Original Research Article DOI: 10.18231/2456-9267.2018.0019 Evaluation of osteonectin as a diagnostic marker of osteogenic bone tumors Murad Ahmad1, Sadaf Mirza2, Kafil Akhtar3,*, Rana K. Sherwani4, Khalid A Sherwani5 1Assistant Professor, 2Resident, 3,4Professor, 1-4Dept. of Pathology, Jawaharlal Nehru Medical College, Aligarh Muslim 5 University, Aligarh, Uttar Pradesh, Professor, Dept. of Orthopaedics Surgery, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India *Corresponding Author: Email: [email protected] Abstract Introduction: Immunohistochemistry plays an important but limited role in the diagnosis of primary bone tumors. Sometimes it is very difficult to differentiate osteosarcomas histologically from other tumors with similar morphology but with different malignant potential and treatment protocol. The correct diagnosis of OSA relies on identification of osteoid production by malignant cells which can be detected by the use of osteonectin. Materials and Methods: The present study was carried out on 200 patients of benign and malignant lesions of bone. After a detailed clinical history and local examination, paraffin section of resected specimens were studied by hematoxylin and eosin and immunohistochemical stain, osteonectin and matrix was graded on a four-tiered grading system with statistical analysis of osteonectin positivity in osteogenic bone tumours and tumour-like lesions. Results: Benign and malignant tumours accounted for 74.6% and 25.0% of the total cases. Osteoid production were seen in 36 cases (100.0%) of osteosarcomas, followed by fibrous dysplasia in 18 cases (100.0%) and osteoid osteoma in 10 cases (100.0%). Both polygonal and spindle shaped tumour cells in osteosarcoma showed Grade 4 positivity with osteonectin. Fibroblastic variant of osteosarcoma showed diffuse (Grade 4) and focal strong staining (Grade 2) alternating with negative areas. Chondroblastic variant of osteosarcoma showed Grade 4+ immunoreactivity in conventional osteosarcoma like areas, whereas chondrocyte- like tumour cells surrounded by chondro-osteoid –like matrix showed well- defined osteonectin staining. Conclusions: Osteonectin is a helpful osteogenic marker for establishing the diagnosis of osteoblastic origin of a tumour that produce no or scarcely any matrix. Keywords: Bone Tumors, Immunohistochemistry, Osteonectin. Introduction diagnosis is necessary for a better outcome. The correct Bone tumours represents only 1.7% of orthopaedic diagnosis of OSA relies on identification of osteoid lesions. Osteosarcoma is the most common type production by malignant cells which can be detected by of bone cancer; a malignant mesenchymal tumour in the use of osteonectin. which the cancerous cells produce bone matrix. It occurs in all age groups but has a bimodal age Materials and Methods distribution. Seventy- five percent occur in person The present study was carried out on 200 patients younger than 20 years, with male preponderance. It is of benign and malignant lesions of bone, attending the also common over 60 years of age.1 Outpatients and Inpatients Departments of Immunohistochemistry plays an important but Orthopaedics Surgery and Pathology, Jawaharlal Nehru limited role in the diagnosis of primary bone tumors. Medical College, Aligarh Muslim University, Aligarh The results should be carefully assessed taking into for a period of 5 years. account the clinical and radiological features of the case A detailed history and a thorough clinical and the morphology on the haematoxylin and eosin examination, including local examination of bony stained sections. The prognostic value of antibodies lesions was carried out along with relevant such as the proliferation marker MIB1 and markers for investigations like histopathology of paraffin embedded multiple drug resistance, may in future, become part of section of specimens using hematoxylin and eosin stain. the standard panel for some primary bone tumors. Histologically proven benign and malignant lesions SPARC (secreted protein, acidic and rich in were selected for the study. Subsequently, serial cysteine), also known as osteonectin or BM-40 sections, were used for the immunohistochemical (basement membrane tumor matrix component), is a analysis, using rabbit antihuman polyclonal antibodies. secreted calcium-binding glycoprotein.2 Rather than Mesenchymal tumours without bone formation like acting as structural components of the extracellular Fibrosarcoma, Rhabdomyosarcoma, Leiomyosacoma matrix, members of this family mediate cell–matrix and Ewing’s sarcoma served as controls. interactions.3,4 Osteonectin immunostaining of tumor cells and Sometimes it is very difficult to differentiate matrix was graded on a four-tiered grading system: 1 = osteosarcomas histologically from other tumors with focal (< 50%) weak staining; 2 = focal strong staining; similar morphology but with different malignant 3 = diffuse (> 50%) weak staining; 4 = diffuse strong potential and treatment protocol. Therefore the correct staining. Statistical Analysis of osteonectin positivity in IP Archives of Cytology and Histopathology Research, April-June, 2018;3(2):93-100 93 Murad Ahmad et al. Evaluation of osteonectin as a diagnostic marker of osteogenic…. osteogenic bone tumours and tumour-like lesions was Most common tumour was giant cell tumour, also calculated which accounted for 45 (22.5%) cases, followed by osteosarcoma 36 cases (18.0%), Fibrous dysplasia 18 Observations cases (9.0%), Aneursymal bone cyst 15 cases (7.5%), Most of the patients in this study had benign osteochondroma 14 cases (7.0%), callus formation 14 tumours, accounting for 74.6% of the cases whereas cases (7.0%), osteoid osteoma 10 cases (5.0%), malignant tumours accounted for 25.0% of the total chondrosarcoma 9 cases (4.5%), enchondroma 8 cases cases. (4.0%). (Table 1) Table 1: Distribution of different types of tumours according to who classification Types of tumours No. of cases Percentage A. Cartilage forming tumours Osteochondroma 14 7.0 Enchondroma 08 4.0 Chondromyxoid fibroma 02 1.0 Chondroblastoma 02 1.0 Chondrosarcoma 09 4.5 Dedifferentiated tumours 02 1.0 B. Bone forming tumours Osteoid osteoma 10 5.0 Osteoblastoma 01 0.5 Osteosarcoma 36 18.0 C. Fibrogenic tumours Fibrosarcoma 02 1.0 D. Primitive Neuroecto-Dermal Ewing’s sarcoma 08 4.0 E. Giant Cell Tumour 45 22.5 F. Notochordal Chordoma 03 1.5 G. Muscle Rhabdomyosarcoma 02 1.0 Leiomyosarcoma 03 1.5 H. Miscellaneous Fibrous dysplasia 18 9.0 Aneursymal bone cyst 15 7.5 Callus formation 14 7.0 Simple bone cyst 03 1.5 Non- ossifying fibroma 03 1.5 Total 200 100 In our study, maximum number of cases of osteoid (100.0%), osteoid osteoma, 10 cases (100.0%), 10 cases production were seen in osteosarcomas, 36 cases (71.0%) each of callus formation and osteochondroma (100.0%), followed by fibrous dysplasia, 18 cases and 9 cases (60.0%) of aneurysmal bone cyst. (Table 2) Table 2: Distribution of different tumours and tumour- like lesions with osteoid production Tumour and tumour like lesions No. of cases Osteoid Osteoid absent present Osteosarcoma 36 36 - Chondrosarcoma 09 - 09 Callus formation 14 10 04 Fibrous dysplasia 18 18 - Giant cell tumour 45 - 45 Aneurysmal bone cyst 15 09 07 Osteoid osteoma 10 10 - Osteochondroma 14 10 04 Osteoblastoma 01 - 01 IP Archives of Cytology and Histopathology Research, April-June, 2018;3(2):93-100 94 Murad Ahmad et al. Evaluation of osteonectin as a diagnostic marker of osteogenic…. Chondromyxoid fibroma 02 02 Enchondroma 08 08 Chondroblastoma 02 - 02 Dedifferentiated tumours 02 02 Fibrosarcoma 02 - 02 Ewing sarcoma 08 - 08 Leiomyosarcoma 03 - 03 Rhabdomyosarcoma 02 - 02 Simple bone cyst 03 - 03 Non- ossifying fibroma 03 03 Chordoma 03 - 03 Total 200 93 107 Two (1.0%) cases of rhabdomyosarcoma were positive immunohistochemical reaction was not only seen, with pleomorphic rhabdomyoblasts with found in the osteoblastic variant, but also in the multinucleation and osteonectin negativity in the fibrosarcomatous and pleomorphic variants of the malignant tumour cells. Eight (4%) cases of Ewing’s osteosarcoma. Nine cases of Osteoblastic osteosarcoma sarcoma was seen, with clusters of small round cells showed predominantly thick anastomosing lace-like with hyperchromatic nucleus with mild karyomegaly pink osteoid formation by malignant tumour cells and scant cytoplasm, infiltrating the bone. All the soft having high N/C ratio and marked nuclear tissue tumours of similar morphological structures pleomorphism, with focal areas of mineralization (Fig. (fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma 1). Both polygonal and spindle shaped tumour cells and Ewing’s sarcoma) were negative for osteonectin, showed Grade 4 positivity with osteonectin (Fig. 2). but Ewing’s sarcoma showed CD99 positivity. Poorly mineralized matrix was weakly stained (Grade 1 Our results showed that all cases of osteosarcoma positivity). (Tables 3 and 4) (100%) reacted with the osteonectin antibody. A Table 3: Distribution of bone tumour and tumours like lesions showing osteonectin positivity Tumour Type No. of cases Osteonectin Positivity No. of positive cases Percentage Osteosarcoma 36 36 100 Osteoblastic 9 09 100 Fibroblastic 03 03 100 Chondroblastic 04 04 100 Telangiectatic 03 03 100 Small cell 03 03 100 Giant cell rich 04 04 100 Anaplastic 02 02 100 Epithelioid 03 03 100 Periosteal 03 03 100 Chondrosarcoma 09 - -- Enchondroma 08 - - Chondromyxoid fibroma 02 - - Chondroblastoma 02 02 100 Differentiated cartilagenous tumours 02 - - Giant cell tumours