Electron Microscopy in the Diagnosis of Small Round Cell Tumors of Bone

Janine K. Mawad, MD Br0u.n & Associates Medical Laboratories, 8060 El Rio, Houston, Texas 77054, USA

Bruce Mackay, MD, PhD, A. Kevin Raymond, MD, and Albert0 G. Ayala, MD Depurtment of Pathology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA

Most small round cell sarcomas of bone occur in children and young adults. The commonest and best known is Ewing's sarcoma, but a number of other neoplasms arising in or spreading to bone can present a similar appearance by light micros- copy and give rise to problems in differential diag- nosis. When Sim et al' published the first account of the bone tumor that they labeled small cell os- teosarcoma, they described the 20 cases as os- seous lesions with histologic features of both Ew- ing's sarcoma and osteosarcoma and suggested that this was a specific entity whose behavior might be worse than that of conventional osteo- sarcoma. Subsequent clinicopathologic have reaffirmed that, although small cell osteosar- coma resembles Ewing's sarcoma, there are dis- tinct differences in the histopathology and behav- ior of the two tumors. Radiographic findings may be h e l p f ~ l , ~ although small cell osteosarcomas have demonstrated considerable variation in their radiographic so that microscopic confirmation is needed to establish the diagnosis. The finding of osteoid associated with the neoplas- tic cells is the definitive criterion, but it may not be detected in a small biopsy specimen.

Another neoplasm that enters into the differen- tial diagnosis is mesenchymal chondrosarcoma, first described in 1959 by Lichtenstein and Bern- stein,6 who suggested that it was derived from primitive mesenchyme that was committed to chondroblastic differentiation. It can occur in child-

and cellular areas containing undiffer- entiated small round cells' are often present. Radiographic examination may show stippled calcifications that suggest the diagn~sis,~ but mi- croscopic examination is again mandatory. lt is sometimes necessary to consider

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Address correspondence to 6 . Mackay

neuroblastoma,

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Small round cell tumors involving bone can present problems in differential diagnosis by light microscopy. In exploring the role of electron microscopy in this situation, seven small cell osteosarcomas and seven mesenchymal chondrosarcornas were examined by electron microscopy and compared with typical and atypical Ewing's sarcomas. There is much overlap in the ultrastructural features of these tumors, but electron microscopy is helpful to establish or confirm a diagnosis of typical Ewing's sarcoma and, if representative matrix is present, of small cell osteosarcoma.

KEY WORDS: bone tumors, osteosarcoma, chondrosarcoma, Ewing's tumor, small round cell tumors.

rhabdomyosarcoma, and lymphoma in the patho- logic evaluation of a small cell tumor that is involv- ing bone.

Other than for Ewing's sar~oma,~ information in the literature about the ultrastructure of small cell tumors that arise in bone is not extensive. As a continuation of an initial assessment of the value of electron microscopy in the differential diagnosis of these entities," we examined 7 small cell osteo- sarcomas and 7 mesenchymal chondrosarcomas of bone and compared our findings with 50 exam- ples of Ewing's sarcoma. The ultrastructural fea- tures of neuroblastoma, rhabdomyosarcoma, and lymphoma have been described on many occa- sions and are not discussed further here.

MATERIALS AND METHODS Seven specimens of small cell osteosarcoma were obtained from the files of the diagnostic electron microscopy service of the M.D. Anderson Cancer Center. In three of the cases two samples were available, one obtained at biopsy and one at sub- sequent surgery. All the tumors had been fixed at the time of accession in buffered glutaraldehyde. Seven mesenchymal chondrosarcomas were also examined: 6 of them had been fixed in glutaralde- hyde and 1 in formalin. Micrographs of 50 previ- ously studied, glutaraldehyde-fixed specimens of Ewing's sarcoma of bone were used for compari- son with the osteosarcomas and chondrosarco- mas. Light microscopic sections of each osteosar- coma and chondrosarcoma were stained with hematoxylin and eosin and with periodic acid- Schiff (PAS) reagent before and after diastase di- gestion.

RESULTS By light microscopy, cellular areas of the small cell osteosarcomas were made up of sheets of polygo-

Ultrastructural Pathology, 18:263-268, 1994 263 Copyright 0 1994 Taylor & Francis

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nal, stellate, and in some areas ovoid to elongated cells with scant cytoplasm. The nuclei were often eccentric, and they varied in shape from round to angulated. Some were slightly cleaved. The chro- matin formed small clumps at the nuclear periph- ery, and one or more inconspicuous nucleoli were present. The solid sheets and clusters of tumor cells were dissected by thin connective tissue par- titions and blood vessels. The amount of osteoid matrix varied markedly among the seven tumors, ranging from total absence in some of the cellular areas to a reticulated network with narrow to broad bands in the sites where it was most abundant. The PAS stain for glycogen was focally positive within the neoplastic cells in five of the tumors. Areas of the mesenchymal chondrosarcomas that had been sampled for electron microscopy were made up of sheets of closely packed small cells without any architectural pattern. The cells were mostly round with bland, central nuclei and scant pale or faintly basophilic cytoplasm. Some corlrained small quan- tities of glycogen.

At low magnification under the electron micro- scope, cells of the small cell osteosarcomas ap- peared more or less round if they were solitary and polygonal when they were forming small nests (Fig. 1) or short cords. Individual cells had smooth surfaces, and the cell membranes of adjacent cells were closely apposed. A few cell junctions were

identified, but they were small and primitive. Nu- clei were central with clumped chromatin and nu- cleoli that were small in some cells and prominent in others. Nuclear profiles were quite variable, ranging from smooth to indented. In one of the tumors, many nuclear contours were irregular. The amount of cytoplasm and therefore the size of the cells was not consistent among the seven tumors, as is evident when Figs. 1 and 2 are compared. Organelles were sparse in one tumor (Fig. 11, but most of the cells in the other tumors contained at least moderate numbers of mitochondria, cister- nae, and a well-developed Golgi complex (Fig. 2). The profiles of granular endoplasmic reticulum were slender or slightly dilated in most of the cells. In a few they were more distended, and some con- tained moderately electron-dense material. Vesi- cles and lysosomes were seen in small numbers. Nonspecific filaments formed ill-defined aggre- gates in a few cells. Although glycogen could often be detected, it was never prominent, and the dis- crete lakes that characterized most of the Ewing's tumor cells were not evident. Foci of condensed ground substance and early deposition of hydroxy- apatite crystals were identified around stromal col- lagen fibrils (Fig. 3).

The cells of the mesenchymal chondrosarcomas were similar in size and shape to those of the small cell osteosarcomas. Their nuclei had smooth pro-

FIG. 1 Small cell osteosarcoma. The cells of this tumor are closely packed in ir- regular groups. Nuclei are moderately irregular, and organelles are sparse, x 4,000.

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Small Cell Tumors of Bone

FIG. 2 Small cell osteosarcoma. The cells of this tumor have more cytoplasm and considerable numbers of organelles, ~3,600.

FIG. 3 An area of stroma from the tumor shown in Fig. 2. There are foci of hy- droxyapatite deposition amid the collagen fibrils, x 18,000.

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266 J . K. Mawad et HI

files, dispersed chromatin, and small nucleoli. The cytoplasm of the chondrosarcoma cells contained organelles in the same proportions as in the small cell osteosarcomas, but on the whole there were fewer mitochondria and less endoplasmic reticu- lum in the chondrosarcomas, and the cisternae were often slender (Fig. 4). The stroma contained collagen with variable amounts of myxoid material, but hydroxyapatite crystals were not seen.

The electron micrographs of Ewing's tumors of bone revealed a range of appearances, but they were fairly consistent and reflected what we con- sider the typical appearance of this tumor in about two thirds of the 50 reviewed cases. In this majority group, the cells were more or less round, and their surfaces were usually smooth, although an occa- sional short filopodium or interdigitation of ap- posed cell membranes was seen. Cell junctions were hard to find and, when visible, were tiny paired densities. Nuclei had smooth profiles with only minor indentations in these typical cases, and nucleoli were small. The cytoplasm contained many free ribosomes, but mitochondria and pro- files of endoplasmic reticulum were sparse. The amount of glycogen was quite variable. Lakes of glycogen were distinctly more prominent in Ew- ing's tumor than in the other two neoplasms, how- ever, and they were present in some cells of most of the tumors and were often extensive, occupying

much of the cytoplasm (Fig. 5). In approximately one third of the 50 cases of Ewing's tumor, the ul- trastructural features were more varied with nu- clear irregularities, larger nucleoli, and, in a few tumors, moderate numbers of organelles in some cells. Stromal collagen and intermingled ground substance was scant in the areas of Ewing's tumors that had been selected for study by electron mi- croscopy. Hydroxyapatite deposition was never seen.

DISCUSSION Published descriptions of these three small cell tu- mors of bone are in agreement that they can be closely similar in their light microscopic appear- ance, and the differential diagnosis is on occasion difficult on paraffin sections alone. Stains for gly- cogen are of limited value. They are likely to be positive in Ewing's tumor if the patient has not un- dergone therapy, and although Sim et al' did not find glycogen in the small cell osteosarcomas they re orted, others have noted its presence. Ayala et

cases. We have found glycogen in small amounts in mesenchymal chondrosarcomas.

lmmunostaining is also of limited value to sep- arate the tumors. There are no specific markers for small cell osteosarcoma or for Ewing's tumor.

al P detected cytoplasmic glycogen in 10 of 27

FIG. 4 Mesenchymal chondrosarcoma. The round cells are intimately apposed. Organelles are present in moderate numbers, x 5,200.

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Small Cell Tumors of Bone 267

FIG. 5 Typical appearance of cells of intraosseous Ewing’s tumor. There are few organelles, but the cytoplasm contains lakes of glycogen, x 7,500.

Swanson et al” performed an immunohistochem- ical analysis of nine cases of mesenchymal chon- drosarcoma using a battery of antibodies, and the tumor cells failed to express S-100 protein, al- though in six cases all components of the tumor cells (small cells, lacunar chondroblasts, and chon- droid matrix) stained for Leu-7 antigen. These in- vestigators noted that the immunophenotype of the tumor resembles that of embryonic cartilage but agreed that immunohistologic studies are not particularly helpful in distinguishing mesenchymal chondrosarcoma from other small round cell le- sions. lmmunoperoxidase studies are of proven value in identifying certain small round cell tumors that can involve bone. Desmin positivity may re- veal a tumor as a rhabdomyosarcoma, and lym- phomas can be recognized with the use of leuco- cyte common antigen and other markers.

From our study and the information in the liter- ature, we believe that electron microscopy has a role, albeit limited, in classifying the small round cell tumors that can arise in or secondarily affect bone. The most distinctive features are seen in typ- ical examples of Ewing‘s tumor, which made up about two thirds of the cases we reviewed from our files. A combination of sparse organelles and ex- tensive lakes of glycogen in bland round cells with inconspicuous nucleoli and barely detectable cell junctions will suggest Ewing‘s tumor. Criteria for the diagnosis of Ewing‘s sarcoma have become

more complex with the description of atypical vari- ants12 in which the cells can have irregular nuclei and prominent nucleoli and can possess more or- ganelles. It is possible that cytogenetic data will assume greater importance in the classification of small round cell tumors of bone?

In contrast to the typical Ewing’s sarcomas, we observed at least moderate quantities of organelles in the cytoplasm of cells of small cell osteosarco- mas and a lesser number in mesenchymal chon- drosarcomas. Dickersin and R~senberg’~ studied four small cell osteosarcomas by electron micros- copy and listed as features a high nuclear-to-cyto- plasmic ratio, poorly differentiated cytoplasm, nu- merous free ribosomes, mitochondria as the next most prevalent organelle, small junctions, and en- velopment of individual and groups of cells by ma- trix. Small cell osteosarcoma expresses its differ- entiation in the form of an osseous matrix that first appears as minute deposits in the stroma between the groups of neoplastic cells, and it is reasonable to assume that the quantity of organelles can be equated with the need to elaborate this matrix. Be- cause of the patchy distribution of the matrix, it may not be evident in a small biopsy specimen, and it was not found in every sample taken for elec- tron microscopy from our cases. The ultrastructural appearance of the matrix is di~tinctive,’~ however, and it is worth searching the available material us- ing semithin sections to detect foci of matrix for

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ultrastructural scrutiny when there is a need to re- solve a differential diagnosis because even the smallest clusters of hydroxyapatite crystals can be recognized with the electron microscope.

CONCLUSION Although small cell tumors of bone usually can be identified by routine light microscopy of ade- quately preserved material, there are occasions when the diagnosis is in question, and this is more likely to be so if only a needle biopsy specimen is available. Electron microscopy can confirm a sus- pected diagnosis of Ewing’s tumor if the typical findings are seen, but it is not possible to distin- guish with any confidence the cells of an atypical Ewing’s tumor from those of small cell osteosar- coma and mesenchymal chondrosarcorna. If stroma is present in the specimen procured for electron microscopy, a diagnosis of small cell os- teosarcoma will be supported by the demonstra- tion of foci of osseous matrix.

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