Histopathology and Molecular Pathology of Bone and Extraskeletal Osteosarcomas

Osteosarcoma has been recognized for almost two centuries and is the most common primary, non-haemopoietic malignant tumour of the skeletal system. It is thought to arise from primitive mesenchymal bone-forming cells and its histologic hallmark is the production of osteoid. Other cell populations may also be present, as these types of cells may also arise from pluripotential mesenchymal cells, but any area of bone or osteoid synthesized by malignant cells in the lesion establishes the diagnosis of osteosarcoma (Acchiappati et al, 1965).


Introduction
Osteosarcoma has been recognized for almost two centuries and is the most common primary, non-haemopoietic malignant tumour of the skeletal system. It is thought to arise from primitive mesenchymal bone-forming cells and its histologic hallmark is the production of osteoid. Other cell populations may also be present, as these types of cells may also arise from pluripotential mesenchymal cells, but any area of bone or osteoid synthesized by malignant cells in the lesion establishes the diagnosis of osteosarcoma (Acchiappati et al, 1965).
It accounts for approximately 20% of all primary malignant bone tumours. In the United States, the incidence of osteosarcoma is 400 cases per year (4.8 per million population <20 y). The incidence is slightly higher in blacks than in whites , in males than in females. It has a bimodal age distribution and a propensity to develop in adolescents and young adults (Dix et al, 1983;Soloviev, 1969;Wilimas, et al, 1977, Dorfman & Czerniak, 1995, with 60% of tumours occurring in patients younger than 25 years of age and only 13% to 30% in patients older than 40 years (de Santos et al, 1982;Huvos, 1986). Osteosarcoma is very rare in young children (0.5 cases per million per year in children <5y).
Osteosarcoma can occur in any bone, but most tumours originate in the long bones of the appendicular skeleton, near metaphyseal growth plates, especially the distal femur (42%, 75% of which in the distal femur), followed by the proximal tibia (19%, 80% of which in the proximal tibia) and proximal humerus (10%, 90% of which in the proximal humerus) (Goes M, 1952). In the long bones the tumour is most frequently centered in the metaphysis (90%), infrequently in the diaphysis (9%) and rarely in the epiphysis (Tsuneyoshi & Dorfman, 1987). Other significant locations are the skull and jaw (8%) and pelvis (8%) (Benson et al, 1984).
The clinicopathologic features of osteosarcoma form the basis of its classification, most importantly including the histologic features, the biologic potential (grade), relation to bone (intramedullary or surface), multiplicity (solitary and multifocal) and the pre-existing state of underlying bone (primary or secondary).

Chondroblastic osteosarcoma
Chondroid matrix is predominant in chondroblastic osteosarcoma, intimately associated with non-chondroid elements. The neoplastic chondrocytes are mostly characterized by severe cytologic atypia and reside in lacunar spaces, hyaline matrix or float singly or in cords in myxoid matrix. Myxoid and other forms of cartilage are uncommon, except in the jaws and in the pelvis.

Fibroblastic osteosarcoma
Ty pically , i s c omposed of f usiform hig hly pleomorphic malignant cells, arranged in a herringbone or storiform pattern, similar to fibrosarcoma or malignant fibrous histiocytoma, with minimal osseous matrix. Although, the degree of atypia is variable, is frequently severe, with numerous mitoses, including atypical forms. In general, the lack of significant amounts of osteoid, bone or cartilage, relegates them to subtypes of fibroblastic osteosarcoma.
Although, there is a tendency for metastases of osteosarcomas to mimic the primary, exceptions are frequent and is a higher than expected incidence of fibroblastic differentiation in metastases.

Small cell osteosarcoma
It comprises 1.5% of osteosarcomas, with slight predilection for females and is composed of small cells with variable degree of osteoid production (Sim et al, 1979). According to the predominant cell pattern, tumours are classified to round cell type or short spindle cell type. Nuclei can be very small to medium sized, with scanty amount of cytoplasm, comparable to those of Ewing sarcoma and to large cell lymphoma respectively. The chromatin distribution can be fine or coarse and the mitoses range from 3 to 5/per high power field-HPF. Lace-like osteoid production is always present, although particular care must be taken to differentiate osteoid from fibrin deposits that may be seen among Ewing sarcoma cells.

Telangiectatic osteosarcoma
When first recognized, telangiectatic osteosarcoma was considered a distinct clinical and pathologic entity (Gaylord, 1903). On the basis of subsequent findings, telangiectatic osteosarcoma was considered a variant of osteosarcoma (Ewing, 1922(Ewing, , 1939.

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It is rare, less than 4% of all cases of osteosarcomas and more frequent in the second decade of life. Although, most frequently affects long tubular bones, has also been noted to arise in extraosseous soft tissues in the forearm, thigh and popliteal fossa (Mirra et al, 1993).
Radiographically, is characterized by typically purely lytic destructive process, without matrix mineralization. Clinically, a pathological fracture is present in one fourth of the cases. On gross examination, there is a dominant cystic architecture in the bone medulla. The cystic space is filled incompletely with blood clot, described as 'a bag of blood'. Histologically, the tumour is characterized by dilated, blood-filled or empty spaces, separated by thin septae, simulating aneurysmal bone cyst. The cystic spaces are lined by benign osteoclast-like giant cells, without endothelial lining. The septae are cellular, containing highly malignant, atypical mononuclear tumour cells with high mitotic activity, including atypical mitoses. The amount of osteoid varies, but usually is fine, lace-like and of minimal amount, although it tends to be more prominent in metastatic foci. Therefore, it is imperative that the entire specimen is meticulously examined histologically.

Low grade central (well differentiated intraosseous/intramedullary) osteosarcoma
It accounts for 1-2% of all osteosarcomas and is the medullary equivalent to parosteal osteosarcoma (Kurt et al, 1990;Unni et al, 1977). There is a tendency for a slightly older age and slightly longer symptomatology in comparison to the conventional osteosarcoma. It is important to recognize this subtype, since the patient tends to do much better, than those with conventional osteosarcoma. It is a low grade fibroblastic osteosarcoma and is histologically identical to parosteal osteosarcoma.
Histologically, contains long, parallel trabeculae or round islands of woven bone intimately associated with a mildly to moderately cellular population of cytologically bland neoplastic spindle cells with variable amounts of osteoid production. Nuclear enlargement and hyperchromasia are generally evident with occasional mitotic figures (1-2 mitoses per 10 high power fields-HPFs). When involving the medullary canal it may be confused with fibrous dysplasia (FD). Radiologically, FD is homogeneous ('ground glass' appearance), whereas the well differentiated osteosarcoma is less so, and may show trabeculations. Furthermore, a permeative growth pattern into the pre-existing lamellar bone or fatty marrow is diagnostic. Microscopically, the trabeculae of FD tend to be rather short and curled, whereas those of well differentiated osteosarcoma are longer and may be arranged in parallel arrays. Again, cytologic atypia should be sought and is diagnostic if found.
Conventional osteosarcomas often have better differentiated, innocuous 'normalized' areas. These should not be underdiagnosed as low grade osteosarcoma, since biologically they behave accordingly to their more aggressive foci.
Desmoplastic fibromas may infiltrate the bone and can be diagnostically challenging. In these cases, osteoid production should be sought. Radiographic evidence of matrix production is helpful in establishing the correct diagnosis. www.intechopen.com

Secondary osteosarcomas
Secondary osteosarcomas are bone forming sarcomas occurring in bones that are affected by preexisting abnormalities, the most common being Paget disease and radiation change.

Paget osteosarcoma
Incidence of sarcomatous change in Paget disease is estimated to 0.7-0.95%, with osteosarcomas representing 50-60% of Paget sarcomas (Haibach et al, 1985;Schajowicz et al, 1983). It is more common in men, with median age of 64 years and is usually observed in patients with widespread Paget disease (70%). Most tumours arise in the medulla. Histologically are high grade sarcomas, mostly osteoblastic or fibroblastic. A great number of osteoclast-like giant cells may be found. Telangiectatic and small cell osteosarcomas have been reported.

Post-radiation osteosarcoma
They constitute 3.4-5.5% of all osteosarcomas and 50-60% of radiation-induced sarcomas. It is estimated that the risk of developing osteosarcoma in irradiated bone is 0.03-0.8% (Huvos et al, 1985;Mark et al, 1994). Children treated with high-dose radiotherapy and chemotherapy are at the greater risk, and the prevalence of post-radiation osteosarcomas is increasing as children survive treatment for their malignant tumour. It can develop in any irradiated bone, with most common locations the pelvis and the shoulder. The modified criteria for postradiation cancer initially promulgated in 1948 by Cahan and associates are as follows : (1) the patient received irradiation, (2) the neoplasm occurred in the radiation field, (3) a latent period of years had elapsed, (4) histologic or radiographic evidence for the pre-existent osseous lesion, a benign tumour or non-bone forming malignancy (Cahan et al, 1948).
Many of the reported cases of osteosarcoma arising in fibrous dysplasia have also been complicated by radiation therapy.

Osteosarcomas in other benign precursors
Other rare instances of secondary osteosarcomas have included cases arising in association with bone infarcts, endoprostheses and already mentioned fibrous dysplasia. Infarct associated sarcomas as well as malignant tumours at the site of prosthetic replacements (Brien et al, 1990), or at the site of prior fixation, most commonly show the histological pattern of malignant fibrous histiocytoma (MFH), with a minority being osteosarcomas.

Parosteal osteosarcoma
It is the most common type of osteosarcoma of the bone surface, although it accounts for about 5% of all osteosarcomas, with a slight female predominance (Okada et al, 1995). It is a low grade, slow growing neoplasm, with a predilection for the posterior aspect of the distal femur, where presents as a hard lobulated mass attached to the underlying cortex with a broad base. Histologically, it is a well differentiated fibro-osseous neoplasm, consisting of well formed bony trabeculae embedded in a hypocellular fibrous stroma. The bony trabeculae can be arranged in parallel strands, simulating normal bone and may or may not show osteoblastic rimming. The intertrabecular stroma is hypocellular with minimal atypia, whereas it can be more cellular with moderate cytologic atypia in 20% of cases. About 50% of the tumours will show cartilaginous differentiation, in the form of hypercellular nodules of cartilage within the tumour substance or as a cup on the surface. When present, the cartilage cup is mildly cellular with mild cytologic atypia and lacks the 'columnar' appearance seen in osteochondromas.
About 15% of the tumours will show high grade spindle cell sarcoma (dedifferentiation), more often at the time of recurrence, in the form of osteosarcoma, fibrosarcoma or MFH (Wold et al, 1984).
Its diagnosis can be very difficult and the differential diagnosis has included diverse entities, such as: myositis ossificans, fracture callus, ossifying haematoma, osteochondroma, extraosseous osteosarcoma, parosteal chondroma, desmoplastic fibroma and osteoma. Therefore, parosteal osteosarcoma, like no other tumour, implies the necessity of close cooperation of all involved disciplines for the correct diagnosis.

Periosteal osteosarcoma
It accounts for less than 2% of all osteosarcomas, more common than high grade surface osteosarcoma, but about one third as common as parosteal osteosarcoma (Unni et al, 1976). Unlike parosteal osteosarcoma, which extends from the cortex like a bony knob, periosteal osteosarcoma tightly encases the bone, like a glove. Unlike other osteosarcomas, it tends to involve the diaphysis. Radiologically, it is a circumferential surface mass, less radiodense than parosteal osteosarcoma. Mineralization occurs as ring-shaped radiodensities or as streaks of reactive bone radiating from the surface. Lesions can be best visualized by MRI scan. A sub-periosteal lesion with a bright white signal is present, indicating its high cartilage content. Histologically, it has the appearance of a moderately differentiated, grade 2/3 chondroblastic osteosarcoma. It consists almost entirely of lobules of cellular, atypical cartilage, with bone formation in the centre of the lobules, separated by thin strands of fibrous tissue. Careful scrutiny of the fibrous component, reveals seams of neoplastic osteoid, usually at the outer surface of the neoplasm, which distinguishes it from surface chondrosarcoma.

High grade surface osteosarcoma
A high grade, bone-forming malignant tumour, arising from the bone surface, which comprises less than 1% of all osteosarcomas. Histologically, is similar to conventional osteosarcoma. Regions of predominantly osteoblastic, chondroblastic, or fibroblastic differentiation may predominate. However all tumours will show high cytologic atypia and lace-like osteoid. The pattern of osteoid production and the high grade cytologic atypia help to separate it from parosteal osteosarcoma. High grade surface osteosarcoma with chondroblastic differentiation may be confused with periosteal osteosarcoma. The degree of cytologic atypia is greater in high grade surface osteosarcoma and the tumours generally show larger spindle cell areas. Finally, unlike dedifferentiated parosteal osteosarcomas, low grade regions are not found in high grade surface osteosarcomas.

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Cortical destruction and invasion into the medullary canal in high-grade juxtacortical (periosteal and high-grade surface) osteosarcomas, is often absent, but when present, is only focal. If extensive, it becomes difficult, if not impossible to distinguish an intramedullary tumour with an eccentric soft tissue component from a surface neoplasm with extensive invasion of the medullary canal.

Extraskeletal osteosarcomas
Extraskeletal osteosarcomas (EOs) or soft tissue osteosarcomas (STOs) are rare sarcomas arising in extraskeletal somatic soft tissue, in which the neoplastic cells produce osteoid and/or bone matrix, therefore recapitulating the phenotype of osteoblasts. By definition, it is a high grade mesenchymal neoplasm that produces osteoid, bone and chondroid material, shows no evidence of epithelial component and is located in soft tissues, without attachment to bone or periosteum, as determined by X-ray findings or inspection during the operative procedure. It is significantly less frequent than its osseous counterpart. They account for 1.2% of soft tissue sarcomas and 4% of osteosarcomas (Bane et al, 1990;Sordillo et al, 1983). They typically arise in the deep soft tissues of the proximal extremities, with most common locations the deep soft tissues of the thigh and buttocks, followed in descending order by the upper limb, retroperitoneum, trunk, head and neck (Chung & Enzinger, 1987;Lee et al, 1995;Lidang et al, 1998;Sordillo et al, 1983). Fewer than 10% are superficial, originating in the dermis or subcutis. Cases of EOs arising in unusual sites have been reported, such as the breast, larynx, thyroid gland, parotid, abdominal viscera, including esophagus, small intestine, omentum majum, liver, heart, the urogenital system, including urinary bladder, ureter the prostate and the penis, pleura, mediastinum, ectopic thymus, pulmonary artery, pilonidal area and aorta (Baydar, et al, 2009;Burke & Virmani, 1991;Kemmer et al, 2008;Loose et al, 1990;Micolonghi et al, 1984;Piscioli et al, 1985;Shui et al, 2011;Silver & Tavassoli, 1998;Trowell & Arkell, 1976;Young & Rosenberg, 1987;Wegner et al, 2010;Greenwood & Meschter, 1989). Fewer than 300 cases have been reported to date and their aetiology is essentially unknown. Unlike osseous conventional osteosarcoma, it typically occurs in older adults, with a peak incidence in the fifth and sixth decades of life, in contrast with skeletal osteosarcomas that most commonly affect young adults (Sordillo et al, 1983). Males are affected more frequently than females at a ratio of 1.9:1. Some cases have been associated with a history of prior therapeutic irradiation at the site of the tumour (Logue & Cairnduff, 1991) and trauma (Allan & Soule, 1971). In one case report of primary osteosarcoma of the urinary bladder, prolonged treatment with immunosuppressive medications, including cyclophosphamide for active systemic lupus erythematosus (SLE) has been reported (Baydar et al, 2009). The diagnosis is generally delayed and prognosis is poor, with most patients died within months of diagnosis and with a cause-specific survival rate at 5 years less than 25%. Clinically and mammographically can mimic a benign tumour. Like most of soft tissue sarcomas, tumours may appear grossly circumscribed, however they are microscopically infiltrative. Imaging studies usually reveal compact calcifications/variable mineralization within the mass. Primary extraosseous osteosarcoma should always be included in the differential diagnosis, in the view of a well demarcated calcified mass on image analysis. In the case of extraskeletal osteosarcoma of the breast, an underdiagnosis as a calcified fibroadenoma, should be avoided.
STOs are usually large, ranging in size between 5 and 10 cm, well-circumscribed, grossly heterogeneous tumours that exhibit areas of haemorrhage and/or necrosis. Microscopically, STOs constitute highly cellular, cytologically pleomorphic, mitotically active sarcomas, spindle cell to epithelioid in appearance. The defining feature is the presence of neoplastic osteoid and/or bone. The latter usually presents in a 'lace like' manner, although solid sheets of amorphous osteoid can also be found. When the bone/osteoid matrix is found only focally in the tumour, largely demonstrates a nonspecific, undifferentiated, spindle cell sarcomatous appearance. Lobules of highly cellular, atypical hyaline/fibrocartilage may also present. Osteoclast-like giant cells have also been described. Various histologic subtypes of bone osteosarcoma can be seen in extraskeletal osteosarcoma. Osteoblastic variant is the most common type, followed by fibroblastic, chondroid, telangiectatic, small cell and welldifferentiated variants. Common to all variants is the production of osteoid, intimately associated with tumour cells, which may be deposited in a lacy, trabecular or sheet-like pattern. Neoplastic bone formation is more prominent in the centre of the tumour with the peripheral areas being more cellular, a reverse pattern of myositis ossificans. In essence, any of the microscopic patterns of high-grade intraosseous osteosarcoma may be seen in EO.
The differential diagnosis should always include spindle cell (sarcomatoid) carcinomas and the exclusion of metastasis from a primary osteosarcoma, as well as carcinosarcomas, malignant tumours with osseous metaplasia and sarcomas. Metaplastic ossification, ranging from osteoid to woven bone formation can be seen in synovial sarcoma, epithelioid sarcoma, liposarcoma and carcinosarcoma. Osteogenic differentiation can also be seen as a phenomenon of dedifferentiation in various soft tissue sarcomas. In the above tumours, other histologic lineages or histologic characteristics of original tumours are usually present. The key to diagnosis, is the identification of the matrix surrounding the tumour cells and lack of epithelial differentiation. Furthermore, except osteoblastic, chondroblastic and fibroblastic differentiation, no other lineages of histologic differentiation should be detectable in EOs. Diagnostic confirmation using immunohistochemical markers is necessary to ensure the absence of an epithelial component and exclude the neoplasms of biphasic origin.

The use of cytology in the preoperative investigation of bone tumours with emphasis in osteosarcoma
Fine needle aspiration (FNA) of bone lesions has been performed ever since the technique was introduced (Coley et al, 1931). It has certain advantages over open biopsy, as it is less disruptive to bone and permits multiple sampling without complications. Its main use is to confirm malignancy. Although, cytomorphology of primary bone tumours has been extensively described in correlation with histology (Hajdu, 1975;Stormby & Akerman, 1973Walaas &Kindblom, 1990White et al, 1988), FNA is not as good for diagnosing primary bone tumours.
There are two main indications of FNA of soft tissue and bone lesions: preoperative diagnosis before definitive treatment and the investigation of lesions suspicious of tumour recurrence or metastasis. Eventhough, the first use has limitations, however, at present, the use of FNA as the diagnostic, pre-treatment tool for musculoskeletal tumours is accepted in many orthopaedic centres, provided that certain requirements are fulfilled and that the final cytologic diagnosis is based on the combined evaluation of clinical data, radiographic findings and cytomorphology (Domansky et al, 2010). One important reason, why FNA is preferred upon open biopsy, is when the preoperative evaluation of a lesion as malignant is of most importance, rather the subtype of the lesion. On the other hand, in the use of neoadjuvant treatment (radio-or chemotherapy), before surgery, the FNA diagnosis is of major importance, analogous to histopathologic examination, in regard to subtype and tumour grade. This is mostly the case with small round cell sarcomas. Regarding bone lesions, FNA may also replace open biopsy in the primary diagnosis. It is the task of the pathologist to distinguish benign and malignant primary bone tumours from metastatic deposits and from the wide range of benign and inflammatory reactive lesions of the bone. Furthermore, the pathologist should give a confident diagnosis of the various benign bone tumours and sarcomas, if open biopsy is to be avoided. Skeletal aspiration is not different from other aspirations, but it is important to remember that intact cortical bone cannot be penetrated by regularly used (22 gauge) needles, something that can be easily done with partly destroyed or eroded bone. A 18-gauge needle can be used in intact bone, under anaesthesia and multiple passes can be performed. Many malignant bone tumours have palpable soft tissue involvement, which can be much easier penetrated by the needle. In any case, it is strongly recommended that the pathologist is familiar with the radiological findings, discuss the best approach to the tumour with the radiologist and that non-palpable lesions are aspirated under image-guided techniques. Essentially, when the aspirated material is technically satisfactory, it is suitable for use of specialized techniques, likewise biopsy material, to assist in the diagnosis.
The FNA cytological findings of osteosarcoma have thoroughly been described (Mertens et al, 1982;Walaas &Kindblom, 1990;White et al, 1988). They include mixture of cell clusters and dispersed cells, pleomorphic, spindle and rounded cells with frequent mitoses, including atypical forms, intercellular tumour matrix of osteoid, benign osteoclast-like giant cells, epithelioid tumour cells, which may be of osteoblastic type, or resemble chondroblasts, in osteoblastic and chondroblastic variants respectively, atypical spindle-shaped cells in fibroblastic subtypes. The presence of osteoid-like material can be 'prominent, or scarce, dissociated, or in association with cell clusters, either as moderate-sized fragments, or not easily discernible globule-like particles'. This material could be associated either with the predominat histologic pattern or with the sampled area represented in the smears. Osteoidlike matrix material has been originally described as 'homogeneous or vacuolated eosinophilic plaques' or pink-purple acellular material in May Grunwald Giemsa (MGG) smears and green-blue in Papanicolaou-stained (PAP) smears. Another cause of difficulty in accurately assessing the presence of osteoid in the smears is that osteoid, cartilage and dense collagen fibers appear very similar in both Diff-Quick and PAP stains. Furthermore, osteoid resists the suction, during aspiration, and when aspirated, the small, disrupted fragments lack the characteristic lattice-like pattern observed in histological sections (Mertens & Langnickel, 1982;Nikol et al, 1998).
FNA has proven to be of value in the preoperative diagnosis of breast EOs (Trihia et al, 2007). Its diagnosis is often delayed because of a desceptively benign clinical and radiologic appearance (Watt et al, 1984). Although differential diagnosis from metaplastic carcinoma may not be possible on cytological grounds alone, FNA has proven to be of value in preoperative diagnosis of malignancy and prompt surgical intervention, as clinically and mammographically these lesions can be underdiagnosed as a benign tumour (Trihia et al, 2007).

The role of immunohistochemistry in the diagnosis of osteosarcoma
The diagnostic algorithm of primary bone tumors is, and always has been, a collaborative effort in which clinical, radiologic, and pathologic findings have to be considered. In the majority of cases, the pathologist can rely exclusively on histopathologic examination to provide an accurate diagnosis. In some cases, however, ancillary studies have to be employed to distinguish entities that share morphologic characteristics.
When evaluating a bone tumor, the pathologist is confronted with several difficulties. Bone tumors are rare entities, and not all pathologists are exposed to bone pathology with the frequency needed to gain the necessary level of diagnostic expertise. Also, certain osseous tumors share histopathologic features and, in many cases, important diagnostic features may not be readily evident in small specimens. Finally, intramedullary lesions often must be decalcified, a process that may be associated with loss in cellular morphologic detail. All of these factors complicate the diagnostic process. Diagnosis for many entities can be reached by the evaluation of histopathologic features alone, or can be interpretated in the context of clinico-radiologic findings, but for others, only a differential diagnosis can be reached without ancillary studies.
Several studies have shown that gentle decalcification methods preserve antigenicity relatively well for the most commonly used markers.
One of the greatest challenges in bone and soft tissue pathology is the reliable recognition of osseous matrix production in malignant lesions. Although several antigens have been explored, unfortunately, little is known about the antigenic specificity of normal bone tissue and bone neoplasms and currently there is no specific marker to distinguish the bone matrix from its collagenous mimics.
Since late 1990's, due to their central function in the process of mineralization, a group of proteins have been proposed for tumor diagnosis: alkaline phosphatase, osteonectin, and osteocalcin. Osteocalcin (OCN) and osteonectin (ONN) have been applied to paraffin sections and been used to highlight osteoid. OCN is one of the most prevalent intraosseous proteins and is produced exclusively by bone-forming cells-osteoblasts and therefore has received special attention as a specific marker (Fanburg et al, 1997(Fanburg et al, , 1999Takada et al, 1992). In the detection of bone-forming tumors, osteocalcin has been associated with 70% sensitivity and 100% specificity, compared with the 90% sensitivity and 54% specificity reported for ONN. Nevertheless, OCN is rarely used in clinical practice. ONN is a protein that is implicated in regulating the adhesion of osteoblasts and platelets to their extracellular matrix, as well as early mineralization and should only be used as part of a panel of reagents, directed at several lineage-related proteins (Serra et al, 1992;Wuisman et al, 1992).
Strong labeling of the osseous isozyme of alkaline phosphatase has been used to distinguish EO from other pleomorphic sarcomas. The major drawback of this marker is that it can only be used on cryostat sections and imprint smears.
Ancillary techniques have a limited role in diagnosing osteosarcoma, as the tumour is largely recognized by its morphologic features. Because of the many varieties of osteosarcoma, diverse tumours are considered in its differential diagnosis.
Currently, immunohistochemistry has limited application in the differential diagnosis of primary bone tumors. In general, osteosarcoma has a broad immunoprofile that lacks diagnostic specificity. Vimentin, OCN, ONN, S-100 protein, muscle protein smooth muscle actin (SMA), neuron specific enolase (NSE) and CD99 are some of the antigens that are commonly expressed. Importantly, some tumours also stain with antibodies to keratin and epithelial membrane antigen (EMA). Also, it's worth noticing, that osteosarcoma usually does not stain with antibodies to factor VIII and CD31.
Osteosarcoma is distinguished from benign tumours and fibro-osseous lesions, by virtue of its infiltrative growth pattern, with the tumour replacing the marrow space and surrounding bony trabeculae, which may also serve as scaffolding for the deposition of neoplastic bone.
Telangiectatic osteosarcoma differs from aneurysmal bone cyst because it contains cytologically malignant cells within the cyst walls, whereas the cells in aneurysmal bone cyst are banal in appearance.
Biopsies of osteosarcoma that lack neoplastic bone can be problematic because its immunophenotype can generate a broad list of differential diagnoses that include Ewing's sarcoma/Primitive Neuroectodermal Tumour-PNET, metastatic carcinoma and melanoma, leiomyosarcoma and malignant peripheral nerve sheath tumour.
The subtype of osteosarcoma that most likely will benefit from the application of an immunohistochemistry panel is the "small-cell" type. The diagnosis of this entity is difficult due to the paucity of osteoid and the similarity to other small round-cell tumors. Although the antigenic profile of small-cell osteosarcoma is unknown, expression of markers specific for other small-cell tumors, help in ruling out this diagnosis.
In these circumstances, immunohistochemical analysis, electron microscopic evaluation and molecular studies may be helpful. Small cell osteosarcoma is distinguished from Ewing's sarcoma by the presence of neoplastic bone and dilated rough endoplasmic reticulum by electron microscopy, as osteosarcoma cells have the features of mesenchymal cells with abundant endoplasmic reticulum and the matrix contains collagen fibres, which may show calcium hydroapatite crystal deposition and the absence of t(11;22) translocation, or its variants, which is diagnostic of Ewing's sarcoma.
MIC-2 gene product (CD99), which is located in the short arm of the sex chromosome, encodes a surface protein, first described in T-cell and null-cell acute lymphoblastic leukemia. Osteosarcoma usually has a diffuse moderate to strong intracytoplasmic staining for CD99. Tumour cells of the small cell variant of osteosarcoma may be positive for CD99, vimentin, osteocalcin, osteonectin, smooth muscle actin, Leu-7 and KP1.
The differential diagnosis of osteosarcoma from other sarcomas (e.g., malignant fibrous histiocytoma, fibrosarcoma) is important because of the specific therapy available for osteosarcoma patients. Most osteosarcomas express vimentin and, according to some authors, some tumors focally express cytokeratin and desmin, although these findings have not been widely confirmed. Bone matrix proteins, such as OCN, alkaline phosphatase, and ONN, are expressed in osteosarcomas. However, their presence has also been detected in chondrosarcomas, Ewing's sarcoma, fibrosarcomas, and malignant fibrous histiocytomas. Caution should also be used in the interpretation of focal expression of a variety of markers (e.g., S-100, actin, epithelial membrane antigen) found occasionally in otherwise typical osteosarcomas. Extraskeletal osteosarcomas of the fibroblastic subtype often have sparse amounts of osteoid and can be differentiated from malignant fibrous histiocytoma on the basis of strong expression of alkaline phosphatase. Chondroblastic osteosarcoma and chondrosarcoma, however, cannot be distinguished immunohistochemically.
The different types of collagen present in the bone matrix are also produced by other tumors and therefore have no application in differential diagnosis. The basic calponin gene, a smooth muscle differentiation-specific gene that encodes an actin-binding protein involved in the regulation of smooth muscle contractility, is expressed in osteosarcomas (Yamamura, et al, 1998).
The list of entities included in the differential diagnosis of MFH is extensive. Immunohistochemistry helps in the distinction of MFH (CD68+, OCN-, alkaline phosphatase-) from leiomyosarcoma (CD68-), malignant neurilemmoma (S-100+) and from fibroblastic osteosarcoma (occasionally positive for both OCN, alkaline phosphatase). The distinction of cytokeratin-positive MFH from sarcomatoid carcinoma may be impossible by immunohistochemistry and is best accomplished by electron microscopy.
Finally, stress fracture and accompanying callus can sometimes be confused with osteosarcoma because the reactive bone and cartilage are deposited around pre-existing bony trabeculae, mimicking an infiltrative pattern of growth. However, the cells in reactive tissues are banal and osteoblastic rimming is usually present.
In conclusion, although immunohistochemistry does not currently play an important diagnostic role in primary bone tumors as it does in soft-tissue counterparts, research efforts to characterize the histogenesis of many of these neoplasias may offer new alternatives for diagnosis in the near future. For the distinction of primary tumors versus metastases of nonosseous origin and for the characterization of a small subset of neoplasias, such as those with small round-cell morphology, immunohistochemistry remains the technique of choice.

Molecular pathology of osteosarcoma
Traditionally, our understanding of osteosarcoma has been largely based on anatomic and histologic features. However, recent studies in the molecular pathology of osteosarcoma have provided new insight into its pathogenesis. Through the identification of molecular pathways of osteosarcoma development and progression, the roles played by mutated tumor suppressor genes, oncogenes, and cell cycle regulatory molecules in bone oncogenesis, differentiation, cell death and cell migration have been explored. Furthermore, numerous cytogenetic abnormalities have been associated with osteosarcoma, including chromosomal amplifications, deletions, rearrangements, and translocations.
Thus, the diagnostic and prognostic significance of molecular aberrations are beginning to be evaluated and novel approaches for therapeutic interventions of osteosarcoma are being developed.

Bone growth, differentiation and osteosarcoma tumorigenesis
It is well known that osteosarcoma has a propensity for developing in bone growth plates characterized by rapid bone turnover during childhood and adolescence (Broadhead et al., 2011;Gelberg et al., 1997) Additionally, patients affected by Paget's disease, a disorder characterized by both excessive bone formation and breakdown, also have a higher incidence of osteosarcoma (Vigorita, 2008). These observations, along with the knowledge that normal bone differentiation process occurs in the epiphyseal growth plates, strongly suggest that osteosarcoma is caused by genetic and epigenetic disturbances in the osteoblast proliferation and differentiation pathways Thomas et al., 2006).
Among these factors, Runx2 is the most important regulator of bone development and serves as a hub to direct progenitors to osteogenic lineage through BMP-induced osteogenesis, synergistically inducing many terminal differentiation markers (Lian et al., 2006;Nakashima et al., 2002;Thomas et al., 2004;Yamaguchi et al., 2000). Additionally, Runx2 associates with p27KIP1 protein and through interaction with the hypophosphorylated form of Rb and transactivation of Osterix transcription factor, promotes terminal cell cycle exit and the formation of a differentiated osteoblastic phenotype (Nakashima et al., 2002;Nishio et al., 2006;Thomas et al., 2004).
Furthermore, the canonical Wnt pathway has been identified to play a crucial role in osteoblast differentiation, as evidenced by the fact that Wnt3a expression leads to cell proliferation and suppression of osteogenic differentiation in adult mesenchymal stem cells (Boland et al., 2004), and that multiple aberrations in the Wnt signaling pathway have been associated with osteosarcoma tumorigenesis (Haydon et al., 2002, Reya & Clevers, 2005, Clevers, 2006. Disruption of the well-coordinated balance between osteogenic progenitors proliferation and differentiation may lead to osteosarcoma development. The defects caused by genetic (eg, activation of oncogenes or inactivation of p53 and RB tumor suppressor genes) and epigenetic alterations may occur at different stages of osteogenic differentiation leading to more or less aggressive tumorigenic phenotypes (Kansara and Thomas, 2007).
Moreover, osteosarcoma cells utilize an alternative lengthening of telomere (ALT) pathway that prevents telomere shortening, allowing the tumor cells to evade senescence and resemble their stem cell progenitors (Wang, 2005).

Osteosarcoma invasion and metastasis
Osteosarcoma is an aggressive tumor with high metastatic potential. Osteosarcoma invasion of bone and metastasis to other organs relies on complex cell-cell and cell-matrix interactions. The www.intechopen.com invasion and metastatic sequence involves the detachment of osteosarcoma cells from the primary site, lysis of bone matrix, local migration, invasion through stromal tissue, intravasation, and extravasation. In this process, interactions between osteosarcoma cells, osteoblasts and osteoclasts are the main events leading to the substantial osteolysis exhibited by some osteosarcomas as a result of increased osteoclastic activity.
During the initial stages of osteosarcoma invasion, TGF-is released from the degraded bone matrix and acts on osteosarcoma cells, stimulating the release of PTHrP, interleukin-6 (IL-6) and interleukin-11 (IL-11) (Quinn et al., 2001). These cytokines then stimulate osteoclasts, facilitating further invasion and release of proresorptive cytokines. Osteoblasts function as mediators in this process of bone resorption. Osteosarcoma cells release endothelin-1 (ET-1), VEGF, and PDGF in response to the hypoxic and acidotic conditions leading to angiogenesis and stimulation of osteoblastic function (Kingsley et al., 2007;Chirgwin et al., 2007). PTHrP and IL-11 also act on osteoblasts, stimulating increased expression of receptor activator of nuclear factor κB ligand (RANKL). RANKL is a main mediator of osteoclast differentiation and activity, and osteosarcoma cells produce RANKL independently (Kinpara, 2000). RANKL activates osteoclasts through binding to RANK on the osteoclast surface. RANK expression is regulated by cytokines IL-1, IL-6, IL-8, tumour necrosis factor-(TNF-), PTHrP, and TGF- (Hofbauer & Heufelder, 1998).
Receptor-ligand binding leads to activation of both NFκB and MAPK pathways, with a resulting increase in nuclear factor of activated T cells (NFATc1) activity. RANK/RANKL also activates the c-Fos component of AP-1, resulting in additional NFATc1 upregulation. NFATc1 then activates the transcription of genes involved in osteoclast activity and maturation (Takayanagi, 2007).
Activated osteoclasts release proteases such as cathepsin K (Cat K) necessary for breakdown of collagen I, osteopontin, and osteonectin (Stoch & Wagner, 2008) aiding the invasion process (LeGall et al., 2007). This protease is essential for osteoclast function in normal bone remodelling and also in pathological states of osteolysis. For patients with high-grade metastatic osteosarcoma, low Cat K levels at the time of diagnosis confers a better prognosis (Husmann et al., 2008).
Invasion of the surrounding tissues by osteosarcoma also involves degradation of the extracellular matrix. Matrix metalloproteinases (MMPs) are principally involved in the breakdown of the extracellular matrix and are regulated by natural inhibitors such as tissue inhibitors of MMPs (TIMPs), RECK, and 2 macroglobulin (Birkedal-Hansen et al., 1993;Chakraborti et al., 2003). In the setting of osteosarcoma, MMPs break down extracellular collagens, facilitating both tumour and endothelial cell invasion. MMPs play also a role in angiogenesis aiding further the metastatic process.
The urokinase plasminogen activator (uPA) and its receptor (uPAR) system is another key regulator of osteosarcoma invasion, which acts as an activator of pro-MMPs. An inverse relationship between uPA levels and overall survival has been demonstrated in osteosarcoma cases (Choong et al., 1996).

Syndromes associated with osteosarcoma
A variety of genetically based diseases and syndromes show a susceptibility to the development of osteosarcoma, such as Paget's disease, Rothmund-Thomson, Bloom,Werner and Li-Fraumeni syndromes, as well as Hereditary Retinoblastoma.
In patients affected by Paget's, a hereditary disorder characterized by rapid bone remodelling leading to dysregulated bone turnover, about 1 % of them will develop osteosarcoma. This percentage accounts for a substantial fraction of the osteosarcoma cases diagnosed over 60 years of age (McNairm et al., 2001). Although the complete pattern of genetic aberrations leading to Paget's disease remains unclear, LOH18CRI located at 18q21-q22 locus has been identified as the major underlying genetic abnormality linked to Paget osteosarcoma (Cody et al., 1997;Good et al., 2002;Hansen et al.,1999;Nelissery et al., 1998 ). Furthermore, the genes SQSTMI at 5q31 and MAPK8 at 5q35qter, which are implicated in IL-1/TNF and RANK signalling pathways, respectively, seem to be involved in Paget osteosarcoma pathogenesis (Kansara & Thomas, 2007).
Rothmund-Thomson, Bloom and Werner syndromes are characterized by germline mutations in RecQ helicases genes whose products are responsible for separation of doublestranded DNA prior to replication (German, 1993;Goto et al., 1996;Hickson, 2003). Among them Rothmund-Thomson syndrome, an autosomal recessive disorder, is related to the highest osteosarcoma incidence (32 %) with a tendency to occur at a younger age Wang et al, 2003;Wang et al., 2005).
Li-Fraumeni syndrome patients are characterized by germline mutations in p53 gene and have an increasesd risk in developing osteosarcomas among other malignancies (Malkin et al., 1990).
Hereditary Retinoblastoma patients have germline mutations in Rb1 tumor suppressor gene and are predisposed to develop osteosarcoma (Araki et al., 1991). Such bone sarcomas are likely to show LOH at 13q and molecular alterations of the Rb1 gene (Andreassen et al., 1993;Wadayama et al., 1994;Wunder et al., 1991).
Recent studies have identified amplifications of chromosomes 6p21, 8q24, and 12q14, as well as loss of heterozygosity of 10q21.1, as being among the most common genomic alterations in osteosarcoma. Furthermore, patients carrying these aberrations had a poorer prognosis (Smida et al., 2010).
Cytogenetic features of gene amplification, such as ring chromosomes, double minutes, and homogeneously staining regions are frequently identified in conventional osteosarcomas (Menghi-Sartorio et al., 2001).
Despite intensive research, no consensus specific chromosomal aberrations that could be used diagnostically in osteosarcoma tumors have been identified.

DNA copy numbers
Osteosarcoma tumors contain multiple random chromosomal aberrations and only a few deletions and amplifications appear common to comparative genomic hybridization (CGH) studies (Atiye et al., 2005;Selvarajah et al., 2008).
In a recent study with 10 osteosarcomas, CGH whole-genome analysis showed changes including: hypomethylation, gain, and overexpression of histone cluster 2 genes at chromosome 1q21.1-q21.3; loss of chromosome 8p21.2-p21.3 and underexpression of DOCK5 and TNFRSF10A/D genes; and amplification-related overexpression of RUNX2 at chromosome 6p12.3-p21.1. Amplification and overexpression of RUNX2 could disrupt G2/M cell cycle checkpoints and bone differentiation leading to genomic instability. Disruption of DOCK5-signaling, together with p53 and TNFRSF10A/D related cell cycle and death pathways, may play a critical role in abrogating apoptosis (Sadicovic et al., 2009). Diploid ploidy pattern by DNA fluocytometry has been reported to be a poor prognostic sign (Kusuzaki et al., 1999).

Loss of heterozygosity (LOH)
Loss of heterozygocity are detected more frequently at the long arms of chromosomes 3, 13 and 18 and at the short arm of chromosome 17 (Kruzelock et al., 1997). As the incidence of LOH is high at 3q26.6-26.3, this area has been suggested to harbour a putative suppressor gene (Kruzelock et al., 1997).

Telangiectatic osteosarcoma
A limited number of cases show highly complex chromosomal changes, and only one case is characterized by trisomy 3 (Bridge et al., 1997;Fletcher et al., 1994;Hoogerwerf et al., 1994). Mutations in the TP53 and RAS genes, LOH at the TP53, CDKN2A and RB1 loci, and amplification of the MDM2 and MYC genes are rare in telangiectatic osteosarcomas (Radig et al., 1998).
Telangiectatic osteosarcoma can be distinguished from aneurysmal bone cyst by the balanced tranlocations between the short arm of chromosome 17 and the long arm of chromosome 16 (Panoutsakopoulos et al., 1999). This rearrangement is characteristic of aneurysmal bone cyst. However, there are many variations on this theme, and at least five different chromosomes can serve as translocation partners with chromosome 17 (Dal Cin et al., 2000;Herens et al., 2001;Wyatt-Ashmead et al., 2001).

Small cell osteosarcoma
No specific cytogenetic abnormalities have been detected in small cell osteosarcoma.
Small cell osteosarcoma can be distinguished from Ewing sarcoma by the presence of neoplastic bone and the absence of the t(11:22) translocation or one of its variants.

Low grade central osteosarcoma
Low grade central (intramedullary) osteosarcoma show minimal chromosomal imbalances compared with the complex cytogenetic aberrations identified in high grade osteosarcoma. Cytogenetic studies revealed ring chromosomes of amplified regions 12q13-15, as well as abnormalities in chromosome 6p, 14 and 15 (Werner et al., 1998).
Comparative genomic hybridization demonstrate recurrent gains in chromosomal regions at 12q13-14, 12p, and 6p21 (Tarkkanen et al., 1998). MDM2, CDK4, and SAS at the 12q13-15 a m p l i c o n h a v e b e e n r e p o r t e d t o b e a m p lified at frequencies of 35%, 65% and 15%, respectively .

Postradiation osteosarcoma
Cytogenetic and DNA copy number changes are complex and similar to those in conventional osteosarcomas . Postradiation osteosarcomas frequently exhibit 3p and 1p chromosomal losses compared to sporadic osteosarcoma which show more gains than losses (Mertens et al., 2000;. In one study, a high (58%) incidence of TP53 mutations was found (Nakanishi et al., 1998).
Comparative genomic hybridization studies indicate 12q13-15 amplified region in the chromosomal rings (Szymanska et al., 1996). The SAS, CDK4, and MDM2 genes are coamplified and overexpressed in the majority of cases (Wunder et al., 1999).
Mutations in RB1 (Wadayama et al., 1994) or microsatellite instability  have not been found to be present in parosteal osteosarcoma.

Extraskeletal osteosarcomas
Clonal chromosomal aberrations with complex patterns have been reported (Mandahl et al., 1989;Mertens et al. 1998). So far, no genetic differences between osteosarcomas of bone and extraskeletal origin have been identified.

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Histopathology and Molecular Pathology of Bone and Extraskeletal Osteosarcomas 23

Molecular genetic alterations
A number of molecular defects in tumor suppressor genes, oncogenes, bone differentiation genes and genes involved in cell migration have been observed in osteosarcomas.

Tumor suppressor genes
The p53 and retinoblastoma (Rb) genes are the major tumor-suppressor genes affected in osteosarcoma (Marina et al., 2004).The p53 gene is mutated in 22% of osteosarcomas (Ta et al., 2009) and its expression seems to be higher in low grade osteosarcomas and correlate with reduced metastatic disease and improved survival . p53 mutation has also been shown to be more common in high-grade conventional osteosarcomas versus low grade central osteosarcomas (Park et al., 2004). However, other studies showed no correlation between survival and the p53 protein, while coexpression of p53 and Pglycoprotein was associated with a poorer prognosis (Lonardo et al., 1997;Park et al., 2001).
In addition to p53, the Rb tumour suppressor has also been implicated in the tumorigenesis of osteosarcoma. Both germ-line and somatic mutations of Rb confer an increased risk of osteosarcoma . Loss of heterozygosity for Rb has been reported to confer both an improved and poorer prognosis for patients (Heinsohn et al., 2007;Wadayama et al., 1994;Feugeas et al., 1996).

Oncogenes
Transcription factors c-Fos and c-Jun are significantly upregulated in high-grade osteosarcomas compared with benign osteogenic lesions and low-grade osteosarcomas (Wu et al., 1990; and are associated with the propensity to develop metastases . Another transcription factor involved in cell proliferation, is c-Myc, whose amplification has been implicated in osteosarcoma pathogenesis and resistance to chemotherapeutic drugs. Overexpression of Myc in bone marrow stromal cells leads to osteosarcoma development and loss of adipogenesis (Shimizu et al., 2010).
Overexpression of MET (Ferracini et al., 1995;Rong et al., 1993) and c-Fos (Wu et al., 1990) has been reported in more than 50% of osteosarcoma cases, whereas c-Myc is overexpressed in less than 15% of cases (Barrios et al., 1993;. c-Myc, c-Fos, and cathepsin L have been shown to be overexpressed in a high proportion of relapsed tumours and metastases Park et al., 1996).
Transforming growth factor beta (TGF-) family proteins are also implicated in osteosarcomagenesis through impairment of osteoblast proliferation, differentiation and cell death. High-grade osteosarcomas are found to express TGF-1 in significantly higher amounts than low-grade osteosarcomas .
IGF (insulin-like growth factor)-I and IGF-II are growth factors that are often overexpressed by osteosarcomas (Rikkof et al., 2009).
Connective tissue growth factor (CTGF) is related to a number of proteins in the CCN family (CTGF/Cyr61/ Cef10/NOVH). This protein family act via integrin signalling pathways (Lau et al., 1999) and, is involved in a diverse range of functions including adhesion, migration, proliferation, survival, angiogenesis, and differentiation. A related protein, CCN3, was found to be overexpressed in osteosarcoma and associated with a worse prognosis (Perbal et al., 2008).
In one study, a substantial percentage (42.6%) of osteosarcomas displayed high levels of HER2/neu (c-erbB2,ERBB2) expression, relative to adjacent normal tissues .
Recently, it was shown that APEX1 gene was amplified in osteosarcomas and that APEX1 expression was an independent predictor of the osteosarcoma local recurrence and/or metastasis (Yang et al., 2010).

Gene expression
Osteosarcoma cells share many similar features to undifferentiated osteoprogenitors, including a high proliferative capacity, resistance to anoikis, and expression of many osteogenic markers, such as CTGF, Runx2, ALP, Osterix, Osteocalcin and Osteopontin Haydon et al., 2007;Luu et al., 2007). Furthermore, the more aggressive osteosarcoma phenotypes often have features of early osteogenic progenitors, while less aggressive tumors seem to be more similar to osteogenic mesenchymal stem cells that have progressed further along the differentiation cascade (Broadhead et al., 2011;He et al., 2010).
Osteogenic differentiation of mesenchymal stem cells can be monitored by using bone morphogenetic proteins (BMPs) and their downstream mediators, such as Id proteins and connective tissue growth factor (CTGF), as early markers, alkaline phosphatase and Osterix as early/middle markers and osteocalcin and osteopontin as late markers of bone formation .
Analysis of the expression of these osteogenic markers in osteosarcoma cells demonstrates a much lower alkaline phosphatase expression in tumor cells when compared to committed osteoblastic cell lines (Harris et al., 1995;Luo et al., 2008). Similarly, the late osteogenic markers osteopontin and osteocalcin are highly expressed in mature, differentiated osteoblasts, but are minimally expressed in both primary OS tumors and OS cell lines (Cheng et al., 2003;Luu et al., 2007). CTGF, a multifunctional growth factor that is normally upregulated at the earliest stages of osteogenic differentiation, also shows elevated basal expression in human osteosarcoma cells (Luo et al., 2004).
Recent studies have revealed that WWOX, a gene involved in bone differentiation, is deleted in 30% of osteosarcoma cases and WWOX protein is absent or reduced in ~60% of osteosarcoma tumors (Del Mare et al., 2011). WWOX associates with Runx2, a key regulator of bone differentiation. Interestingly, Runx2 has a very low expression in osteosarcoma cell lines. Runx2 also associates with BMPs, Rb, and p27KIP1 playing a crucial role in cell cycle and bone differentiation regulatory pathways. Thus, it is natural that any alterations would lead to uncontrolled proliferation and loss of differentiation. Accordingly, high-grade osteosarcomas show decreased expression of p27KIP1, while lower-grade tumors have detectable p27KIP1 levels (Thomas et al., 2004).
Bone morphogenetic proteins (BMPs) such as bone morphogenetic protein-6 and bone morphogenetic protein receptor 2 are expressed in more than 50% of osteosarcomas and related to poor prognosis (Gobbi et al., 2002;Guo et al., 1999;Yoshikawa et al., 2004).
As above mentioned, Wnt pathway plays a crucial role in osteoblast differentiation and elevated levels of -Catenin, an important regulator of the Wnt pathway, are correlated with osteoprogenitor proliferation and osteosarcoma metastasis (Iwaya et al., 2003). In addition, osteosarcoma tumors overexpressing LRP5, a Wnt co-receptor, are associated with a poorer prognosis and decreased patient survival (Hoang et al., 2004). Furthermore, Wnt mutations causing excessive Wnt (Wingless and int) signalling are associated to decreased survival and increased metastatic capacity (Hoang et al., 2004;Iwaya et al., 2003;Kansara and Thomas, 2007).
Overexpression of Ezrin, a protein involved in cell movement, correlates with poor outcome in paediatric osteosarcoma (Khanna et al., 2004) and according to animal studies, is overexpressed in highly metastatic osteosarcoma compared to tumors with lower metastatic potential (Khanna et al., 2000).
MMP2 and MMP9 were overexpressed in osteosarcoma cells and associated with the ability of the cells to metastasize (Bjornland et al., 2005). Increased expression of membrane-type MMP1 has been correlated with poor prognosis (Ushibori et al., 2006) and upregulation of TIMP1 is associated with poor clinical outcome (Ferrari et al., 2004).
Recent studies have identified a close association between the aberrant methylation of histone cluster 2 genes, p14ARF/CDKN2A, p16 and Ras effector homologue (RASSF1A) genes and osteosarcoma tumorigenesis (Hou et al., 2006;Rao-Bindal & Kleinerman, 2011). It is believed that hypermethylation and subsequent transcriptional silencing of tumor suppressor genes contributes to the neoplastic process by increasing the mutation rate (Jones & Baylin, 2002;Wajed et al., 2001).

Epilogue
Understanding of the molecular pathogenesis of osteosarcoma has advanced considerably over recent decades. The processes involved in osteosarcoma oncogenesis have been outlined above with emphasis on the disruption of bone differentiation machinery.
However, the study of pathogenic mechanisms is in itself not enough. Translational studies are critical if an effective treatment for osteosarcoma is to arise from this understanding of osteosarcoma molecular pathology. If osteosarcoma results from bone differentiation defects, future research should focus on identifying relevant biomarkers and therapies targeting cellular differentiation thereby avoiding complications associated with conventional chemotherapy and be most effective in treatment of this debilitating tumor.