As current treatment regimens utilize presurgical chemotherapy, it is unusual to encounter a resection specimen that has not been altered by treatment. Grossly, conventional osteosarcomas typically form large destructive tumors that transect the cortex, extend into soft tissue, and contain variable degrees of necrosis, calcification and hemorrhage (Fig. 4.3). Even without presurgical treatment, necrosis may be extensive.

Histologically, diagnosis of conventional osteosarcoma requires two fundamental elements: atypical mesenchymal cells and tumoral (as opposed to reactive) osteoid or bone. Osteoid may be quite focal. The prominence of these features varies by patient and by microscopic field, a fact important to consider with small biopsies. Usually, conventional osteosarcoma is a straightforward diagnosis. Most typically, malignant mesenchymal cells are dense and mitotically active, with hyperchromatism, cytologic atypia, and pleomorphism (Fig. 4.4). Tumor cells may be spindled, round, or polymorphic; giant cells, markedly pleomorphic cells (monstrocellular cells), and atypical mitotic figures are common. Osteoid comprises a pink, dense, amorphous material abutting tumor cells, and fibrosis and atypical chondroid matrix are also common. Distinction of osteoid from other types of collagenous matrix can be difficult and subjective. In general, a fibrillar matrix is not osteoid but rather non-osteoidal collagen. Occasionally cartilage may dominate, and demonstration of osteoid production may require careful searching [18]. Tumoral osteoid is often wispy and lace-like but may appear as larger deposits with bone production (Fig. 4.5).

In the sclerosing osteosarcoma variant, abundant osteoid matrix totally envelopes tumor cells, giving them the appearance of benign intralacunar osteocytes. The decreased cellularity obscures the malignant nature of the lesion, especially in limited biopsy material [18]. Close attention to the tumor interface with surrounding bone can be helpful in difficult cases, as the leading edges of the tumor often show increased cellularity with atypical cells [18], but such areas may not be present in limited biopsy material. The background reveals variable degrees of hemorrhage, tumoral necrosis and calcification.

Depending upon the predominant matrix component, conventional osteosarcomas have historically been placed into categories of osteoblastic (predominant osteoid and bone production), chondroblastic (predominant chondroid production) or fibroblastic (spindle cells with only focal matrix production) (Fig. 4.6). However, the value of these descriptive categories is debatable, as there can be significant variation in different areas of a given tumor, and moreover, these histologic subtypes appear to have no prognostic significance [9].

Rare types of osteosarcoma that present difficulties in diagnosis include epithelioid and osteoblastoma-like tumors. Epithelioid osteosarcoma contains large round to polyhedral undifferentiated cells that resemble epithelial cells and produce little osteoid (Fig. 4.7). They may have prominent nucleoli and on occasion can be cohesive, mimicking an epithelial malignancy or angiosarcoma, so that careful search for osteoid becomes necessary to establish the diagnosis [18]. Cytokeratin positivity makes this distinction even more problematic (see below). Osteoblastoma-like osteosarcoma contains broad bony trabeculae and osteoblastic rimming that mimics osteoblastoma; atypical osteoblasts may also be present. However, it shows infiltration of surrounding bone and atypical mitoses [18].

Immunohistochemical stains: In most cases a firm diagnosis of conventional osteosarcoma only requires routine H&E staining. Immunohistochemistry has limited utility and is mainly used to exclude tumors with better defined and more specific immunohistochemical profiles. The only consistently positive stain in general clinical use is vimentin, and smooth muscle actin may be positive as well [11]. Immunohistochemistry is most helpful in biopsies of high grade primary bone tumors with cellular pleomorphism, necrosis, and hemorrhage, but lacking tumoral osteoid or bone. Such tumors usually represent osteosarcoma, as few other primary bone tumors of childhood have these features [19] CD99 is helpful in ruling out Ewing sarcoma, as the staining pattern in osteosarcoma tends to be cytoplasmic and granular, as opposed to membranous. Rarely osteosarcomas stain with cytokeratin, which can be a pitfall in distinguishing them from synovial sarcoma, angiosarcoma, and metastatic carcinoma [20].

Diagnostic molecular genetics: No specific translocations or mutations define osteosarcoma, so that molecular cytogenetic studies mainly exclude other entities. Osteosarcomas usually exhibit very complex numerical and structural chromosomal aberrations. Some are recurring but none are consistent enough to be helpful in diagnosis [21]. Common abnormalities include loss of chromosomes 9, 13, and 17, gain of chromosome 1, partial loss of 6q, and chromosomal rearrangements of 11, 19, and 20 [22]. Li-Fraumeni syndrome and hereditary retinoblastoma syndrome markedly increase risk for developing osteosarcoma and, respectively, result in loss of RB1 on chromosome 13q and TP53 on chromosome 17p. A significant percentage of sporadic osteosarcomas also show alterations of these tumor suppressor genes [23].

Prognostic and post-treatment issues: According to SEER, the overall 1994–2003 5-year survival rate in children and adolescents was 61.6 %, with distant hematogenous metastases (most commonly lung) or local disease recurrence associated with much poorer survival [9, 24]. Pelvis or vertebral tumors also have adverse prognostic significance [9, 25].

Pathologic examination of the definitive (post-chemotherapy) resection specimen for margin status and degree of necrosis plays an important role in clinical care. As these specimens are often large and require extensive decalcification and fixation, proper handling to ensure good histologic detail and preservation of important margins is not trivial. In our institution, such specimens are handled in the following manner: Upon receipt in the pathology laboratory, the fresh specimen is photographed and X-rayed, and when necessary, orientation is confirmed with surgical personnel. The entire specimen is inked. The specimen is then frozen at −70 °C. Depending on tumor location, the frozen specimen is sequentially sectioned along the longitudinal axis most likely to demonstrate the greatest tumor area. We obtain band saw sections at approximately 5 mm intervals, usually yielding up to 25–30 slabs. The slabs are then fixed and decalcified. Afterwards one or more slabs having the greatest tumor area is mapped, photocopied, and completely submitted along with bone and soft tissue margins (Fig. 4.8). Using the grid map, the pathologist examines each block and evaluates necrosis, expressed as a percentage of the total tumor surface area. Positive margins at time of definitive surgery are associated with a poor prognosis [24]. With effective chemotherapy, extirpation of malignant cells in osteoblastic and chondroblastic areas leaves a background of acellular matrix, often containing empty lacunae or pyknotic “ghost” cells (Fig. 4.9). Fibroblastic areas tend to be replaced by bland granulation and myxoid tissue (Fig. 4.10) [26]. Using current COG protocols, patients are stratified to either “favorable” or “standard” responses, the forming having necrosis >90 %. Unfortunately, such assessments involve a high level of subjectivity. Large areas of the tumor bed composed of either sheets of tumor cells or expanses of acellular matrix are usually straightforward. However, post-chemotherapy specimens that contain foci of chemoresistant cells are typically less cellular than before therapy. Scattered pleomorphic cells with smudgy nuclei (considered to be viable for purposes of grading) [27] often persist in microscopic fields that are otherwise necrotic. In these situations, assignment of a particular percentage necrosis becomes inherently imprecise, and the literature gives little practical guidance. For additional information, the reader is advised to consult the excellent reference by Raymond et al. [28].

Small cell osteosarcoma is rare, accounting for less than 2 % of osteosarcomas. These lesions are small blue cell tumors that demonstrate osteoid production (sometimes focal) but otherwise have features suggestive of Ewing sarcoma. Like conventional pediatric osteosarcoma, it usually arises in long bones and most commonly occurs in the second decade. It may have a slightly worse prognosis than conventional osteosarcoma, although its relative infrequency and controversial diagnostic criteria have raised questions about its behavior.

Telangiectatic osteosarcoma is a rare (4 % of osteosarcomas) high grade subtype of osteosarcoma, with similar demographics regarding age and tumor location [32]. Approximately a quarter of patients present with pathologic fracture. Originally thought to have a worse prognosis than conventional osteosarcoma, survival figures with modern chemotherapeutic regimens are now comparable [33].

Low-grade central osteosarcoma accounts for less than 2 % of osteosarcomas and has a favorable prognosis with complete surgical resection [37]. It only occurs in long bones. Histologically, it resembles parosteal osteosarcoma, with bony trabeculae separated by a hypocellular fibrous stroma containing minimally to moderately atypical spindle cells. Mitotic activity is always present but may be low. Cytologic atypia, infiltration, and permeation distinguish low-grade central osteosarcoma from benign processes such as fibrous dysplasia and chondromyxoid fibroma [37]. Few studies have investigated the molecular genetics of this rare neoplasm. In one study of six patients, comparative genomic hybridization showed recurrent gains at 12q13-14, showing genetic as well as histologic relatedness to parosteal osteosarcoma [38].

Surface osteosarcomas arise from the periosteum or cortex, with minimal or no involvement of the underlying medullary cavity. The three major types are parosteal osteosarcoma, periosteal osteosarcoma, and high grade surface osteosarcoma. Parosteal osteosarcoma is a low grade tumor that accounts for 4 % of osteosarcomas in general but is rare in childhood. Most cases occur in young adults [39]. The most common site is the distal posterior femur, with occasional examples in other long bones. Origin in flat bones is exceptional. With total resection, the prognosis of these slow growing tumors is excellent, although there is a 10 % risk of distant metastatic disease [18]. Unlike conventional osteosarcoma, these tumors tend to be painless and usually only have symptoms of a mass or restriction of movement [18].

Periosteal osteosarcoma is an intermediate grade surface osteosarcoma characterized by its mainly cartilaginous matrix. Less common than parosteal osteosarcoma, it accounts for fewer than 2 % of all osteosarcomas. Most cases occur in adolescents and young adults. The metaphyseal–diaphyseal regions of long bones such as the tibia and femur are most commonly involved [45], but involvement of the flat bones is rare. Disease-free survival after adequate treatment is >80 %, with most recurrences and deaths occurring within 3 years of diagnosis [46].

High grade surface osteosarcoma is a high grade osteosarcoma arising from the surface of the bone. Extremely rare (<1 % of all osteosarcomas), it has the clinical presentation, demographic distribution, and prognosis of conventional osteosarcoma. The most common site of involvement is the midshaft of the femur [48].

Ewing sarcoma (ES) is the second most common primary malignancy of bone in childhood. Although first described as a primary undifferentiated bone tumor, it also occurs in soft tissue and may exhibit variable degrees of neuroectodermal differentiation, designated “primitive neuroectodermal tumor” (PNET). Ewing sarcomas and PNETs involving either bone or soft tissue share identical recurrent translocations. No longer biologically distinct, these entities have now been subsumed into the category of “Ewing’s sarcoma family of tumors” (ESFT’s) [52]. ESFTs are most common in whites, rare in African and African-American children, and slightly more common in males than females [53]. They most commonly affect adolescents but occur at any age, even infancy [54]. Usually they arise from the diaphysis of long bones or the pelvis, but any bone can be involved. [55]. ESFTs are clinically aggressive, and prior to the advent of chemotherapy nearly all patients died of their disease. With current treatment regimens, the overall survival of patients with localized disease is 75 %. However, one quarter of patients have metastatic disease at diagnosis and a poor prognosis [56]. Tumors commonly metastasize to lung; other sites include bone marrow and other bones. Although pain with or without pathologic fracture is the most common presenting symptom, patients may show signs and symptoms mimicking infection [55].