Parosteal osteosarcoma Parosteal osteosarcoma Low grade central osteosarcoma Low grade central osteosarcoma Undifferentiated recurrences of known osteosarcomas Undifferentiated recurrences of known osteosarcomas Non bone-forming intra-osseous sarcomas Non bone-forming intra-osseous sarcomas

11. 11 Results: normal bone (1) Strong expression in lining cells and osteoblasts

12. 12 Results: normal bone (2) Weak expression in stromal cells adjacent to osteoblast seams OsteocytesPredominantlynegative

13. 13 Results: callus No expression in cartilage No expression in fibroblasts Strong expression in osteoblasts

14. 14 Results: periosteal reaction No expression in outer fibrous layer Strong expression in bone-forming areas

15. 15 Results: aneurysmal bone cyst Strong expression on Surface layer of septal structures Background stromal Population negative

16. 16 Results: fibrous dysplasia Spindle cell component positive – most strongly adjacent to areas of bone formation Similar results in osteofibrous dysplasia

17. 17 Results: non-ossifying fibroma Some expression in spindle cells adjacent to bone Similar results in desmoplastic fibroma

18. 18 Results: osteoid osteoma Nidus osteoblasts positive positive Similar results in osteoblastoma

19. 19 Results: giant cell tumour Focal weak expression by spindle cell component Osteoclasts negative

20. 20 Results: high grade osteosarcoma Most tumour cells positive Strong expression in osteoblastic areas Loss of expression in chondroblastic areas

21. 21 Results: parosteal osteosarcoma Variable expression in spindle cell component – stronger adjacent to areas of osteoid Similar results in low grade central osteosarcoma

22. 22 Results: telangiectatic osteosarcoma Focal expression In undifferentiated Tumour cells

23. 23 Results: undifferentiated recurrence of high grade osteosarcoma Areas of expression in the absence of osteoid formation Similar results in some undifferentiated intra-osseous sarcomas

24. 24 Results - summary by cell type: non-neoplastic Normal osteoblasts – always positive Normal osteoblasts – always positive Normal osteocytes – mostly negative Normal osteocytes – mostly negative Normal periosteal spindle cells – positive in areas of active bone formation Normal periosteal spindle cells – positive in areas of active bone formation Normal bone marrow stromal cells - positive in areas of active bone formation Normal bone marrow stromal cells - positive in areas of active bone formation Normal osteoblasts – always positive Normal osteoblasts – always positive Normal osteocytes – mostly negative Normal osteocytes – mostly negative Normal periosteal spindle cells – positive in areas of active bone formation Normal periosteal spindle cells – positive in areas of active bone formation Normal bone marrow stromal cells - positive in areas of active bone formation Normal bone marrow stromal cells - positive in areas of active bone formation

25. 25 Results - summary by cell type: neoplastic Neoplastic osteoblasts – consistently positive Neoplastic osteoblasts – consistently positive Neoplastic osteocytes – variably positive Neoplastic osteocytes – variably positive Neoplastic spindle cells with osteogenic potential – variably positive Neoplastic spindle cells with osteogenic potential – variably positive Neoplastic spindle cells without evidence of osteogenic potential – sometimes positive Neoplastic spindle cells without evidence of osteogenic potential – sometimes positive Neoplastic osteoblasts – consistently positive Neoplastic osteoblasts – consistently positive Neoplastic osteocytes – variably positive Neoplastic osteocytes – variably positive Neoplastic spindle cells with osteogenic potential – variably positive Neoplastic spindle cells with osteogenic potential – variably positive Neoplastic spindle cells without evidence of osteogenic potential – sometimes positive Neoplastic spindle cells without evidence of osteogenic potential – sometimes positive

26. 26 ConclusionsConclusions CD56 is consistently strongly expressed by normal and neoplastic osteoblasts CD56 is consistently strongly expressed by normal and neoplastic osteoblasts CD56 expression also occurs in non- osteogenic spindle cells in bone and bone lesions – it may not be a specific marker of osteoblastic potential for diagnostic purposes CD56 expression also occurs in non- osteogenic spindle cells in bone and bone lesions – it may not be a specific marker of osteoblastic potential for diagnostic purposes CD56 may be a biologically important molecule in osteoblast function CD56 may be a biologically important molecule in osteoblast function CD56 is consistently strongly expressed by normal and neoplastic osteoblasts CD56 is consistently strongly expressed by normal and neoplastic osteoblasts CD56 expression also occurs in non- osteogenic spindle cells in bone and bone lesions – it may not be a specific marker of osteoblastic potential for diagnostic purposes CD56 expression also occurs in non- osteogenic spindle cells in bone and bone lesions – it may not be a specific marker of osteoblastic potential for diagnostic purposes CD56 may be a biologically important molecule in osteoblast function CD56 may be a biologically important molecule in osteoblast function