Osteosarcoma is a primary bone tumor composed of cells which, at least focally, produce osteoid and tumoral bone. Osteosarcoma is the most frequent primary nonhematopoetic malignant bone tumor, accounting for approximately 20% of all primary bone sarcomas. The majority of the patients are older children, adolescents, and young adults, but up to 30% of osteosarcomas occur in patients older than 40. Osteosarcoma is rare in infants. Male patients are affected slightly more frequently than females, with equal distribution among races. The most common symptoms are pain and a palpable bone mass, often with a soft tissue extension.

Most osteosarcomas arise around the knee, followed by the humerus and pelvic bones. Approximately 7–10% of osteosarcomas arise in the craniofacial bones (mandible and maxilla). Any bone in the body may harbor an osteosarcoma, but this neoplasm is rare in the spine and in the small bones of hands and feet.

The most frequent site of osteosarcoma in the long bones is metaphysis (approximately 90% of cases) followed by diaphysis and, rarely, epiphysis. Osteosarcomas show a wide variety of microscopic features, biologic potential, and relation to bone: intramedullary or surface. The most common type is the conventional, high-grade osteosarcoma and its histological osteoblastic and chondroblastic subtypes, accounting for 75–85% of all osteosarcomas. High-grade conventional type is also the most common target for FNA examination. Less common variants are periosteal, parosteal, and juxtacortical osteosarcoma, central low-grade osteosarcoma, high-grade surface osteosarcoma, and teleangiectatic and small cell osteosarcoma. Infrequent secondary osteosarcomas are associated with severe Paget disease, previous bone infarction, chronic osteomyelitis, metallic bone implants, and pre-existing numerous benign bone neoplasms such as enchondroma, osteochondroma, giant cell tumor, fibrous dysplasia, osteoblastoma, and bone cysts. Osteosarcoma may be associated with some genetic syndromes such as Rothmund-Thomson, Bloom, Werner, Li-Fraumeni, and Bilateral retinoblastoma. (6.5, 6.6, 6.7, 6.8, and 6.9)

FNA smears of the osteoblastic subtype of osteosarcoma display variable cellularity, but often are hypercellular (Table 6.1). A mixture of single cells and loosely or somewhat cohesive clusters of moderately to highly pleomorphic rounded, ovoid, and polygonal tumor cells in a hemorrhagic background is a common pattern in smears. Most tumor cells have a moderate or abundant amount of well-demarcated cytoplasm that often contains small vacuoles. The nuclei have a coarse chromatin and one or more, often prominent, macronucleoli (Figs. 6.5 and 6.6 ). The nuclei may be eccentrically placed, and such tumor cells resemble atypical osteoblasts. In the chondroblastic subtype the most common cell types are similar to those of osteoblastic osteosarcoma, but nuclei are often central and the nucleoli smaller and less prominent. A variable number of pleomorphic, often multinucleated, tumor cells is almost always visible in smears irrespective of subtype (Fig. 6.7 ). Osteoclast-like cells, necrosis, calcifications, and mitoses are also frequent findings in smears of high-grade osteosarcomas (Figs. 6.8, 6.9 , and 6.10). A background matrix is present in most of the smears, and strands of tumor matrix stained pinkish-violet-grey and red-violet-grey in Diff-Quik and May-Grünwald-Giemsa stains is considered to represent osteoid. This matrix occurs also as thin strands separating cells that lay in clusters (Figs. 6.11 and 6.12). Smears of chondroblastic subtype display a chondro-myxoid background matrix surrounding single tumor cells and small fragments of acellular cartilage or cartilage containing atypical chondrocytes (Fig. 6.13). Smears of fibroblastic osteosarcomas are dominated by atypical spindle cells with ovoid or fusiform nuclei showing coarse chromatin and small nucleoli. Smears of teleangiectatic osteosarcoma are bloody and hypocellular with scattered pleomorphic tumor cells. Aspirates from rare small cell osteosarcomas are hypercellular and tumor cells resemble those of Ewing sarcoma (Fig. 6.14). Aspirates from low-grade osteosarcomas are usually hypocellular and difficult to evaluate cytologically with respect to malignancy and histological type of neoplasm.

Ancillary studies have a limited role in the diagnosing of osteosarcomas, which are largely diagnosed by synthesis of imaging and routinely stained histological sections or FNA smears. Osteosarcoma cells express a broad immunoreactivity that lacks diagnostic specificity. Many osteosarcomas express, at least focally, vimentin, osteonectin, osteocalcin, S-100 protein, keratins, EMA, desmin, muscle specific actin, SMA, and CD99. With regard to establish osteoblastic differentiation, tumor cells of osteosarcoma display strong and diffuse cytoplasmic alkaline phosphatase (ALP) staining , either in air-dried FNA smears or imprint preparation of biopsy specimen (Fig. 6.15) [16, 17]. The architecture of the tumor tissue and strands of osteoid are often present in the cell block sections (Fig. 6.16).

Reactive osteoblastic proliferations such as fracture callus and pseudomalignant myositis ossificans are the most important benign mimics of osteosarcoma in FNA smears [18–20]. Correlation of cytological features with imaging studies and clinical history is very important in such cases, as is applying distinctive cytomorphological criteria in the evaluation of smears, because smears of high-grade osteosarcomas display in the vast majority of cases firm signs of malignancy, compared to smears of callus fracture and myositis ossificans. Other benign mimics of osteosarcoma are giant cell tumor (GCT) and osteoblastoma/epithelioid osteoblastoma. Smears of GCT of bone display double-cell populations of clustered spindle cells and multinucleated osteoclastic cells, but in osteosarcoma, osteoclastic giant cells are randomly distributed in the smears, whereas in GCT, giant cells are often situated in the periphery of cohesive clusters of spindle cells. The radiographic appearances and cytologic features of osteoblastomas, including osteoblast-like cells containing round-to-oval, eccentric nuclei with a fine chromatin pattern and a smooth nuclear membrane, admixture of bland spindle cells, and an absence of significant atypia and mitoses, help to distinguish these neoplasms from osteosarcoma. An important differential diagnosis of high-grade chondroblastic osteosarcoma is high-grade chondrosarcoma. Like chondrosarcoma, smears of chondroblastic osteosarcoma may contain an abundance of atypical cartilage in addition to more undifferentiated pleomorphic malignant cells. Some of the tumor cells, however, show features of atypical osteoblasts, which make the possibility of chondroblastic osteosarcoma more likely than high-grade chondrosarcoma (Fig. 6.13). In addition, strong cytoplasmic positivity of ALP and osteoid present in air-dried smears or cell block sections facilitates diagnosis of chondroblastic osteosarcoma (Figs. 6.15 and 6.16). Undifferentiated pleomorphic sarcoma, leiomyosarcoma, and, less frequently, other subtypes of sarcoma, arising primarily in bone as well as metastatic depositions of malignant melanomas, carcinomas, and manifestation of large-cell anaplastic lymphoma, constitutes other differential diagnoses of osteosarcoma. Antibodies to keratin, EMA, melanoma markers, CD45, CD30, ALK, although not always entirely specific, may help to render a correct diagnosis. As mentioned earlier, clinical data, imaging, and ALP staining are all important adjuncts in the examination of smears of osteosarcoma.

Osteosarcoma is the primary bone sarcoma in which FNA cytology has been most thoroughly investigated, in some large series [16, 21–39] and multiple case reports published to date. Due to their destructive growth and extension to the surrounding soft tissues, the majority of high-grade osteosarcomas are easy accessible for FNA. When the cortex is intact but FNA sampling technique is desired, trocar with a drill is necessary to penetrate the cortical bone. After cortical penetration, FNA samples can be taken from the target lesion using a coaxial technique supporting the use of a single needle-insertion point even when performing several punctures. FNA cytology is an efficient technique in the diagnosis of high-grade osteosarcoma in conjunction with imaging and appropriate clinical data. FNA is less useful in the evaluation of low-grade osteosarcoma, often providing aspiration smears that are non-specific or diagnostically insufficient. The optimal use of FNA technique as a diagnostic tool in the evaluation of osteosarcomas requires the referral of patients to specialized orthopedic-oncologic centers with multidisciplinary expertise in the examination and treatment of bone sarcoma.

In the experience of our institution, cases of intraosseous osteosarcomas without destruction of the cortical bone are difficult to examine by FNA alone [16]. In such cases we use to supplement FNA with core needle or, rarely, surgical biopsy in order to obtain sufficient material.

The clue to the cytological diagnosis of conventional high-grade osteosarcoma in routinely stained smears is the presence of intercellular osteoid and osteoblast-like tumor cells. Osteoid is best appreciated in MGG-stained smears (Figs. 6.11 and 6.12). Differentiating osteoid from collagenous matrix can sometimes be difficult. The architecture of the tumor tissue with clearly visible strands of osteoid are often present in the cell block sections. Electron microscopic examination is a well-established method for defining osteoid in fine needle aspirates. We believe, however, that ALP staining has been underestimated in many studies of FNA of osteosarcoma. Strong intracytoplasmic ALP staining in intact tumor cells confirms their osteoblastic differentiation.

MPNST usually arises in older children, adolescents, and young adults, especially in those with NF1 syndrome. Tumors are frequently localized in the extremities, trunk, and head and neck. MPNSTs are fusiform masses centered on a large nerve [40]. Trunk localizations, large tumor size, high grade, and recurrences are adverse prognostic factors.

Histologically, well-differentiated MPNST are composed of spindle-shaped cells with fascicular growth and Schwannian tissue. In anaplastic types, tumors are composed of anaplastic and polymorphous cells. However, rhabdomyoblastic (Triton tumor), glandular (glandular MPNST), melanocytic (pigmented MPNST), and epithelioid differentiations are also described [40].

Smears in well-differentiated MPNST are cell-rich and stroma rich [41–44]. Smears are hypercellular, composed of small spindle-shaped cells with wavy and comma-like naked nuclei. A mixture of cell clusters, fascicles, dispersed cells, fibrillar background, variable presence of pleomorphic and/or multinucleated cells, mitotic figures, and epithelioid cells are detected. Polymorphous or roundish cells may also be found. The general impression of malignancy may not be obvious (Figs. 6.17 and 6.18).

In anaplastic MPNST, stroma is less abundant. Cellular material is rich and composed of large, atypical, polymorphous, clearly malignant cells. Necrosis, mitotic figures, and apoptotic bodies are noted. Spindle-shaped cells are usually present, but atypical.

Smears in Triton tumor may contain many large, rhabdoid cells with eccentric nuclei.

Smears in melanocytic MPNST show cells with melanin. Finally, smears in epithelioid MPNST are composed of large, epithelioid cells mimicking carcinoma or melanoma.

Immunohistochemical studies show partial positivity for S100 protein and GFAP. Rhabdoid areas are positive for muscular markers. Melanocytic markers are always negative. Keratin may be positive in glandular cells.

Well-differentiated MPNST should be differentiated from atypical (ancient) Schwannoma (neurilemmoma) and other spindle cell childhood sarcomas, including monophasic synovial sarcoma and dermatofibrosarcoma protuberans (if superficial) [41, 42, 45–48]. Anaplastic MPNST should be differentiated from other anaplastic sarcomas. Triton tumor should be differentiated from rhabdomyosarcoma [49]. Finally, epithelioid MPNST should be differentiated from malignant melanoma, synovial sarcoma, and/or carcinoma.