Small Round Cell Tumors of Soft Tissue and Bone
Shi Wei, MD, PhD; Gene P. Siegal, MD, PhD
 Context.—Small round cell tumors of soft tissue and
bone constitute a divergent group of neoplasms. These
lesions often demonstrate overlapping clinical and radio-
logic characteristics and share histomorphologic and
sometimes immunophenotypic similarities, but they typi-
cally have diverse prognostic outcomes, thus warranting
different clinical management. Recent advances in molec-
ular and cytogenetic techniques have identified a number
of novel molecular alterations contributing to the diversity
of these lesions. This state-of-the-art knowledge has
enhanced our understanding of these diseases.
Objective.—To provide an overview of the current
concepts in the classification and diagnosis of small round
cell tumors of soft tissue and bone, focusing on salient
histologic features, key immunophenotypic characteristics,
and recent molecular genetic advancements.
Data Sources.—Data were obtained from pertinent
S mall round cell tumors (also known as small, blue, round
cell tumors) constitute a divergent group of neoplasms.
These tumors are morphologically alike and characterized as
highly aggressive malignant tumors composed of relatively
small, round, hyperchromatic and monotonous undifferen-
tiated cells with an increased nuclear to cytoplasmic ratio.
The differential diagnosis of these tumors is particularly
difficult because of their undifferentiated or primitive
character. They can occur in any age group but demonstrate
different age peaks for given tumor types.1 Although there
are always exceptions, some tumors primarily affect the
skeleton, such as Ewing sarcoma and small cell osteosar-
coma, whereas others typically occur in somatic soft tissue,
including rhabdomyosarcoma and desmoplastic small round
cell tumor. Moreover, the overlapping histologic, immuno-
histochemical, and molecular features create diagnostic
challenges despite significant clinical and prognostic differ-
ences as well as therapeutic indications.
Accepted for publication December 11, 2020.
Published online February 26, 2021.
From the Departments of Pathology (Wei, Siegal) and Genetics
(Siegal), O’Neal Comprehensive Cancer Center, University of
Alabama, Birmingham.
This work was funded in part by the Haley’s Hope Memorial
Support Fund and the Thomaus Logan RAID Fund. The authors have
no relevant financial interest in the products or companies described
in this article.
Presented in part at the Seventh Princeton Integrated Pathology
Symposium; May 16, 2020; Plainsboro, New Jersey.
Corresponding author: Shi Wei, MD, PhD, Department of
Pathology, University of Alabama at Birmingham, NP 3545, 619
19th Street South, Birmingham, AL 35249-7331 (email: swei@
uabmc.edu).
Arch Pathol Lab Med—Vol 146, January 2022
peer-reviewed English-language literature and firsthand
experience from the authors as practicing bone and soft
tissue pathologists.
Conclusions.—Immunohistochemistry plays a vital role
in rendering a specific diagnosis or narrowing the
differential diagnosis in small round cell tumors of soft
tissue and bone. Molecular genetic studies are often
needed, especially for those lesions with unusual histologic
features, an uncommon immunoprofile, and/or unusual
clinical presentation. Accurate diagnosis of these tumors
necessitates recognition of salient histologic features,
judicious and astute use of ancillary studies, and correla-
tion with the clinical and radiologic characteristics to
guide clinical decision-making.
(Arch Pathol Lab Med. 2022;146:47–59; doi: 10.5858/
arpa.2020-0773-RA)
Recent molecular genetic advances have identified a
growing list of round cell sarcomas, thus having revolu-
tionized the diagnosis of sarcomas and provided insight into
potential therapeutic targets as well as prognostic biomark-
ers. The research to that end has also resulted in the
reclassification of a number of previously established tumor
types, as illustrated in the most recent edition of the World
Health Organization (WHO) classification of soft tissue and
bone tumors. Herein, we provide an overview of the current
concepts of small round cell tumors of soft tissue and bone,
focusing on salient histologic features, key immunopheno-
typic characteristics, and recent molecular genetic advance-
ments.
EWING SARCOMA AND SO-CALLED EWING-LIKE
SARCOMAS
Ewing Sarcoma
The Ewing sarcoma/primitive neuroectodermal tumor
(PNET) family of tumors was historically thought to be 2
different entities, with the latter demonstrating neuroecto-
dermal differentiation. It is now generally recognized that
Ewing sarcoma of bone, extraosseous Ewing sarcoma,
PNET, and Askin tumor (PNET arising in the chest wall)
represent one and the same neoplastic process. These
tumors share the common genetic abnormalities character-
ized by fusions involving a member of the TET (TLS/EWSR1/
TAF15) or FET (FUS/EWSR1/TAF15) family of genes and a
member of the ETS family of transcription factors. The most
common cytogenetic abnormality is the t(11;22)(q24; q12)
translocation, which results in EWSR1-FLI1 fusion tran-
script (~85%), followed by t(21;22)(q22; q12), which results
Small Round Cell Tumors—Wei & Siegal 47
Figure 1. Selective Ewing and ‘‘Ewing-like’’ sarcomas. A, Ewing sarcoma of soft tissue demonstrates solid sheets of uniform, small, blue, round cells
with scant cytoplasm and minimal extracellular matrix. B, Atypical Ewing sarcoma arising in the kidney shows larger, atypical epithelioid cells. C,
Admantinoma-like Ewing sarcoma of the thyroid manifests lobules of primitive, small round cells. D through F, CIC-rearranged sarcoma displays
variably sized round cells with focal prominent myxoid stroma (D), moderate nuclear pleomorphism (E), and diffuse WT1 expression (F)
(hematoxylin-eosin, original magnifications 3200 [A through D] and 3400 [E]; original magnification 3400 [F]).

in a EWSR1-ERG transcript (~10%). The remaining cases
demonstrate alternative translocations involving either
EWSR1 or FUS and other ETS family members, including
ETV1, ETV4, and FEV, each accounting for ,1% of cases.1
Ewing sarcoma is the second most common malignant
tumor of bone in children and young adults, with a peak
incidence during the second decade of life. Those occurring
in patients older than 30 years more often affect soft tissue.2
When in long bones, it typically arises in the diaphysis or
metadiaphysis, although any bone in the skeleton can be
affected. An ill-defined, osteolytic (and rarely osteosclerotic)
lesion with an ‘‘onion-skin’’–like multilayered periosteal
reaction is the most characteristic radiologic presentation.
Extraskeletal Ewing sarcoma (about 12% of all cases) has a
wide distribution of anatomic sites, including visceral
organs.2,3
48 Arch Pathol Lab Med—Vol 146, January 2022
Ewing sarcoma typically appears as solid sheets of
uniform, small blue round cells with scant clear or
eosinophilic cytoplasm and minimal extracellular matrix
(Figure 1, A). In some cases, the tumor cells are larger and
more pleomorphic, known as atypical Ewing sarcoma
(Figure 1, B). So-called admantinoma-like Ewing sarcoma
typically occurs in the head and neck region (Figure 1, C).2,4,5
Diffuse membranous expression of CD99 is characteristic for
Ewing sarcoma (~95%) but not specific. Focal expression of
cytokeratin can also be seen.1 FLI1 and ERG are often
expressed in those with the corresponding gene fusions,
albeit not specifically, in addition to their utility as
endothelial markers. FLI1 is also expressed in a number of
lymphomas, including lymphoblastic lymphoma (also
CD99þ), anaplastic large cell lymphoma, and angioimmu-
noblastic T-cell lymphoma, as well as a subset of
Small Round Cell Tumors—Wei & Siegal
melanomas, carcinomas of various organs, and other sar-
comas, such as synovial sarcoma.6 ERG is also positive in a
number of other malignancies, including acute myeloid
leukemia, epithelioid sarcoma, and a subset of prostate car-
cinoma.7–9 NKX2.2, a homeodomain-containing transcrip-
tion factor that plays a critical role in neuroendocrine/glial
differentiation, is a target of EWSR1-FLI1 and has been
shown to be a relatively sensitive marker with a suboptimal
specificity. It is also positive in Ewing sarcoma with an
EWSR1-ERG gene rearrangement, and thus may be helpful
in distinguishing Ewing sarcoma from its histologic mimics.10
Genetic analysis is often required for the diagnosis of
Ewing sarcoma. Fluorescence in situ hybridization analysis
using a break-apart probe is highly sensitive in detecting
EWSR1 rearrangements but does not identify its transloca-
tion partner, whereas reverse transcription–polymerase
chain reaction analysis is a more specific test in identifying
the EWSR1-FLI1 fusion gene but has suboptimal sensitivity
(54%) in formalin-fixed, paraffin-embedded tissue.11 Virtu-
ally all current commercial sarcoma fusion panels include
EWSR1.
Ewing sarcoma displays a high propensity for distant
metastases, with the most common sites being lung and
bone. The prognosis of Ewing sarcoma has been improved
with current multimodal therapeutic strategies.
Round Cell Sarcoma With EWSR1–Non-ETS Fusions
There is an emerging subgroup of small round cell
sarcomas that possess EWSR1 or FUS rearrangements and
share various degrees of similarity with Ewing sarcoma
clinically, histomorphologically, and immunophenotypically
(ie, CD99 and NKX2.2). However, these tumors lack the
pathognomonic molecular hallmark of Ewing sarcoma
because their fusion partners are invariably non-ETS
transcription factor family members. The reported cytoge-
netic alterations include EWSR1-NFATc2, FUS-NFATc2,
EWSR1-PATZ1, EWSR1-SMARCA5, and EWSR1-SP3.12,13
These tumors have been previously regarded as Ewing-like
sarcomas.
There is a wide age distribution for this group of tumors,
and each of them has a small number of cases. EWSR1-
NFATc2 and FUS-NFATc2 sarcomas predominantly affect
bones, with a strong male predominance (5:1). EWSR1-
PATZ1 sarcomas arise in deep soft tissue, and the same
fusion has also been identified in gliomas.14 They are mostly
treated similarly to Ewing sarcoma, with generally unfavor-
able outcomes. Given the limited data, it remains to be
determined whether these tumors represent one or more
stand-alone pathologic entities or are better classified as
variants of Ewing sarcoma.
CIC-Rearranged Sarcoma
There have been new emerging undifferentiated round
cell sarcomas that clinically and histologically mimic Ewing
sarcoma but fail to demonstrate any of the aforementioned
molecular genetic abnormalities characteristic of Ewing
sarcoma. In 2006, two cases of ‘‘Ewing-like sarcoma’’ were
found to harbor a recurrent t(4;19)(q35;q13) translocation,
which resulted in fusion between CIC (Capicua Transcrip-
tional Repressor), a human homolog of Drosophila capicua
that encodes a high-mobility group box transcription factor,
and DUX4, a double homeodomain gene.15 The latter is
located within a D4Z4 repeat array in the subtelomeric
region of 4q35. A similar D4Z4 repeat array has been
Arch Pathol Lab Med—Vol 146, January 2022
identified on 10q26, and the fusions of CIS to the DUX4
gene on 10q26 have also been found subsequently.16 To
date, CIC-DUX4 fusion is the most frequent genetic
alteration in EWSR1/FUS-negative undifferentiated small
round cell tumors, whereas a number of other fusion
partners for CIC have been recently identified, including
FOXO4, LEUTX, NUTM1, and NUTM2A.17
CIC-rearranged sarcomas primarily occur in the deep soft
tissue of the limbs and trunk; less commonly affect the head
and neck, retroperitoneum, pelvis and visceral organs; and
rarely arise in bone. There is a wide age distribution at
presentation, ranging from 6 to 81 years, but they more
commonly occur in young adults.
Histologically, CIC-rearranged sarcomas are typically
composed of solid sheets of small round cells, with (at least
in part) a lobulated growth pattern. Focal myxoid stroma is
reportedly seen in one-third of cases (Figure 1, D). A minor
component of spindled or epithelioid cells is not uncom-
mon. Most cases show minor nuclear pleomorphism,
although a moderate degree of cytologic atypia may be
rarely present, including coarse chromatin and prominent
nucleoli (Figure 1, E). These tumors may have variable CD99
immunoreactivity, ranging from negative through focal and/
or weak to diffuse and/or strong. Expression of WT1 and
ETV4 is seen in 90% to 100% of tumors, and thus is useful in
distinguishing CIC-rearranged sarcomas from their histo-
logic mimickers (Figure 1, F). Other rarely expressed
markers include NUT (CIC-NUTM1 fusion), cytokeratin,
S100 protein, calretinin, ERG, and myogenic makers.17
Most CIC-rearranged tumors have a dismal response to
Ewing sarcoma regiments and have aggressive outcomes
when compared to Ewing sarcoma matched for age and
stage. Distant metastasis is frequent, most commonly to the
lung.
Sarcoma With BCOR Genetic Alterations
Another distinct entity of round cell sarcoma is charac-
terized by reproducible BCOR genetic alterations. In 2012, a
fusion of the BCOR (BCL6 corepressor) and CCNB3 genes,
which are nonadjacent genes on the X chromosome, was
identified in 4 cases of round cell sarcoma of bone lacking
the canonical TET-ETS translocation.18 Additional gene
fusions involving BCOR have also been subsequently found,
including BCOR-ITD (internal tandem duplication), BCOR-
MAML3, ZC3H7B-BCOR, BCOR-KMT2D, and CIITA-
BCOR.19–22
BCOR-CCNB3 sarcomas more frequently arise in bone
than soft tissue, have a striking predilection for children and
young adults (.90% younger than 20 years), and are male
predominant. BCOR-ITD tumors occur preferentially in the
somatic soft tissue of the trunk, abdomen, and head and
neck, sparing the extremities.23 The BCOR-ITD fusion and,
less frequently, YWHAE-NUTM2B (both resulting in up-
regulation of BCOR), frequently occur in infantile round cell
sarcoma, clear cell sarcoma of the kidney, and so-called
primitive myxoid mesenchymal tumor of infancy.19
Histologically, sarcomas with BCOR alterations consist of
uniform, primitive small round or spindle cells in solid
sheets or a nesting pattern, with varying myxoid or
collagenous stroma. Strong and diffuse nuclear BCOR
expression is seen in virtually all tumors but is not specific,
because it can be seen in other tumors, such as synovial
sarcoma.19 CD99 expression is variable (~50%). SATB2,
TLE1, and cyclin D1 can also be expressed. Cyclin B3 is also
expressed in BCOR-CCNB3 sarcomas but not other BCOR-
Small Round Cell Tumors—Wei & Siegal 49

Figure 2. Round cell sarcomas producing bone or cartilage matrix. A,
Small cell osteosarcoma, areas with no identifiable osteoid/bone matrix.
B, Small cell osteosarcoma producing lacelike tumor osteoid, with a
hemangiopericytoma-like growth pattern. C, Mesenchymal chondro-
sarcoma characterized by a biphasic appearance composed of hyaline
cartilage and sheets of small, round, blue cells (hematoxylin-eosin,
original magnifications 3200 [A], 3100 [B], and 340 [C]).
rearranged tumors.23 The prognosis in BCOR-CCNB3
sarcomas is similar to Ewing sarcoma, whereas the
outcomes of other tumors in the BCOR family are not well
characterized.
50 Arch Pathol Lab Med—Vol 146, January 2022
SMALL ROUND CELL SARCOMAS PRODUCING
CARTILAGE OR BONE MATRIX
Small Cell Osteosarcoma
Small cell osteosarcoma has long been considered as a
stand-alone subtype of osteosarcoma.24 It has been now
merged into conventional osteosarcoma as a histologic
variant by the most recent edition of the WHO classification
of soft tissue and bone tumors.25 This rare variant,
reportedly comprising approximately 1.5% of all osteosar-
comas, has a wide age distribution but is more commonly
seen in the second decade of life. Most instances occur in
the metaphysis or diaphysis of long bones. The clinical and
radiologic features of small cell osteosarcoma are similar to
those of conventional osteosarcomas, whereas it reportedly
has a slightly worse prognosis than the latter.24,25
The histomorphologic features of small cell osteosarcoma
are those of combined osteosarcoma and Ewing sarcoma.
The lesional cells are typically round to oval and, less
frequently, short-spindled, and generally have hyperchro-
matic nuclei and scanty cytoplasm (Figure 2, A). The nuclear
size may vary from as small as those of Ewing sarcoma to as
large as those comparable to large cell lymphoma. Nucleoli
are rarely seen and are typically inconspicuous, if present.
The presence of a focal hemangiopericytoma-like growth
pattern is a frequent finding. The diagnosis of small cell
osteosarcoma requires identification of osteoid/bone matrix
production, which is frequently subtle and more commonly
arranged in a lacelike fashion (Figure 2, B).4 Mitotic figures
are variably present. Tumor cells may variably express CD99
and, rarely, keratin and epithelia membranous antigen
(EMA), thus representing a diagnostic pitfall. Special AT-
rich sequence-binding protein 2 (SATB2), a transcription
factor essential for osteoblastogenesis, is a very sensitive
marker but lacks specificity. It can be of help in distinguish-
ing small cell osteosarcoma from Ewing sarcoma in the
absence of unequivocal tumor-producing osteoid/bone, as it
is only rarely (1.3%) expressed in the latter.26
To date, in keeping with other conventional osteosarco-
mas, no recurrent molecular genetic abnormalities have
been detected in small cell osteosarcoma.27 Interestingly, the
t(11; 22)(q24; q12) translocation was reported in 1 case of
small cell osteosarcoma in 1990.28 Subsequently, another
case of small cell osteosarcoma with an EWSR1 rearrange-
ment was also detected by interphase fluorescence in situ
hybridization.29 An EWSR1-CREB3L1 fusion transcript was
identified in a multifocal small cell osteosarcoma, and the
corresponding chimeric gene was confirmed by sequencing
of the genomic breakpoint between EWSR1 and CREB3L1.30
It is not clear if a bone-forming round cell sarcoma
harboring an EWSR1 rearrangement should be classified
as small cell osteosarcoma or Ewing sarcoma. Some
authorities believe that cases with this morphology and
EWSR1 translocation should be considered as Ewing
sarcoma with divergent differentiation. Investigation of their
possible relationship may provide further insight into the
molecular mechanisms of the 2 tumor types.
Mesenchymal Chondrosarcoma
Mesenchymal chondrosarcoma is a biphasic tumor
comprising primitive round cells and well-differentiated
hyaline cartilage. This high-grade tumor, accounting for 2%
to 4% all chondrosarcomas, may occur at any age, with a
peak incidence in the second and third decades of life. The
tumor more commonly arises in bone but may also
Small Round Cell Tumors—Wei & Siegal
primarily affect extraskeletal sites. There is a widespread
skeletal distribution, more frequently occurring in the axial
skeleton, including craniofacial bones, ribs, ilium, and
vertebrae. The meninges are one of the most common
extraskeletal sites. Visceral organs, such as kidney, can also
be rarely involved. Skeletal lesions are primarily lytic and
destructive, with poor margins on imaging studies, often not
significantly different from conventional osteosarcoma.31
The histologic hallmark of mesenchymal chondrosarcoma
is a biphasic appearance composed of sheets of primitive
small round cells admixed with islands of well-differentiated
hyaline cartilage (Figure 2, C). The former simulates Ewing
sarcoma or lymphoma, whereas the cartilage nodules may
mimic normal chondrogenesis in the growth plate, or those
from a well-differentiated cartilaginous neoplasm. A hem-
angiopericytoma-like growth pattern can be seen in the
small cell zones, as can subtle osteoid production. The
transition between the 2 components may be abrupt or
gradual.1,32
The small cells are variably positive for CD99. SOX9, a
master regulator of chondrogenesis, is positive in both small
cell and cartilaginous components in virtually all cases, but it
is negative in other small cell malignancies, and thus it may
serve as a useful tool in the differential diagnosis of small
round cell tumors.33
The defining molecular signature of mesenchymal chon-
drosarcoma is a recurrent HEY1-NCOA2 fusion in almost all
well-characterized cases, representing an in-frame fusion of
HEY1 exon 4 to NCOA2 exon 13 at the mRNA level,
probably arising through intrachromosomal rearrangement
of chromosome 8q.34 This fusion was absent in other
subtypes of chondrosarcoma. Interestingly, t(1;5)(q42;q32)
as the sole karyotypic aberration was reported in a single
case of mesenchymal chondrosarcoma lacking a HEY1-
NCOA2 fusion. This resulted in a novel fusion between the
IRF2BP2 gene and the transcription factor CDX1 gene,
suggesting the genetic heterogeneity in this tumor.35
Notably, IDH1/2 mutations frequently harbored in central
and periosteal chondromas and chondrosarcomas have not
been identified in mesenchymal chondrosarcoma.36,37
Mesenchymal chondrosarcoma is an aggressive neoplasm
with a poor prognosis. Surgical resection followed by
adjuvant chemotherapy has been considered the standard
of care treatment. Young age and craniofacial bone
involvement are reportedly associated with a slightly better
prognosis.31
RHABDOMYOSARCOMA
Rhabdomyosarcoma (RMS) constitutes the largest cate-
gory of soft tissue sarcomas in children and young adults.
The subtypes with prominent small round cell morphology
include embryonal RMS (ERMS), alveolar RMS (ARMS),
and, rarely, spindle cell/sclerosing RMS, whereas pleomor-
phic RMS typically does not demonstrate a small round cell
morphology.
Embryonal Rhabdomyosarcoma
Embryonal RMS is the most common subtype of RMS,
typically affecting children younger than 10 years and
occasionally occurring in adolescents, with a predilection
in white individuals. The head and neck region and
genitourinary system are the common sites of involvement.
The tumor may infrequently arise in other sites, including
Arch Pathol Lab Med—Vol 146, January 2022
the abdomen, retroperitoneum, and biliary tract, whereas it
rarely affects the extremities.38
The tumor contains primitive mesenchymal cells housed
within a loose myxoid stroma, admixed with variably
differentiated rhabdomyoblasts, which demonstrate elon-
gation, cytoplasmic eosinophilia, and sometimes cross-
striation in cells with terminal differentiation (Figure 3, A).
Poorly differentiated tumors may be composed exclusively
of solid sheets of primitive round cells (also called a dense
pattern), thus resulting in confusion with ARMS.39 Heter-
ologous cartilaginous differentiation, albeit rare, is a well-
recognized phenomenon (Figure 3, B). Botryoid ERMS
refers to those with a linear distribution of tumor cells
abutting an epithelium-lined surface (Figure 3, C). So-called
anaplastic ERMS is characterized by the presence of
markedly atypical cells, not to be confused with pleomor-
phic RMS that typically occurs in elderly individuals.
Although immunoreactivity may be variable, ERMSs are
always positive for desmin, a key subunit of the intermediate
filament in cardiac, skeletal, and smooth muscles, as well as
myogenin and MyoD1, both of which are transcription
factors involved in myogenesis. It is noteworthy that
nonspecific cytoplasmic MyoD1 staining is not infrequent,
and it may be misinterpreted as positive. Aberrant
expression of other markers, such as cytokeratins and
S100 protein, can also be seen.40 There are no reproducible
molecular genetic alterations identified for ERMS.
Alveolar Rhabdomyosarcoma
Alveolar RMS occurs more commonly in the second and
third decades of life and more often affects the deep soft
tissue of extremities, followed by the head and neck,
paraspinal, and perineal regions.41 The tumor is typically
composed of a monotonous population of primitive,
hyperchromatic round cells arranged in nests divided by
fibrous septa, thus giving rise to an alveolar growth pattern
(Figure 3, D). The solid variant of ARMS is densely cellular
but lacks septa and thus may be confused with the dense
pattern of ERMS.39 Rhabdomyoblastic differentiation may
be seen, but not often, and when present it is often a minor
component. Other occasional histologic features include
multinucleated giant cells and clear cell morphology.
The molecular signature of ARMS is a t(2;13)(q35;q14) or,
less frequently, t(1;13)(p36;q14) translocation, resulting in
PAX3-FOXO1 or PAX7-FOXO1 fusion genes, respectively.42
Compared with ERMS, myogenin expression in ARMS is
typically stronger and diffuse, whereas MyoD1 expression
may be focal. Expression of focal keratin or neuroendocrine
markers can be occasionally seen (Figure 3, E). This should
not be confused with olfactory neuroblastoma when in a
sinonasal location, because the latter typically demonstrates
diffuse expression of neuroendocrine markers. ARMS is
clinically more aggressive than ERMS, thus, it is important
to distinguish the former from the latter.38
Spindle Cell/Sclerosing Rhabdomyosarcoma
As its name implies, spindle cell/sclerosing RMS demon-
strates a spindle cell and/or sclerosing morphology. This
variant most commonly involves the head/neck region
followed by the extremities in adults, but it arises more
frequently in the paratesticular region in boys. It may also
rarely occur in visceral organs, the retroperitoneum, and
bone.43
The molecular genetic abnormalities identified in this
subtype are complex but can be largely categorized into 3
Small Round Cell Tumors—Wei & Siegal 51
Figure 3. Rhabdomyosarcomas. A, Embryonal rhabdomyosarcoma exhibiting primitive rounded to spindled cells as well as numerous
differentiating rhabdomyoblasts. B, Embryonal rhabdomyosarcoma with cartilaginous differentiation. C, The botryoid pattern of embryonal
rhabdomyosarcoma showing a variably cellular tumor with condensation beneath an epithelium-lined surface. D, Alveolar rhabdomyosarcoma
consisting of monotonous round cells with an ‘‘alveolar’’ growth pattern. E, Focal neuroendocrine differentiation of alveolar rhabdomyosarcoma as
demonstrated by antisynaptophysin immunohistochemistry. F, Sclerosing rhabdomyosarcoma displaying clusters of single cells in a sclerotic
collagenized stroma (hematoxylin-eosin, original magnifications 3200 [A, C, and D], 3100 [B], and 3400 [F]; original magnification 3200 [E]).

groups. The congenital/infantile spindle cell RMS shows
VGLL2/NCOA2/CITED2 rearrangements, resulting in gene
fusions, including SRF-NCOA2, TEAD1-NCOA2, VGLL2-
NCOA2, and VGLL2-CITED2. Further, the MYOD1
p.Leu122Arg gene mutation is typically seen in young
adults. Important to note is that recurrent genetic alterations
have not been identified in a significant proportion of cases.
Interestingly, EWSR1/FUS-TFCP2 and MEIS1-NCOA2 fu-
sions have been recently identified in a number of
intraosseous RMSs, showing distinct morphology and
immunophenotype.44 Thus, further classification of this
subtype is emerging.
Histologically, spindle cell RMS is characterized by the
proliferation of spindle cells arranged in sheets, fascicles, or
a herringbone pattern, as is commonly seen in leiomyosar-
52 Arch Pathol Lab Med—Vol 146, January 2022
coma or fibrosarcoma, with variable degrees of nuclear
pleomorphism and mitotic activities. Rhabdomyoblastic
differentiation can be observed occasionally. In sclerosing
RMS, the tumor cells are arranged in various patterns, such
as nests, cords, trabeculae, or microalveoli, and they are
embedded in a distinctive sclerotic/hyalinized stroma that
may simulate tumor osteoid in osteosarcomas. Primitive
undifferentiated cells with hyperchromatic, round nuclei
and a high nuclear to cytoplasmic ratio may constitute a
variable proportion of the tumor (Figure 3, F). This, along
with the lack of prominent sclerotic stroma, may be
mistaken for other undifferentiated small round cell
sarcomas. Moreover, a distinctive epithelioid morphology
has also been observed in intraosseous RMS.44 Spindle cell/
sclerosing RMSs typically demonstrate variable expresion of
Small Round Cell Tumors—Wei & Siegal

Figure 4. Round cell sarcomas with a prominent sclerotic stroma. A,
Desmoplastic small round cell tumor showing nests of round cells
separated by a densely collagenized stroma. B, Sclerosing epithelioid
fibrosarcoma displaying bland, uniform, small, round, epithelioid cells
lacking significant cytologic atypia resembling lobular carcinoma of the
breast. C, Another example of sclerosing epithelioid fibrosarcoma
exhibiting a pseudoalveolar growth pattern (hematoxylin-eosin, original
magnifications 3100 [A and C] and 3200 [B]).
Arch Pathol Lab Med—Vol 146, January 2022
desmin, limited myogenin, and strong positivity for MyoD1.
Aberrant expresison of cytokeratin and ALK has been
reportedly seen in intraosseous RMS.
The prognosis of this group of tumors is highly variable.
Congenital/infantile spindle cell RMSs harboring gene
fusions have a favorable clinical course. MYOD1-mutant
tumors are associated with poor survival outcomes.43
ROUND CELL SARCOMAS WITH A PROMINENT
SCLEROTIC STROMA
Desmoplastic Small Round Cell Tumor
Desmoplastic small round cell tumor is a distinct
translocation-related sarcoma characterized by small round
cells with polyphenotypic differentiation, prominent stromal
desmoplasia, and an EWSR1-WT1 gene fusion. It primarily
affects children and young adults, with a striking male
predilection. The tumor more commonly arises in the
abdomen, retroperitoneum, or pelvis, with widespread
serosal implants.45
The neoplastic cells may vary in size and shape, and they
may rarely be spindled or epithelioid. They are typically
arranged in irregularly contoured nests embedded in a
prominent desmoplastic stroma, with occasional glandlike
or rosettelike structures (Figure 4, A). Tumor necrosis,
frequent mitoses, and cystic degeneration are common.
Multiphenotypic differentiation is a distinctive feature of
desmoplastic small round cell tumor. Therefore, markers for
epithelial, muscular, and neural differentiation may be
variably immunoreactive, including antibodies directed
against cytokeratins, EMA, desmin, and neuron-specific
enolase. Myogenin and MyoD1 biomarkers are negative.
Selective nuclear expression of WT1 using antibodies raised
against the C-terminus, but not the N-terminus, is a
characteristic immunophenotype.46,47 Dotlike perinuclear
staining with desmin and coexpression of cytokeratin can
be seen in both desmoplastic small round cell tumor and
Wilms tumor, whereas dual immunoreactivity for the WT1
N-terminus and C-terminus is seen in the latter.40,48
The detection of an EWSR1-WT1 rearrangement resulting
from a t(11;22)(p13;q12) translocation is diagnostic for
desmoplastic small round cell tumor but not always needed
for cases with characteristic histomorphology and immu-
nophenotype. The clinical outcomes are generally poor.
Sclerosing Epithelioid Fibrosarcoma
Sclerosing epithelioid fibrosarcoma (SEF) is a distinctive
fibroblastic neoplasm characterized by epithelioid tumor
cells arranged in nests, cords, or sheets embedded within a
prominent sclerotic collagenized stroma. It most commonly
arises in deep soft tissue of extremities followed by the
shoulder, trunk, and head and neck regions, but it may
rarely occur in the visceral organs or bone.49,50
The tumor is typically composed of small, bland, and
uniform epithelioid cells with round to ovoid nuclei and
eosinophilic or clear cytoplasm. It lacks significant cytologic
atypia, thus sometimes closely mimicking lobular carcinoma
of the breast (Figure 4, B). The lesional cells are arranged in
sheets, nests, cords, or, rarely, fascicles. Pseudoalveolar or
pseudoglandular growth may also be seen (Figure 4, C).
Less cellular areas may have myxoid or fibrous stroma.
Marked nuclear pleomorphism, increased mitotic activities,
and necrosis are uncommon features. There is a small subset
of SEF cases showing overlapping histologic, immunophe-
notypic, and molecular characteristics with low-grade
Small Round Cell Tumors—Wei & Siegal 53
fibromyxoid sarcoma (LGFMS).51 This observation leads
some authorities to propose a potential relationship
between these 2 tumors.
The most distinctive immunophenotype of SEF is the
expression of mucin 4 (MUC4) (~90%), similar to that in
LGFMS. Focal, weak expression of EMA can be seen,
whereas staining for cytokeratins is typically negative. Most
SEFs harbor EWSR1-CREB3L1 fusions. Less frequently,
EWSR1 may exchange with FUS or PAX5, and CREB3L1
may be replaced by CREB3L2, CREB3L3, or CREM.52 So-
called hybrid SEF/LGFMS cases more frequently display a
FUS-CREB3L2 fusion identical to that observed in most
LGFMS.51 Pure SEFs have a more aggressive clinical course
than LGFMSs.
ROUND CELL TUMORS WITH A PROMINENT MYXOID
STROMA
The presence of a prominent myxoid stroma is not
uncommon in many tumor types, some of which have
round cell morphology, as described herein.
Myxoid Liposarcoma
Myxoid liposarcoma (which consolidates with the entity
formerly known as round cell liposarcoma) is characteris-
tically composed of uniform, round/ovoid, nonlipogenic
cells and small lipoblasts. The lesional cells are set in a
prominent myxoid stroma with a curvilinear capillary
vascular network that gives rise to a ‘‘chicken-wire’’
appearance. The tumor most frequently affects young adults,
with the peak incidence in the fourth to fifth decades of life.
It typically arises in the deep soft tissue of extremities, may
have synchronous or metachronous multifocal disease, and
spreads hematogenously to unusual locations, such as
retroperitoneum or bone.53,54
Histologically the tumors are typically lobulated, and the
cellularity is usually moderate but varies from region to
region. There may be large mucin pools in paucicellular
areas, thus imparting a pulmonary edema-like growth
pattern. The small round cells lack cytologic atypia. The
lipoblasts range from rare to abundant, are usually small,
and are often univacuolated, mimicking signet ring cells
(Figure 5, A). The pathognomonic molecular genetic
alteration of myxoid liposarcoma is a t(12;16)(q13;p11)
translocation leading to FUS-DDIT3 fusion transcripts
(.95%). A EWSR1-DDIT3 gene fusion resulting from
t(12;22)(q13;q12) substitutes in a very small subset of
tumors.53
High-grade tumors have .5% hypercellularity, less
myxoid matrix, and increased nuclear pleomorphism and
mitotic activity, and are associated with a significantly
higher rate of distant metastases or death. Pure high-grade
lesions lack myxoid matrix and thus may be indistinguish-
able from other undifferentiated round cell sarcomas,
requiring molecular studies for diagnostic confirmation
(Figure 5, B).
Extraskeletal Myxoid Chondrosarcoma
Extraskeletal myxoid chondrosarcoma is characterized by
small, uniform cells with round to oval nuclei, set in an
abundant chondromyxoid matrix. The tumor typically arises
in the deep soft tissue of proximal extremities and limb
girdles in middle-aged adults. Extraskeletal myxoid chon-
drosarcoma may also involve the soft tissue of other
anatomic sites including the head and neck, abdomen,
54 Arch Pathol Lab Med—Vol 146, January 2022
and pelvis.55 Rare cases of primary extraskeletal myxoid
chondrosarcoma of bone have been described.56
Extraskeletal myxoid chondrosarcoma characteristically
demonstrates a multilobulated growth pattern, in which
the neoplastic cells are interconnected with each other to
form cords, strands, or clusters (Figure 5, C). The tumor is
distinctively hypocellular and hypovascular and lacks well-
developed hyaline cartilage. The tumor cells exhibit bland,
round, oval, or bipolar nuclei, and moderate eosinophilic or
vacuolated cytoplasm, and some have prominent rhabdoid
features. In the rare examples of the hypercellular variant,
the cells often show epithelioid cytomorphology and an
elevated nuclear grade.40
There are no specific immunohistochemical markers for
extraskeletal myxoid chondrosarcoma. The tumors may
variably express S100 protein, CD117, and, rarely, cytoker-
atin and neuroendocrine markers. Those with rhabdoid
features may show loss of INI1. A t(9;22)(q22;q12)
translocation resulting in an EWSR1-NR4A3 fusion has
been found as the sole anomaly, whereas a number of other
rare fusion partners for NR4A3 have been recently
identified, including TAF15, TCF12, TFG, FUS, or
HSPA8.57–59 Extraskeletal myxoid chondrosarcoma is usually
associated with a prolonged survival despite of high rates of
local and distant recurrence.
Mixed Tumors/Myoepithelial Neoplasms
Myoepithelial tumors of soft tissue constitute a group of
neoplasms closely related to their salivary gland counter-
parts, namely myoepithelioma, myoepithelial carcinoma,
and mixed tumor (pleomorphic adenoma). These tumors
show variable growth patterns and demonstrate a wide
spectrum of histologic features, including epithelioid,
spindled, plasmacytoid, vacuolated (formerly classified as
parachordoma), and undifferentiated round cell morphology
(Figure 5, D). Mixed tumors show a ductal component. The
stroma may be myxoid or hyalinized. Myoepithelial
carcinoma is defined by increased cytologic atypia, often
along with high mitotic activity and necrosis. Cartilaginous
or osseous dedifferentiation can be seen. Expression of
myoepithelial markers is variable, and the most frequently
expressed markers include cytokeratin, EMA, S100 protein,
SOX10, calponin, p63, and glial fibrillary acidic protein
(GFAP). Loss of INI1 has been observed in a subset of
myoepithelial carcinomas. Approximately half of myoepi-
thelial tumors harbor an EWSR1 rearrangement, with a
number of fusion partners identified, including POU5F1,
PBX1, ZNF444, KLF17, ATF1, and PBX3. Rarely, an alternate
FUS rearrangement with similar partner genes is seen. A
PLAG1 rearrangement has been identified in mixed tumors
similar to that seen in their salivary gland counterparts.60
Poorly Differentiated Sarcomas With Round Cell
Morphology and Myxoid Stroma
Round cell morphology, along with a spectrum of other
histologic features, is not common in various poorly
differentiated sarcomas. Among them, poorly differentiated
synovial sarcoma is such an example. The latter may show
variable histologic features, including epithelioid, fascicular
growth of spindle cells (reminiscent of malignant peripheral
nerve sheath tumor), and hyperchromatic small round cells
resembling Ewing sarcoma (Figure 5, E).61 On the other
hand, myxoid stromal changes are not uncommon in
synovial sarcoma. They are usually focal but can occasionally
become a predominant feature, resulting in alternating
Small Round Cell Tumors—Wei & Siegal
Figure 5. Selective common and uncommon round cell tumors with a prominent myxoid stroma. A, Myxoid liposarcoma composed of uniform,
round nonlipogenic cells and small lipoblasts set in a prominent myxoid stroma with a ‘‘chicken-wire’’ vascular network. B, A pure high-grade myxoid
liposarcoma lacking myxoid matrix, and thus indistinguishable from other undifferentiated round cell sarcomas. C, Extraskeletal myxoid
chondrosarcoma characterized by hypocellular and hypovascular bland, round, ovoid, or bipolar cells arranged in cords, strands, or clusters in a
prominent chondromyxoid matrix. D, Myoepithelial carcinoma showing plasmacytoid and round cell morphology, with a prominent myxoid stroma.
E, Poorly differentiated synovial sarcoma consisting of undifferentiated small round cells with a hemangiopericytoma-like growth pattern. F, Poorly
differentiated and myxoid synovial sarcoma with an arrangement of small round cells in retiform cords or microcysts (hematoxylin-eosin, original
magnifications 3200 [A, C through F] and 3200 [B]).

hypocellular and relatively hypercellular areas and arrange-
ments of cells in retiform cords or microcysts.62 The myxoid
stroma may rarely become prominent, making the diagnosis
extremely challenging (Figure 5, F). In such cases, the more
typical histologic features of synovial sarcoma may be better
recognized in more cellular areas at the periphery of the
lesion.
HEMATOLOGIC NEOPLASMS AND METASTASES
It is doubtless that hematologic neoplasms frequently
originate in bone. Solitary plasmacytoma of bone and
plasma cell myeloma, along with metastases, are always top
differential diagnoses for lytic lesions without identifiable
Arch Pathol Lab Med—Vol 146, January 2022
matrix on imaging in middle-aged and older adults.
Although the recognition of plasmacytic origin is mostly
straightforward given the characteristic cytomorphologic
features of plasma cells, poorly differentiated plasmacyto-
mas and those with anaplastic morphology may be
problematic (Figure 6, A). Of important note, membranous
CD138 reactivity for neoplastic cells in bone is not a
definitive marker for plasmacytic origin unless a diagnosis of
hematologic disease has been established.63 Additional
ancillary studies, such as expression of MUM1 and/or j or
k light chain restriction, may be needed for further
diagnostic confirmation.
Small Round Cell Tumors—Wei & Siegal 55
Figure 6. Selective primary hematologic neoplasms of bone and metastasis of nonepithelial round cell neoplasms to bone. A, Poorly differentiated
plasmacytoma. B, Therapy-related myeloid sarcoma in a patient with metastatic lobular breast cancer to the bone. C, Metastatic medulloblastoma. D,
Metastatic melanoma. E, Metastatic well-differentiated neuroendocrine tumor of the pancreas. F, Metastatic epithelioid gastrointestinal tumor
(hematoxylin-eosin, original magnification 3200).

Primary lymphoma of bone is not uncommon, accounting
for about 7% of malignant bone tumors. Approximately 5%
of all extranodal lymphomas originate in bone. The most
common site is femur, followed by spine and pelvic bones.
Lymphoma of bone may involve a single anatomic site or
multiple bones. Most lymphomas of bone are diffuse large
B-cell lymphomas. Extranodal Burkitt lymphomas common-
ly involve bone. T-cell lymphomas of bone are rare but do
occur, such as anaplastic large cell lymphoma. Rarely,
therapy-related myeloid sarcoma can arise in bone (Figure
6, B). Further detailed descriptions of primary bone
lymphomas are beyond the scope of this review.
It is of utmost importance to keep in mind that bone
represents one of the most common distant sites of tumor
metastasis, of which carcinomas are much more common
56 Arch Pathol Lab Med—Vol 146, January 2022
than sarcomas. Virtually all types of carcinomas may relapse
in bone, some of which are primarily of small round cell
morphology, such as small cell carcinoma, lobular carcino-
ma of the breast, and Merkel cell carcinoma. It is
noteworthy that adenocarcinoma of some organs (ie,
prostate) may recur locally or distantly as small cell
carcinoma after treatment.64 When a metastatic lesion
exhibits a round cell morphology, however, the differential
diagnosis is wide-ranging, including (but not limited to)
neuroblastoma, Wilms tumor, retinoblastoma, medulloblas-
toma, hepatoblastoma, melanoma, well-differentiated neu-
roendocrine tumor, and gastrointestinal stromal tumor,
along with the aforementioned entities (Figure 6, C through
F). Thus, pertinent clinical history is crucial in reaching a
correct diagnosis.
Small Round Cell Tumors—Wei & Siegal
 Clinicopathologic Characteristics of Selective Small Round Cell Tumors
Tumor Type Clinical Characteristics Salient Histologic Features Key Immunophenotypes Molecular Genetics
Ewing sarcoma Children and young adults; Uniform small round cells CD99, NKX2.2, FLI1, ERG Gene fusion involving
long bones . other with scant cytoplasm and EWSR1/FUS and a
skeleton; extraskeletal minimal extracellular member of ETS family
involvement more matrix (FLI1 . ERG . others)
common in middle-aged
or elderly
Round cell sarcoma Variable Similar to Ewing sarcoma Variable; CD99 and EWSR1-NFATc2, FUS-
with EWSR1–non- NKX2.2 more common NFATc2, EWSR1-
ETS fusions PATZ1, EWSR1-
SMARCA5, EWSR1-SP3
CIC-rearranged Wide age range but more Round cells (rarely spindled WT1, ETV4, CD99 (þ/) CIC-DUX4 (rarely other
sarcoma common in young adults; or epithelioid) without fusion partners for CIC)
deep soft tissue of the significant nuclear
limbs and trunk but rarely pleomorphism; variably
involving visceral organs myxoid
and bone
Sarcoma with BCOR Children and young adults; Round or spindled cells; BCOR, SATB2, TLE1, BCOR-CCNB3, BCOR-
genetic alterations male predilection; bone solid or nested; variably cyclin D1, cyclin D3 ITD, BCOR-MAML3,
. soft tissue myxoid (BCOR-CCNB3), ZC3H7B-BCOR,
CD99 (þ/) BCOR-KMT2D, CIITA-
BCOR
Small cell Metaphysis or diaphysis of Combined osteosarcoma SATB2, CD99 (þ/) No recurrent molecular
osteosarcoma long bones and Ewing sarcoma; genetic alteration;
hemangiopericytoma-like EWSR1 rearrangement
growth; lacelike osteoid/ rarely reported
bone matrix
Mesenchymal Wide age distribution, peak Biphasic tumor comprising CD99, SOX9 HEY1-NCOA2
chondrosarcoma in the second and third small round cells and
decades; bone . soft hyaline cartilage
tissue
Embryonal Typically first decade, Primitive mesenchymal cells, Desmin, myogenin, MyoD1 No reproducible
rhabdomyosarcoma predilection in white variable rhabdomyoblasts, molecular genetic
individuals; more common loose myxoid stroma alterations
in head/neck region and
genitourinary system
Alveolar Second and third decades, Monotonous small round Desmin, myogenin (strong/ PAX3-FOXO1, PAX7-
rhabdomyosarcoma more commonly affecting cells, alveolar growth diffuse), MyoD1 FOXO1
deep soft tissue of pattern
extremities
Sclerosing More common in head/neck Primitive cells (þ/ Desmin (variable), MyoD1 MYOD1 mutation (small
rhabdomyosarcoma region or extremities rhabdomyoblasts) with (strong), myogenin subset)
(adults) and paratesticular various growth patterns (limited)
location (children) and sclerotic stroma;
epithelioid cells (in bone)
Desmoplastic small Children/young adults, male Small round cells with Multiphenotypic EWSR1-WT1
round cell tumor predilection; commonly prominent stromal differentiation (epithelial/
involving abdomen, pelvis, desmoplasia muscular/neural); desmin
or retroperitoneum (dotlike), WT-1 (C-
terminus)
Sclerosing epithelioid Deep soft tissue and rarely Small, bland, uniform MUC4 EWSR1 (FUS/PAX5)-
fibrosarcoma visceral organs or bone epithelioid cells and CREB3L1 (CREB3L2/
fibrotic stroma CREB3L3/CREM)
Myxoid liposarcoma Young adults, peak in the Uniform round nonlipogenic None FUS-DDIT3 (.95%),
fourth to fifth decades; cells and small lipoblasts; EWSR1-DDIT3
deep soft tissue of myxoid stroma, ‘‘chicken-
extremities wire’’ vascular network
Extraskeletal myxoid Middle-aged adults; deep Small, uniform cells in S100 (variable) EWSR1-NR4A3 (rarely
chondrosarcoma soft tissue cords, strands, or clusters; other fusion partners
multilobulated growth for NR4A3)
pattern; abundant
chondromyxoid matrix

Arch Pathol Lab Med—Vol 146, January 2022 Small Round Cell Tumors—Wei & Siegal 57
 SUMMARY
Primary small round cell tumors of soft tissue and bone
constitute a diverse group of neoplasms sharing many
histomorphologic similarities and immunophenotypes, and
they may have overlapping clinical and radiologic charac-
teristics but diverse clinical outcomes, as illustrated in the
Table. These lesions may also demonstrate largely overlap-
ping histomorphologic features with hematologic disorders
and metastatic diseases, thus resulting in diagnostic
challenges. Although the pathogenesis of a subset of these
tumors remains to be ascertained, state-of-the-art knowl-
edge has enhanced our understanding of these tumors.
Although numerous molecular alterations have contributed
to the diversity of these lesions, recent molecular and
genetic advancements have led to the reclassification of
some of these entities with potential novel therapeutic
targets emerging, which have significantly improved treat-
ment modalities and prognostic outcomes. Immunohisto-
chemical analyses provide useful tools in characterizing
these lesions and narrowing the differential diagnosis.
However, interpretation of biomarkers requires a cautious
and judicious approach. Molecular genetic studies are often
needed, especially for those with an unusual histomorphol-
ogy and typical histologic features but unusual clinical
presentation (ie, age or anatomic site), or with an
uncommon immunoprofile. Thus, correlation with the
clinical and radiologic characteristics, recognition of salient
histologic features, and astute use of ancillary studies are
critical to reach the correct diagnosis and guide clinical
decision-making.
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