CHAPTER 16 Bone

Clinical aspects

Needle aspiration has been used in the investigation of bone lesions ever since the technique was introduced. Coley and Ellis included several examples of primary and metastatic bone malignancies in their original paper.¹ Fine needle biopsy (FNB) and cytodiagnosis of bone lesions are now well-established methods in many centers. It is not possible to penetrate intact cortical bone or sclerotic lesions using a truly thin needle (22–23 gauge) but partly destroyed or ‘moth-eaten’ cortical bone can often be penetrated. New devices, such as the Bone Biopty instrument, a coaxial biopsy system with an eccentric drill,² have made it possible to drill through intact cortical bone and to insert the fine needle into the lesion through the drilled canal. FNB has the advantage over open biopsy of being less disruptive to bone, permitting multiple sampling without complications and leaving no scar. There is no risk of infection if simple sterility is observed. FNB is a simple outpatient procedure, cost effective and rapid. Its primary purpose is not only to obtain a morphologic diagnosis of benignity or malignancy, to investigate suspected bone secondaries, or sometimes to aspirate material for a bacteriologic diagnosis from osteolytic lesions, radiologically suspected for osteomyelitis, but also, if possible, to replace open or coarse needle biopsy in the diagnosis of primary bone tumors before treatment. Ancillary diagnostic techniques such as cytochemistry, immunocytochemistry, flow cytometric phenotyping, DNA ploidy analysis, molecular genetic analysis and, in some special situations, electron microscopy are important supplements to cytodiagnosis of specific tumor entities before definitive treatment.

Spinal lesions with threatening cord compression are a medical emergency and FNB can rapidly resolve the differential diagnosis between osteomyelitis, metastatic malignancy and a lymphoproliferative malignancy. The diagnosis of Langerhans cell histiocytosis (eosinophilic granuloma) and of plasmacytoma is, in most cases, relatively easy and the distinction between non-Hodgkin’s lymphoma and other small cell malignancies (primary or secondary) is possible. FNB is well suited to the investigation of the cause of pathological fractures, but the proliferating osteoblasts in a fracture callus must be recognised as such. Our experience, and that of others, in the type-specific diagnosis of primary bone lesions is rather substantial, covering all of the malignant bone tumors except adamantinoma, and most of the benign tumors. Definitive diagnosis is possible since the cytomorphology of primary bone tumors, benign as well as malignant, has now been described in detail in correlative cytological/histological studies ³ and in single monographs and reviews^4,⁵

The correct interpretation of smears from a bone lesion requires access to the full clinical and radiological assessment, just as in histological diagnosis of a formal bone biopsy. The cytopathologist should refrain from giving a definitive diagnosis when the results of the radiologic investigation are not known.

Accuracy of diagnosis

Adequate material was obtained in 86–97% in four series, comprising in total 1453 cases.^3,⁶^–⁹ The diagnostic accuracy in the diagnosis of metastases is very high, in the order of 90%. Its value in the diagnosis of primary bone lesions varied between 90% and 97% in the above cited series.

Complications

Complication are few. We have more than 30 years’ experience of FNB of bone lesions in our musculoskeletal tumor centre in Lund University Hospital and have never seen any severe complication. Brief pain at needling is not uncommon but significant hemorrhage is unusual, as is pneumothorax after biopsy of rib or spine lesion. FNB of spinal lesions carries the additional, albeit rare, complication of neurological damage.¹⁰ FNB of bone lesions in patients with a low platelet count or a bleeding disorder must be discussed with the clinician in charge before proceeding to biopsy, and the importance to clinical management of a cytological diagnosis must be decided.

Technical considerations

Fine needle biopsy guided by fluoroscopy or computed tomography (CT) in nonpalpable lytic lesion is a rapid and safe method to obtain a morphologic diagnosis of lesions anywhere in the skeleton, particularly in the spine.^5,^11,¹² Some pain is usually felt at aspiration. Local anesthesia prevents periosteal pain and is recommended in conjunction with fluoroscopy or CT guidance, as several passes with the needle may be necessary to obtain a representative specimen. Bone aspirates are often heavily bloodstained and methods similar to those used in processing hematological bone marrow aspirates are useful to concentrate the cell material. The use of a watch glass to select tissue fragments and of thrombin-clot preparations for paraffin sections and histology are of particular value (see Chapter 2). Both air-dried MGG smears and wet-fixed smears for H&E or Pap should be made. MGG smears are particularly valuable in assessing bone marrow elements and chondroid, myxoid or osteoid material; wet-fixed smears are superior for nuclear detail, particularly in tissue fragments (‘microbiopsies’).

Cytological findings

Normal structures

• Hematopoietic tissue,

The cytopathologist must be familiar with the appearance of cellular marrow. Taken individually, the nuclear features of immature marrow cells may be disturbingly similar to malignant cells, but the mixture of all cell types allows easy identification. The irregular multilobed nucleus of the megakaryocytes in particular can cause alarm if the site of origin is unsuspected. MGG-stained smears are much superior to wet-fixed H&E or Pap smears in identifying hematopoietic or lymphoid cells. Bone marrow cells are frequently found, particularly in aspirates from the ribs, vertebrae and sacrum.

Osteoblasts are commonly seen in aspirates from all kinds of bone lesions. They present either as single cells or as small groups or runs. They have a characteristic eccentric nucleus which sometimes seems to protrude from the cytoplasm. The nucleus is round or oval and often contains a central nucleolus. The cytoplasm is dense, amphophilic or basophilic with a central clear area or ‘Hof’ separated from the nucleus (Fig. 16.1). Reactive osteoblasts in a fracture callus or in benign lesions such as reactive periosteitis may show marked anisokaryosis and prominent nucleoli (Fig. 16.2). Osteoclasts are large cells which possess at least 10–15 uniform nuclei and have abundant cytoplasm with a similar texture to osteoblasts and well-defined borders. A fine, pink cytoplasmic granularity is visible in MGG-stained smears (Fig. 16.3). Normal cartilage does not smear well as it is very cohesive. Flecks or clumps of cartilage may be removed from joint surfaces or costochondral junctions. These show bright red or magenta staining with MGG and are pale and translucent with Pap. Scalloped or fibrillar edges are a feature (Fig. 16.4). Free chondrocytes are almost never seen, but the cells are visible within lacunae in the chondroid matrix. They have small, pale, rounded nuclei and poorly stained cytoplasm, often presenting as a halo. They are best visualised in wet-fixed smears. The chondroid matrix stains heavily with MGG and obscures the fine structure of the cells (Fig. 16.5). A rare but important contamination of smears from spinal FNBs is clusters of mesothelial cells which may be sampled if the lesion is missed by the needle. Such cells may be mistaken for metastatic deposits of well-differentiated carcinoma, but usually do not have a bloody background (Fig. 16.6).

Fig. 16.1 Osteoblasts

Cluster of cells with a plasma cell-like appearance; abundant cytoplasm and eccentric nuclei; ‘Hof’ separated from nucleus is visible in several cells. Smear derived from an area of moderate osteoblastic activity and new bone formation (H&E, HP).

Fig. 16.2 Osteoblasts

Reactive osteoblasts (florid reactive periosteitis) showing marked anisokaryosis and enlarged nucleoli. A cytoplasmic ‘Hof’ is visible (MGG, HP oil).

Fig. 16.3 Osteoclast

A fine, cytoplasmic granularity is visible in MGG-stained smears (MGG, HP).

Fig. 16.4 Normal cartilage

Scalloped or fibrillar edges are one of the features of normal cartilage (MGG, HP).

Fig. 16.5 Normal cartilage

The chondroid matrix stains heavily with MGG and obscures the fine structure of the cells (MGG, HP).

Fig. 16.6 Mesothelial cells

A rare but important contamination of smears from spinal FNBs (MGG, HP).

Inflammatory processes

Osteomyelitis

Smears from bacterial osteomyelitis are dominated by abundant neutrophils but also contain other inflammatory cells and macrophages. The aspirate may look like pus from any other type of non-specific acute inflammatory process (Fig. 16.7). Culture of the aspirated material is the most valuable part of the procedure if an infectious lesion is suspected. Clusters of epithelioid histiocytes with characteristic banana- or bean-shaped nuclei and indistinct cell borders and granular (caseous) necrotic material provoke a strong suspicion of tuberculosis (Fig. 16.8). In our experience, Langhans-type giant cells are difficult to find. Again, the need for cultural evidence is emphasised.

Fig. 16.7 Osteomyelitis

Histiocytes with vacuolated cytoplasm and neutrophils (MGG, HP).

Fig. 16.8 Suspicion of tuberculosis

Clusters of epithelioid histiocytes with banana- or bean-shaped nuclei and indistinct cell borders (MGG, IP).

Neoplasms

Metastatic carcinoma

Criteria for diagnosis

♦ Foreign cell population,

♦ Cell clusters; acinar or gland-like structures,

♦ Cells showing criteria of malignancy.

As with sites such as lymph nodes, diagnosis is usually easy, especially if the histologic type of the primary tumor is known and sections are available for comparison with the smears. Descriptions of specific types of metastatic neoplasms are not included here; their cytological appearances have generally been discussed elsewhere.

Problems in differential diagnosis

♦ Osteoblasts resembling mucin-secreting cells. The cytoplasmic clearing typical of osteoblasts may resemble a mucin vacuole to the uninitiated. Sometimes clustering or lining up of these cells may enhance the epithelial-like appearance.

♦ Smeared marrow elements resembling anaplastic carcinoma. Caution should be exercised in reporting poorly smeared or poorly preserved material. Bone marrow cells are fragile and heavy smearing pressure can cause clumping that may resemble aggregates of small cell or other anaplastic carcinoma. As mentioned, megakaryocytes may also be misinterpreted as large, bizarre multinucleated malignant cells. It should be remembered that metastatic carcinoma usually replaces marrow; therefore, a mixture of marrow and new growth are uncommon.

♦ Anaplastic plasmacytoma may appear as a pleomorphic tumor in which only few cells show typical features of plasma cells. Poor smearing may result in clumping of tumor, cells which may resemble epithelial clusters.

♦ The distinction between chordoma and clear cell or mucinous carcinoma, and between high-grade chondrosarcoma and clear cell carcinoma, pose specific problems.

♦ Excessive blood. This problem is commonly encountered in metastatic malignancies as well as in primary bone tumors. The non-aspiration technique may provide better samples.

Solitary plasmacytoma; myeloma

Criteria for diagnosis

♦ Many plasma cells,

♦ Single cell presentation,

♦ Variable cell differentiation,

The diagnosis is obvious if there is a uniformly dispersed population of plasma cells including multinucleate and pleomorphic forms. The eccentric nucleus, with its speckled or clock-face chromatin, and the abundant amphophilic cytoplasm make recognition easy. A pale or clear cytoplasmic zone near the nucleus is another distinctive cytologic feature (Fig. 16.9).

Fig. 16.9 Multiple myeloma

Pure population of atypical slightly pleomorphic plasma cells; the eccentric nucleus and pale or clear cytoplasmic zone near the nucleus are distinctive cytologic features (MGG, HP).

Problems in differential diagnosis

♦ Excessive blood. Aspirates from solitary plasmacytoma are often very hemorrhagic and neoplastic plasma cells may be few in numbers.

♦ Reactive benign plasmacytosis may be difficult to differentiate from plasmacytoma in hemorrhagic aspirates with low numbers of normal-looking or slightly atypical forms.

♦ Admixture of marrow elements. The dilemma of deciding whether the proportion of plasma cells in marrow is high enough to justify a diagnosis of multiple myeloma is seldom a worry since usually only lytic lesions formed by solid tumors will be aspirated. Admixture with marrow elements is rarely seen in these cases.

♦ Anaplastic plasmacytoma. As mentioned above, anaplastic pleomorphic plasma cells are likely to cause confusion when a primary bone origin is unsuspected.

♦ It may be difficult to distinguish large B-cell non-Hodgkin’s lymphoma with immunoblastic features from poorly differentiated plasmacytoma.

♦ Osteoblasts resembling plasma cells. However, it is important to remember that, unlike osteoblasts, the clear cytoplasmic zone in plasma cells is next to the nucleus and that osteoblast nuclei are more eccentrically located than are plasma cell nuclei (nuclei seem to protrude from the cytoplasm).

Neoplastic plasma cells are monoclonal (express monotypic immunoglobulin). This feature is possible to demonstrate in cell block or cytospin preparations. Neoplastic plasma cells, including most anaplastic forms, express CD138 and CD79A.

Malignant lymphoma

Focal bone involvement, usually in form of lytic lesions and most commonly involving the spine, pelvis and the long bones, is uncommon., Primary non-Hodgkin’s lymphoma comprises about 3–7% of the primary malignant bone tumors. Primary Hodgkin’s lymphoma in bone is extremely rare. Most primary non-Hodgkin’s lymphomas are diffuse large cell B-cell lymphomas. Lymphoplasmacytic lymphoma, anaplastic large cell lymphoma and precursor lymphoma (lymphoblastic lymphoma) have also been reported; the two last mentioned may occur in children and adolescents. Cytological criteria for diagnosis are the same as in other sites (see Chapter 5). Case series of primary non-Hodgkin’s lymphoma of bone have been presented.^13,¹⁴ The important differential diagnosis between precursor lymphoma and classical Ewing’s sarcoma is discussed on page 424.

Langerhans cell histiocytosis (histiocytosis X, eosinophilic granuloma)

Criteria for diagnosis

♦ Large histiocytes with vesicular nuclei of irregular shape, sometimes binucleated,

♦ Variable numbers of eosinophils,

♦ Giant cells of histiocytic type.

Langerhans cell histiocytosis presents as lytic and often well-defined lesions. Most cases are seen in children. The lesions may be solitary (most cases) or multiple. The long bones (femur and humerus) and the skull are the most common sites in children, and the pelvic bones and ribs in adults. The cytological pattern of Langerhans cell histiocytosis is fairly characteristic and may be diagnostic. The typical histiocytes have moderately larger and paler nuclei than those seen in common inflammatory processes. Generally reniform, the nuclei have a distinct irregular and folded outline (Fig. 16.10A). Coffee-bean nuclei has been reported to be typical of this lesion (Fig. 16.10B). The chromatin is entirely bland and nucleoli small. The cytoplasm is abundant and pale and has fairly well-defined borders. It is often vacuolated. In exceptional cases the Langerhans cells may show signs of phagocytosis. Multinucleated cells of similar type are commonly present. These can be quite large and may resemble osteoclasts in wet-fixed smears.

Fig. 16.10 Langerhans cell histiocytosis (eosinophilic granuloma)

(A) Lytic bone lesion, many histiocytes with irregular reniform pale nuclei, some eosinophils (H&E, IP); (B) Coffee-bean nuclei have been reported to be typical of this lesion (H&E, HP, Oil)

Large number of histiocytes can occasionally be found in aspirates of chronic osteomyelitis, but these are of the common, smaller type seen in the usual inflammatory processes and are mixed with neutrophils, lymphocytes and plasma cells.

The Langerhans cell histiocytes express S-100 protein and CD1-antigen (Fig. 16.11). According to the Histiocyte Society the definitive diagnosis of Langerhans cell histiocytosis is based on the demonstration of Birbeck granules by electron microscopy or positivity for CD1 antigen.¹⁵ The examination of conventionally stained material gives a presumptive diagnosis. The cytological features of Langerhans cell histiocytosis has been recorded in several series.¹⁶^–¹⁸