Anatomic Pathology / Cytopathology of Small Cell Osteosarcoma
Small Cell Osteosarcoma
Cytopathologic Characteristics and Differential Diagnosis
Justin A. Bishop, MD,1 Chung H. Shum, MD, PhD,1 Sheila Sheth, MD,2 Paul E. Wakely Jr, MD,3
and Syed Z. Ali, MD1,2
Key Words: Small cell osteosarcoma; Bone; Cytopathology; Fine-needle aspiration
DOI: 10.1309/AJCPO07VGDZCBRJF
CME/SAM
Upon completion of this activity you will be able to:
• describe the typical clinical and radiographic features of small cell
osteosarcoma.
• list 3 cytomorphologic features of small cell osteosarcoma.
• produce a differential diagnosis based on the cytology of small cell
osteosarcoma.
• discuss the role of immunohistochemistry and other ancillary tools in
arriving at the diagnosis of small cell osteosarcoma.
Abstract
Small cell osteosarcoma may present a challenging
primary diagnosis on cytologic assessment owing
to its rarity and its morphologic similarity to other
small round blue cell tumors. Five cases of small
cell osteosarcoma from our cytopathology archives
were identified and reviewed and cytologic features
elaborated. Three cases were fine-needle aspirations
from bony lesions in the classic location for
osteosarcoma (2 distal femur and 1 proximal tibia),
and 2 aspirations were from metastases. Common
cytomorphologic features included relatively small to
intermediate cell size, high nuclear/cytoplasmic ratios,
round nuclei, minimal anisonucleosis, finely granular
nuclear chromatin, fine cytoplasmic vacuoles, and only
rare osteoid. Small cell osteosarcoma shares many of
the well-described cytomorphologic features of classic
osteosarcoma, but the relatively small cells, round
hyperchromatic nuclei, and scant osteoid constitute the
common denominator. Correlation with radiographic
findings and ancillary tests can aid in definitive
diagnosis.
756 Am J Clin Pathol 2010;133:756-761
756 DOI: 10.1309/AJCPO07VGDZCBRJF
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The increasingly widespread use of preoperative fine-
needle aspiration (FNA) of bone lesions necessitates the
recognition of even rare primary osseous neoplasms. The
appropriate classification of a primary bone neoplasm may
drastically alter the planned therapy for the lesion.
Small cell osteosarcoma is an exceedingly rare tumor,
estimated to account for less than 1% of all cases of osteo-
sarcoma.1 Small cell osteosarcoma was first described as a
neoplasm having microscopic features of osteosarcoma and
of Ewing sarcoma in that small cells cytomorphologically
similar to those seen in Ewing sarcoma were found in a histo-
logic background of osteoid.2 Owing to its unique histologic
features, it can have a broad differential diagnosis. Only rare
accounts of the cytopathologic findings on FNA exist in the
literature.1,3
Materials and Methods
The cytopathology archives of The Johns Hopkins
Hospital, Baltimore, MD, and Ohio State University Medical
Center, Columbus, were searched, and all cases of small cell
osteosarcoma were retrieved. FNA was performed using a
22- or 25-gauge needle under ultrasound or computed tomog-
raphy scanning with on-site evaluation by a cytopathologist.
The smears were air dried and wet fixed in 95% ethanol and
stained with rapid Romanowsky and Papanicolaou stains,
respectively. FNA smears were reviewed for cytomorpho-
logic characteristics, including presence or absence of osteoid,
necrosis, pleomorphism, cytoplasmic processes, cytoplasmic
vacuolization, mitoses/apoptosis, multinucleation, anisonucle-
osis, nuclear shape, chromatin characteristics, and nucleolar
© American Society for Clinical Pathology

prominence. Available immunoperoxidase stains and flow
cytometric analyses were reviewed as well, along with the
clinical and radiologic findings in each case.
Results
Patient Demographics and Clinical and Radiologic Data
Five cases of small cell osteosarcoma were identified in
a 19-year period (1990-2009). The ages of the patients ranged
from 11 to 37 years. Three cases were FNAs from bony
lesions in the classic location for osteosarcoma (2 distal femur
and 1 proximal tibia) with a size range of 4.5 to 14 cm (mean,
10.2 cm). One case was a peripancreatic soft tissue metastasis
from a primary paravertebral cervical mass, and another case
was a metastasis to the L1 vertebral body from a primary
femoral tumor. Clinically, all patients originally had pain
ranging in duration from 1 to 20 months. Two of the patients
initially noted the pain following a sports injury, and 2 of the
patients complained of night pain.
Radiographically ❚Image 1❚, the 3 primary bone cases
were described as having prominent periosteal reaction. One
case was heterogeneously contrast-enhancing, another case
was permeative, and yet another case had a blastic appearance.
One case displayed evidence of neoplastic bone formation. All
3 cases were radiographically “suspicious” for osteosarcoma.
Follow-up information was available for 4 patients.
Three of the patients had metastatic disease, and the fourth
patient had a local recurrence. All 4 patients were treated with
A B
❚Image 1❚ Small cell osteosarcoma. A and B, Anteroposterior and lateral plain radiographs of left knee in a 15-year-old boy. A
4.5-cm blastic lesion superimposing the lateral tibial metaphysis with a thick, active, periosteal reaction projecting lateral to the
tibial margin.
© American Society for Clinical Pathology
Anatomic Pathology / Original Article
resections and preoperative and postoperative chemotherapy.
Of the 4 patients, 2 died of disease (4 and 3 years after diagno-
sis). Another patient continued to have progressive metastatic
lung disease as of September 2009, and the fourth patient was
free of disease as of July 2009.
Cytopathologic Features
Smears were generally cellular. Common cytomorpho-
logic features observed included relatively small to inter-
mediate-sized cells, high nuclear/cytoplasmic ratios, round
nuclei, minimal anisonucleosis, hyperchromasia, finely gran-
ular nuclear chromatin, fine cytoplasmic vacuoles, and only
rare osteoid ❚Image 2❚, ❚Image 3❚, ❚Image 4❚, ❚Image 5❚, and
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❚Image 6❚. Osteoid had a variegated appearance in the smears
and appeared mostly as faint purple or metachromatic mate-
rial (rapid Romanowsky stain) surrounding individual cells or
small cell groups in a lacy manner. In contrast with collagen,
the osteoid had a more wispy or lacy quality with ill-defined
borders lacking naked fibroblastic nuclei. Osteoid was diffi-
cult to identify on Papanicolaou stain, where it appeared as a
pale green substance with embedded neoplastic cell nuclei.
All cases displayed numerous mitoses and abundant kary-
orrhectic nuclei reflecting high cell turnover and rapid tumor
growth. Frank cellular necrosis was not observed. Features
not commonly observed included prominent nucleoli, necro-
sis, cytoplasmic processes, spindled nuclei, nuclear molding,
and multiple small nucleoli. Scant focal osteoid was identified
in 3 cases, as were occasional cells with moderate amounts
of spindled to epithelioid cytoplasm in 2 cases, representing
some heterogeneity in the tissue sampled.
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Bishop et al / Cytopathology of Small Cell Osteosarcoma
Flow cytometry was performed in 2 cases, and immu-
noperoxidase studies were done in 3 cases using the
standard method owing to morphologic similarities to
lymphoid lesions and other small round blue cell tumors.
Flow cytometry displayed the presence of a nonlymphoid
❚Image 2❚ Small cell osteosarcoma, fine-needle aspiration.
The smear shows small to intermediate-sized malignant
cells with scant blue cytoplasm, minimal anisonucleosis,
high nuclear/cytoplasmic ratios, and mitosis (4 o’clock) (rapid
Romanowsky, ×400).
❚Image 4❚ Small cell osteosarcoma, fine-needle aspiration.
The smear shows tight clustering of small malignant cells
with a high nuclear/cytoplasmic ratio and nuclear molding and
occasional prominent nucleoli. Despite the tight molding, the
cells have a lymphocyte-like appearance (rapid Romanowsky,
×600).
758 Am J Clin Pathol 2010;133:756-761
758 DOI: 10.1309/AJCPO07VGDZCBRJF
phenotype (CD45–), and immunoperoxidase failed to stain
the small malignant cells for CD99 (O13), bcl2, pancyto-
keratins, and neuroendocrine markers. The standard flow
cytometric panel included CD45, CD71, CD33, HLA-DR,
CD19, CD20, κ, λ, CD3, CD56, and CD5. Histopathologic
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❚Image 3❚ Small cell osteosarcoma, fine-needle aspiration. A
loose fragment of relatively small malignant cells (compare
with a leukocyte at 1 o’clock) with scant pale cytoplasm, finely
granular chromatin, and inconspicuous nucleoli. Background is
clean and devoid of osteoid (Papanicolaou, ×400).
❚Image 5❚ Small cell osteosarcoma, fine-needle aspiration.
The malignant osteoblasts have round nuclei, high nuclear/
cytoplasmic ratios, finely granular chromatin, and conspicuous
cytoplasmic vacuoles. The cytomorphologic features
illustrated here are indistinguishable from Ewing sarcoma/
primitive neuroectodermal tumor (rapid Romanowsky, ×400).
© American Society for Clinical Pathology
 Anatomic Pathology / Original Article

correlation was available for all cases as a follow-up exci-

sion (3 of 5) or an antecedent resection of a newly aspirated
metastasis (2 of 5). Histologically, small cell osteosarcoma
displayed sheets of uniform cells with round nuclei and
minimal cytoplasm ❚Image 7❚ and ❚Image 8❚. All resections
showed at least focal osteoid production that was often lace-
like (Image 6).
Discussion
Small cell osteosarcoma is extremely rare; even at 2
highly specialized tertiary care academic hospitals only 5
cases were identified in a 19-year period. This rarity is similar
to that seen in previously reported series.2,4-6
The demographics of small cell osteosarcoma are similar
to those of conventional osteosarcoma.1,7 In our series, pain

A B

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❚Image 6❚ Small cell osteosarcoma, fine-needle aspiration. A and B, The malignant osteoblasts are small and display nuclear
monotony with inconspicuous nucleoli. The characteristic “lacy” osteoid is present, which appears as faintly metachromatic
(with rapid Romanowsky stain) and pale green (with Papanicolaou stain) amorphous substance. When osteoid is present, its
accurate identification and distinction from collagen and other matrix-type substances can be extremely helpful for the diagnosis
of small cell osteosarcoma (rapid Romanowsky, ×400).

❚Image 7❚ Small cell osteosarcoma, fine-needle aspiration. ❚Image 8❚ Small cell osteosarcoma, histologic section. A
Cell-block section shows loosely placed sheet of small, syncytium of small, round, and uniform malignant cells
uniform cells with round nuclei, finely granular chromatin, and embedded in a loose, pale pink stroma and fine lacy osteoid
few mitotic figures. Osteoid is not seen (H&E, ×200). (2 o’clock) (H&E, ×100).

© American Society for Clinical Pathology Am J Clin Pathol 2010;133:756-761 759

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Bishop et al / Cytopathology of Small Cell Osteosarcoma
was the most common clinical manifestation (all cases), with
pain at night seen in 2 cases. Three of the cases originated
from the classic location for osteosarcoma (2 distal femur and
1 proximal tibia), whereas one was a paravertebral primary
with peripancreatic soft tissue metastasis and another was a
femur primary with a vertebral metastasis. The histories of
small cell osteosarcoma were available in the 2 metastatic
cases at the time of FNA evaluation. Because of the rarity of
this lesion, details about the optimal treatment and prognosis
of small cell osteosarcoma compared with conventional osteo-
sarcoma are not clear.1
Radiologically, most small cell osteosarcomas show a
mixed lytic and blastic pattern, often with soft tissue exten-
sion.1,5-7 In our series, a common feature was a periosteal
reaction (all 3 primary cases). A helpful radiographic
feature of small cell osteosarcoma previously described is
the presence of new bone formation within the tumor; this
helps to distinguish it from Ewing sarcoma, particularly if
mineralization is seen in areas of soft tissue extension.1,5,8
The zonal mineralization pattern described in earlier reports
of small cell osteosarcoma was not observed in the cases in
this small study.
Cytologically, the most common features in our series
were small to intermediate-sized cells, high nuclear/cyto-
plasmic ratios, focal pleomorphism, round nuclei, and dark
finely granular chromatin, followed by cytoplasmic vacuoles,
focal and minimal anisonucleosis, nuclear molding, and scant
osteoid. Cytologic features that are typical of conventional
osteosarcoma but were not observed include macronucleoli,
prominent spindled morphologic features, plasmacytoid mor-
phologic features, cellular necrosis, and relatively abundant
osteoid.
Grossly, small cell osteosarcoma generally has a fleshy
appearance that is seen in many highly cellular neoplasms.1
In tissue sections, 3 histologic patterns have been described:
Ewing sarcoma–like (the most common), lymphoma-like, and
small spindle cell.1,5,7 The presence of osteoid is required for
diagnosis of small cell osteosarcoma; it is often focal, making
the diagnosis often extremely difficult.1,5,7 Although classi-
fied as an osteosarcoma based on its production of osteoid, the
histomorphologic features of small cell osteosarcoma differ
from those of conventional osteosarcoma in that the cells are
smaller with less cytoplasm and more uniform.
As alluded to earlier, the differential diagnosis of small
cell osteosarcoma includes other small round blue cell tumors,
including various non-Hodgkin lymphomas, small cell car-
cinoma, neuroblastoma, mesenchymal chondrosarcoma, and
Ewing sarcoma/primitive neuroectodermal tumor (PNET).1,6,7
The cells of malignant lymphoma generally have larger nuclei
than in small cell osteosarcoma, often with vesicular chroma-
tin, irregular nuclear membranes, prominent nucleoli, a lack
of cellular cohesion, and lymphoglandular bodies, features not
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generally seen in small cell osteosarcoma. In addition, immu-
noperoxidase staining for common leukocyte antigen and
flow cytometry can effectively exclude a lymphoproliferative
process. Small cell carcinoma manifesting as a bony mass,
particularly in a young patient, would be extremely uncom-
mon.6 In addition, a lack of immunoperoxidase staining for
cytokeratins and neuroendocrine markers like synaptophysin
and chromogranin would exclude this possibility.
Neuroblastoma metastatic to bone may also mimic small
cell osteosarcoma with its small blue cell morphologic fea-
tures and proclivity to young patients, particularly if the pres-
ence of a primary adrenal tumor is not known. The presence
of Homer Wright rosettes or pseudorosettes supports a diag-
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nosis of neuroblastoma over small cell osteosarcoma. In addi-
tion, neuroblastomas are immunoreactive for synaptophysin
and chromogranin but are virtually never immunoreactive for
CD99 (O13).
Although mesenchymal chondrosarcoma has areas of
small round blue cell morphologic features, it characteristi-
cally has “staghorn,” hemangiopericytoma-like vessels and
areas of differentiated cartilage, features not generally seen
in small cell osteosarcoma.1,7 However, if these features are
not present on the smears owing to sampling, mesenchymal
chondrosarcoma can be very difficult to differentiate from
small cell osteosarcoma.
The most difficult differential diagnosis is between
small cell osteosarcoma and Ewing sarcoma/PNET. Indeed,
the histologic appearance of Ewing sarcoma/PNET—sheets
of monotonous, small, round cells with minimal cyto-
plasm—can be identical to that of small cell osteosarcoma.
In addition, in the absence of mineralization, true osteoid
of small cell osteosarcoma can be very difficult to differ-
entiate from collagen or fibrin that can be seen in Ewing
sarcoma/PNET.6 If present, any significant degree of cell
spindling in the tumor effectively eliminates the diagnosis
of Ewing sarcoma/PNET.6 The presence of Homer Wright
rosettes and pseudorosettes supports the diagnosis of Ewing
sarcoma/PNET over small cell osteosarcoma, although as
mentioned, these features may also be seen in metastatic
neuroblastoma.7 Ewing sarcoma/PNET (as well as mesen-
chymal chondrosarcoma) is usually strongly immunoreac-
tive for CD99 (O13); unfortunately, small cell osteosarcoma
can also be positive for CD99, so positive staining with this
marker is not helpful. However, a negative result with this
immunoperoxidase marker supports the diagnosis of small
cell osteosarcoma over Ewing sarcoma/PNET.1,7 In addi-
tion, if molecular testing can be performed, the t(11;22)
(q24;q12) translocation is diagnostic for Ewing sarcoma/
PNET. However, in the absence of osteoid, it may not be
possible to definitively differentiate small cell osteosarcoma
and Ewing sarcoma/PNET in limited material obtained by
FNA. Overall, in our series, flow cytometry (2 cases) and
© American Society for Clinical Pathology

immunoperoxidase staining (3 cases) were helpful in exclud-
ing potential mimics in some cases.
The most important reason to be able to recognize small
cell osteosarcoma is the effect of this diagnosis on guiding
appropriate therapy. Although the treatment of small cell
osteosarcoma has not been optimized (owing to its rarity),
small cell osteosarcoma is most often treated like conven-
tional osteosarcoma, with neoadjuvant chemotherapy fol-
lowed by surgical resection.1,6 Mesenchymal chondrosarcoma
is not treated with chemotherapy, and the chemotherapeutic
protocols for small cell carcinoma, non-Hodgkin lympho-
mas, Ewing sarcoma/PNET, and osteosarcoma are differ-
ent. Specifically, osteosarcoma is most often treated with a
regimen of cisplatin, doxorubicin, and high-dose methotrex-
ate,9 whereas Ewing sarcoma/PNET is most often treated
with doxorubicin, cyclophosphamide, vincristine, and dac-
tinomycin.10 However, in cases in which a tumor cannot be
definitively placed into the small cell osteosarcoma or Ewing
sarcoma/PNET category, authorities recommend treating the
tumor as a Ewing sarcoma/PNET.1,6
Small cell osteosarcoma shares some of the previously
described cytomorphologic features of classic osteosarcoma,
but a common denominator is the predominance of small to
intermediate-sized cells with scant osteoid. Clinical and radio-
graphic parameters, in conjunction with cytomorphologic,
flow cytometric, and immunoperoxidase studies, are helpful
in the definitive diagnosis of small cell osteosarcoma.
From the Departments of 1Pathology and 2Radiology, The Johns
Hopkins Hospital, Baltimore, MD; and 3Pathology, The Ohio
State University, Columbus.
© American Society for Clinical Pathology
Anatomic Pathology / Original Article
Address reprint requests to Dr Ali: Dept of Pathology, The
Johns Hopkins Hospital, 600 N Wolfe St, Path 406, Baltimore, MD
21287.
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