Osteosarcoma : an immunophenotypic study for characterization and behavior

Background: Osteosarcoma is the most common primary bone tumor occurring in second and third decades of life with a second peak later. Biopsy (needle or incision) is necessary for diagnosis along with imaging modalities (X-ray, CT scan etc) and serology. Due to diagnostic dilemma in certain cases and for prognosis of patients, immunohistochemistry is increasingly used. Aims: To assess the pathologic features and determinants of osteosarcoma in patients of the Indian subcontinent that would put an insight into its appearance and behavior. Methods and Material: Forty cases of biopsy proven osteosarcoma were selected over a period of three years. Histopathology was done for tumor typing, along with serology (pre and post-operative serum alkaline phosphatase). In all cases TNM staging and immunohistochemistry for antibodies to Osteonectin (ON) (diagnosis), S100 (differentiation), Ki 67 and Her2 (prognosis) was done. Results: Serum alkaline phosphatase was high in 37 (92%) cases initially and remained high in metastatic and recurrent lesions. Osteonectin was positive in 38 (95%) cases, S100 in 31 (77%), Ki 67 showed overlapping labeling indices between 4.8-18.8% and Her2 showed more positivity in higher stage tumors. Conclusions: Biopsy (along with imaging) is mandatory to diagnose osteosarcoma. Osteonectin is a good immunohistochemical marker to differentiate osteosarcoma from its mimics. For prognostication, serum alkaline phosphatase, post chemotherapy tumor necrosis (more than 90%), lack of Her2 expression are good parameters. S100 and Ki67 were found to have limited role in diagnosis and prognosis of osteosarcoma. DOI: http://dx.doi.org/10.3329/bjms.v13i4.13573 Bangladesh Journal of Medical Science Vol.13(4) 2014 p.443-448


Introduction:
Osteosarcoma is the most common primary bone tumor occurring in second and third decades with another peak after 40 years 1,2 .Most osteosarcomas arise de novo in the metaphysis of long bones.It has large number of mimics like giant cell tumor, chondrosarcoma, fibrosarcoma and fracture callus.Hence correct diagnosis remains the cornerstone for effective management.Imaging techniques (X-ray, CT scan) and serology (alkaline phosphatase, lactate dehydrogenase) aid in the diagnosis.But biopsy (needle or incision) still remains the gold standard 3,4 .Diagnosis is being rendered more objective by the use of immunohistochemistry for accuracy

Aims:
This study has been done to assess the pathologic features and determinants of osteosarcoma in patients of the Indian subcontinent that would put an insight into its appearance and behavior.

Material and methods:
The procedures followed were in accordance with the ethical standards of the institutional ethical committee on human experimentation.The study was done over a period of three years from April 2008 to March 2011.Forty cases of biopsy proven osteosarcoma were selected.TNM staging was done in all.Histopathological examination was done by routine method of formalin fixation, paraffin embedding and Hematoxylin and Eosin staining (Fig 1 ).
ferentiation of sarcoma), Ki67 (for detecting tumor proliferation) and HER2 (for prognosis) using monoclonal antibodies (except S100) and Novolink polymer detection system detection (RE7140-K) of Novacastra.Immunohistochemistry was done by polymer method following microwave antigen retrieval.Deep brown stain in varying cellular counterparts was considered positive for a particular stain.Osteonectin (clone15G12) and S100 (RTU-S100p) (cytoplasmic) staining intensity was noted in tumor area ( Out of 25,246 all new cases in our institution over a period of 3 years, 15,634 cases (61.9%) were proved to be malignant.Primary malignant bone tumor (PMBT) was detected in 296 cases (1.9% of all malignancies).Osteosarcoma was confirmed in 89 cases (30% of PMBTs).Forty (45%) cases of osteosarcoma could be followed up at least for 6 months and were selected; 59 cases (55%) either died prematurely or were lost on follow up.Of the 40 subjects male female distribution was 3:1.TNM staging of the selected cases showed: Stage I, 7 cases (17.5%),Stage II, 5 (12.5%),Stage III, 15 (37.5%) and Stage IV, 13 (32.5%).Pre-and postchemotherapy serum alkaline phosphatase (SAP) levels for all the stages, showed initial greater values for higher stage tumors (Table 1).In all stage tumors, response to chemotherapy was associated with reduced levels apart from Stage II tumors which showed no significant change.One unresponsive Stage IV tumor showed higher post-chemotherapy SAP levels.Out of 40 cases, the histological types and their frequencies were as follows: Osteoblastic -24; Chondroblastic -11; Fibroblastic-4; Small cell -1.The sites involved were: Lower limb -31; Upper limb -8; Hand (metatarsal) -1.IHC with Osteonectin (ON) showed staining in Stage IV tumors (8/13 cases) were recorded as 3+ and three were Her2 negative (Table 3).Evaluation of post-chemotherapy tumor necrosis In conclusion, biopsy (along with imaging) is mandatory to diagnose osteosarcoma.ON is a good immunohistochemical marker to differentiate osteosarcoma from its mimics.For prognostication, serum alkaline phosphatase, post chemotherapy tumor necrosis (more than 90%), lack of Her2 expression are good parameters.S100 and Ki67 were found to have limited role in diagnosis and prognosis of osteosarcoma.

TABLE 2 : IHC for confirmation Osteonectin* (Total 40 cases) S100 (Total 40 cases)
overlap between Stage II, III and IV tumors.IHC with Her2/neu showed Stage I and II tumors (11/12 cases) to be negative and none of these were 3+.

TABLE 4 : Post-chemotherapy tumor necrosis
However, there is no consensus as to any prognostic variable that might be used to stratify patients before the onset of therapy 18 .The strongest association with local recurrence in patients undergoing limb-sparing resection is chemotherapy response, followed closely by surgical margins.PicciP et al concluded chemotherapy-induced tumor necrosis is one of the most important prognostic factors for local control of patients with osteosarcoma 19 .Post chemotherapy tumor necrosis correlated well with the stage of the tumor in our study.Most of the patients with lower stage disease developed >90% necrosis of tumor following chemotherapy (stage I -57% and stage II -60%) whereas in minority of cases of higher stage disease developed the same (stage III -27% and stage IV -8%).
but also in three of the osteoblastic type, one of the low-grade central type, and in the solitary giant cellrich type 10 .Gao FX found positive reactions to S-100 protein, collagen type IV, UEA-1 and factor XIII in 26, 20, 36 and 13 of the total 48 cases respective-