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Recent advances in the

molecular diagnosis of
paediatric soft tissue
sarcomas

improve treatment. The establishment of cooperative groups,

such as the Childrens Oncology Group (COG) in the United
States and the International Society of Peadiatric Oncology
(SIOP) in Europe has done much to ameliorate this situation.
These cooperative group studies and their predecessors have
clarified much of what we now know about these tumours,
through central review, biology studies and standardized multidisciplinary therapeutic trials spanning numerous member
institutions. In addition, they have demonstrated the pivotal role
of accurate pathologic diagnosis in optimizing therapy towards
the best possible clinical outcome.
The diagnostic spectrum of soft tissue tumours is vast, and
this review of molecular diagnostic advances will focus upon soft
tissue malignancies that are prone to develop in children.
Historically, paediatric soft tissue sarcomas have been classified
according to their degree and type of cellular differentiation; so
for example tumours that displayed histologic and ultrastructural
evidence of skeletal myogenesis were placed into the rhabdomyosarcoma category and tumours that displayed spindle cell
morphology were placed into the fibrosarcoma/myofibrosarcoma category. Small round cell tumours that displayed no
evidence of specific cytologic differentiation were relegated to
the categories of extraosseous Ewing sarcoma or undifferentiated
sarcoma. Early on, it was clear that strictly morphologic diagnosis, even when buttressed by histochemical staining and
electron microscopy was of limited value, as tumours with
similar histologic features could have markedly different clinical
behaviours and outcomes. Moreover, the form of cellular
differentiation did not translate into an identifiable cell of
origin, which continues to remain elusive in most paediatric
sarcomas. Even with immunohistochemistry many paediatric
sarcomas remain diagnostic challenges, as different tumours
may have only subtle differences in protein expression, some
have only limited reactivity with commercially available antibodies, and there is considerable overlap, lack of specificity and
variability in how tumours express proteins, as well as significant subjectivity in how immunohistochemical stains are
interpreted.
With the advent of karyotyping, it became clear that paediatric sarcomas could be divided into two distinct groups: those
that had recurrent chromosomal translocations, and those with
complex karyotypes lacking recurrent translocations. Morphologically, translocation associated sarcomas were characterized
by more uniform cell populations, whereas those without
translocations were more likely to be pleomorphic. Cytogenetics
became a powerful tool for diagnosis, and in some cases for
prognostication as well. This cytogenetic approach offered a new
way to look at paediatric sarcomas, and was the foundation upon
which molecular diagnostic pathology has been built. However,
classic cytogenetic analysis, in which tumour cells are grown,
metaphase chromosomes are stained, and banding patterns
analyzed, suffers from significant limitations. Among these are
difficulties in growing cells from many solid tumours, potential
contamination, prolonged turn-around time, and labour intensity. With the advent of standard molecular diagnostic techniques such as reverse transcriptase-polymerase chain reaction
(RT-PCR) and fluorescence in-situ hybridization (FISH), many of
these problems inherent with classic cytogenetics have been
mitigated.

Bruce R Pawel

Abstract
Sarcomas comprise a significant portion of solid tumours diagnosed in
children. As they commonly are composed of primitive round or spindled
cells, morphologic diagnosis can be difficult, and ancillary studies are
often necessary. In recent years, progress has been made in understanding the cytogenetic and molecular changes underlying many of
these tumours, and molecular techniques have emerged from strictly
being research tools to becoming standard diagnostic tests. Paediatric
sarcomas fall into two categories: those with characteristic underlying
translocations, and those without. Translocation associated paediatric
sarcomas include, but are not limited to alveolar rhabdomyosarcoma,
Ewings family of tumours, desmoplastic small round cell sarcoma, synovial sarcoma, infantile fibrosarcoma, clear cell sarcoma of soft tissue, and
low-grade fibromyxoid sarcoma. Sarcomas without translocations tend to
be more pleomorphic in appearance, and include embryonal rhabdomyosarcoma, malignant rhabdoid tumour and epithelioid sarcoma. This
review will describe the molecular and immunohistochemical methods
most applicable in diagnosing these soft tissue malignancies.

Keywords molecular techniques; pathology; paediatrics; sarcoma;

translocations

Introduction
Peadiatric solid tumours can be challenging to diagnose, both
due to their relatively rarity and frequently challenging
morphologic features. In contrast to adults, in whom the
majority of solid tumours are carcinomas of epithelial origin,
solid malignancies occurring outside of the central nervous
system in children are almost entirely composed of embryonal
tumours and soft tissue sarcomas, with carcinomas appearing
only exceptionally. Nevertheless, as the overall incidence of
cancer is much lower in children than in adults, even the more
common peadiatric sarcomas are relatively rare, and may only
be sporadically encountered by most pathologists in general
practice. Even in tertiary peadiatric referral centres, these
tumours are seen infrequently, with lack of experience
hampering both diagnosis and attempts to standardize and

Bruce R Pawel MD is Assistant Professor of Pathology and Laboratory

Medicine at the University of Pennsylvania School of Medicine and The
Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Conflicts of interest: none declared.

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REVIEW

Molecular techniques applicable to paediatric sarcoma diagnosis

ARMS is histologically typified by a monotonous population

of primitive appearing tumour cells, and the term alveolar
refers to the tendency of these loosely cohesive cells to separate
from each other, resulting in clustering of tumour cells along
fibrous septae, in a fashion reminiscent of alveoli of the lung
(Figure 1a,b). In many cases, this essentially artifactual change is
not evident, and ARMS may present histologically as formless
sheets of cells. Pleomorphism is usually minimal, and differentiated strap cells are not seen. In ERMS, tumour cells display
a wider range of differentiation than in ARMS. Resembling stages
in embryonal skeletal muscle development, cells in ERMS span
the range of primitive stellate cells, spindled cells, cells analogous to myotubes, and cells with copious eosinophilic cytoplasm
(so-called strap cells and tadpole cells). At low power, the most
typical finding in ERMS is alternating zones of loose mesenchyme and denser spindled regions (Figure 1c,d). In hollow visci,
such as the bladder, some tumours show a macroscopic grapelike growth pattern, with a histologic cambium-layer formed
by a condensation of tumour cells beneath the native epithelium;
this is the botryoid variant of ERMS. Spindle forms of ERMS
which resemble a fibroma or neurofibroma can also be
encountered.
By immunohistochemical methods, both ARMS and ERMS
will show expression of muscle related antigens such as desmin
and muscle specific actin. However, the most sensitive and
specific immunohistochemical test for RMS is for myogenin,
a skeletal muscle transcription factor not expressed in mature
skeletal muscle.5 The percentage of cells expressing myogenin is
significantly higher in ARMS than in ERMS, which may have
diagnostic value, although overlap exists.6e10 Combining data
from several studies, Morgenstern et al found that over 80% of
cases diagnosed as ARMS displayed nuclear myogenin positivity
in over 50% of tumour cells, whereas only 25% of ERMS did.11
Diffuse myogenin positivity is also a marker of poor prognosis,
independent of fusion status and histology.12 In addition to
myogenin, immunohistochemistry for another skeletal muscle
transcription factor, MyoD1, has also been applied to RMS, but is
less sensitive. Finally, immunohistochemistry for PAX-5,
a member of the gene family involved in ARMS translocations, is
also a useful discriminator in the differential diagnosis between
ARMS and ERMS. In a recent study, PAX-5 nuclear expression
was not seen in any ERMS, and was detected in 67% of ARMS.13
Unlike ARMS, ERMS is not associated with a specific translocation. ERMS tumours are associated with multiple numeric
and structural chromosomal changes, most commonly involving
extra copies of chromosomes 2, 8 and 13. Loss of heterozygosity
at 11p15 (the BeckwitheWiedemann region) has also been
described.4
ARMS is associated with two gene fusions which are not seen
in other tumours: PAX3eFOXO1 and PAX7eFOXO1 (FOXO1
previously known as FKHR); these fusion genes correlate with
translocations t(2;13)(q35;q14) and t(1;13)(p36;q14) respectively14(Figure 2). Rare instances of variant PAX3 fusions have also
been described in ARMS, including fusions with AFX, NCOA1
and NCOA2.15 PAX3eFOX01 fusions are approximately three
times more common than PAX7eFOX01 fusions, whereas
PAX7eFOXO1 fusions appear to confer a better prognosis. Both
of these chimeric fusion products are more potent in activating
transcription than PAX3 or PAX7 alone. In approximately

Reverse transcriptase-polymerase chain reaction (RT-PCR) is an

exquisitely sensitive RNA based assay capable of copying gene
sequences of interest up to a billion-fold, so that they can be
detected in clinical material.1 As the DNA breakpoints in translocations occur within introns and not in exons, RNA chimeric
transcripts have a more consistent structure and are more suitable for probing.2 In this methodology, RNA transcripts are
reverse transcribed into complimentary DNA (cDNA), which
can then be amplified. Since RNA is much more labile than DNA,
this method generally requires the availability of fresh or frozen
tumour tissue. Formalin fixed paraffin embedded tissue has been
successfully used; however it is less reliable, the methodology is
more tedious, and the probability of failure is much higher due to
the poor RNA quality inherent in this material.3
Fluorescent in-situ hybridization (FISH) is a DNA based
method which can be applied to frozen tissue, fixed tissue and to
imprint preparations. There are a number of different variations of
FISH, and its amenability to use on paraffin embedded tissue has
made FISH a first line molecular test modality for paediatric
sarcoma diagnosis in many laboratories. Unlike routine karyotyping, which requires metaphase spreads, FISH can be applied
to interphase, non-dividing cells, greatly increasing its applicability in archived tissue. Split signal, or break-apart FISH is widely
used in present day practice, and employs two probes, each
marked with a different fluorescent dye, and each specific to
a flanking region of a translocation break point. In normal cells, the
two probes would overlap and give a fused signal, whereas if the
probes flank a translocation, the two signals would be separated.
With this split-apart strategy, the necessity for primers specific to
a chimeric fusion (as in RT-PCR) is obviated, as any rearrangement
of that specific gene would be detected, without regard to the
specific translocation partner.1 For example, a split-apart FISH
probe for the EWS gene would reveal an abnormal signal pattern in
any of the Ewings family of tumours that harbour an EWS translocation, as well as other non-Ewings tumours with EWS translocations, such as desmoplastic round cell tumours and clear cell
sarcomas. Since FISH does not employ an amplification step, its
sensitivity is generally less than RT-PCR. In general, FISH can
detect fusions involving as few as 5% of cells in a sample.1 Another
form of FISH involves fused signal probes, which are specific for
the two target genes involved in a given translocation.3

Rhabdomyosarcoma
Rhabdomyosarcoma is the most common soft tissue sarcoma of
childhood. Under the current WHO classification, rhabdomyosarcomas are classified into distinct subgroups that have markedly differing clinical behaviour and require different forms of
treatment.4 The two major forms are alveolar rhabdomyosarcoma (ARMS), which is an aggressive tumour with a poor
prognosis, and embryonal rhabdomyosarcoma (ERMS), which is
generally more favourable. In their classic forms, these two
tumours are histologically distinct; unfortunately many cases
present with ambiguous histologic features, and morphologic
distinction may be difficult even for experienced paediatric
pathologists. Since most ARMS are associated with specific gene
fusions, molecular genetic testing is a valuable adjunct to rhabdomyosarcoma diagnosis.

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a Alveolar rhabdomyosarcoma. Note the discohesive nature of the primitive rhabdomyoblasts lining fibrovascular septae. When present, giant
cells are a helpful feature in distinguishing ARMS from embryonal rhabdomyosarcoma (haematoxylin & eosin). b Typical myogenin staining pattern
in ARMS; most tumour nuclei are positive. c Embryonal rhabdomyosarcoma, with alternating cellular and myxoid areas (haematoxylin & eosin).
d typical myogenin staining pattern in ERMS, only a minority of tumour cell nuclei stain.
Figure 1

20e25% of cases with alveolar histologic features, neither of

these translocations can be identified.16,17 Using data from gene
expression profiling and loss of heterozygosity analysis, it

appears that these fusion-negative ARMS (i.e. tumours with

alveolar histology) have genetic profiles indistinguishable for
ERMS.18 Whether or not they behave in a clinically similar
manner to ERMS is still open to question. In addition to RT-PCR
using specific probe sets, detection of FOXO1 related fusions
using break-apart FISH probes on paraffin embedded specimens
has been shown to be both relatively sensitive and specific.19

Chromosomal translocations in ARMS

Ewings family of tumours

Ewing sarcoma (ES) is the second most common primary
malignancy of bone and soft tissue in childhood, with a peak
incidence in adolescence. Morphologically, Ewing sarcoma/
primitive neuroectodermal tumour (PNET) can be considered the
prototype of the small round blue cell tumour of childhood. In
its classic form, Ewing sarcoma manifests as sheets of undifferentiated small cells which form no particular architectural
pattern, except for the occasional production of neuroectodermal
type rosettes (Figure 3). Although first described as a primary
bone tumour, this tumour also occurs in soft tissue, and has
variable degrees of neuroectodermal differentiation. By expression profiling it appears that Ewing tumour cells are similar to
mesenchymal progenitor cells.20,21 The various modes of
presentation, and occasional expression of neuroectodermal

der(13)
der(13)

Chrom 13

Chrom 13

Chrom 2
der(2)

t(2;13)(q35;q14)

Chrom 1 der(1)

t(1;13)(q36;q14)

Figure 2 Chromosomal translocations in alveolar rhabdomyosarcoma

(Courtesy of Dr. Frederic Barr, University of Pennsylvania School of
Medicine).

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REVIEW

features led to a myriad of designations for this tumour, such as

extraosseous Ewing sarcoma for soft tissue tumours, Askin
tumour for tumours occurring in the thoracopulmonary region,
and primitive neuroectodermal tumour for tumours displaying
neuroectodermal differentiation, such as formation of rosettes.
With the advent of molecular genetic diagnostics, it became clear
that all of these entities shared recurrent translocations, and they
are no longer felt to be biologically distinct, and have been
subsumed into the category of Ewings family of tumours
(EFTs).
Given the undifferentiated nature of this tumour, differential
diagnosis with undifferentiated examples of other paediatric
malignancies can be challenging. In particular, it is difficult, if
not impossible to distinguish Ewing sarcoma from lymphoblastic
lymphoma, solid variant ARMS, and undifferentiated neuroblastoma, without the judicious application of immunohistochemistry. In addition to vimentin, Ewing sarcoma
characteristically displays strong perimembranous staining with
CD99; tumours that lack such staining should not be considered
Ewing sarcoma in the absence of confirmatory molecular data.
Although sensitive, CD99 staining is unfortunately non-specific,
as other tumours such as lymphoblastic lymphomas, rhabdoid
tumours, and synovial sarcomas also may express CD99. Also to
be borne in mind is that Ewing sarcoma may express cytokeratins. In cases of suspected Ewing sarcoma, a reasonable antibody panel might include LCA and Tdt to rule out lymphoblastic
lymphoma, as well as a muscle marker such as myogenin to rule
out rhabdomyosarcoma.
Ewing sarcoma is characterized by translocations involving
the EWS gene on chromosome 22, and these can be interrogated
with both FISH and RT-PCR assays. Amongst the paediatric
sarcomas, Ewing sarcoma appears to have the greatest variety of
fusion patterns, resulting in considerable complexity in testing
and interpretation. The most common translocation, present in
approximately 85e90% of ES is 11;22, involving the EWS gene
on chromosome 22 and the FLI1 gene on chromosome 11
(Figure 4). EWS is a widely expressed normal protein

t(11;22) Translocation in Ewings Sarcoma

15
14
13
12
11
11
12

13
12
11
11
12

13

Der 22

14
21
22

13

Normal 22

23
24
25

Figure 3 a Ewing sarcoma. Primitive cells arranged in formless sheets

(haematoxylin & eosin). b CD99 strong perimembranous staining in Ewing
sarcoma. c Dual colour FISH with break-apart EWS probe, performed on
interphase nuclei in a case of Ewing sarcoma. Note one normal fusion
signal and separation of the other green and red probes (Courtesy of
Dr. Paul J. Zhang, University of Pennsylvania School of Medicine).

DIAGNOSTIC HISTOPATHOLOGY 17:1

Normal 11
Der 11
Figure 4 The t(11;22) translocation, the most common translocation in
Ewings sarcoma (Courtesy of Dr. Frederic Barr, University of Pennsylvania
School of Medicine).

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transcription factor whose exact role is unclear22, and FLI1 is

a highly regulated lineage restricted member of the ETS family of
transcription factors.23 Breakpoints vary among the four introns
of EWS and the six introns of FLI1, and as a result the fusion
transcripts are also variable in size, which must be considered
when interpreting diagnostic RT-PCR.24 The most common
fusion is EWS exon 7 to FLI1 exon 6, designated Type 1, followed
by fusion of EWS exon 7 to FLI1 exon 5, designated Type 2. Type
1 fusions may have a better prognosis than type 2 fusions,
although this is still a matter of some debate.25,26 Approximately
5e10% of ES carry an alternate translocation, that of 21;22, in
which the fusion partner for EWS is the ERG gene on chromosome 21. ERG is also in the ETS family of transcription factors
and is closely related to FLI1. In addition to EWSeFLI1 and
EWSeERG, there are a number of less common EWS translocations to other ETS transcription family members that have
been described: t(2;22) which translocates EWS to FEV, t(7;22)
which translocates EWS to ETV1, and t(17;22) which translocates
EWS to E1AF. In addition to fusions involving the EWS gene, rare
cases of ES have been described that involve the FUS gene on
chromosome 16 (FUSeERG and FUSeFEV).27,28 Finally, it is
apparent that rare small cell undifferentiated tumours that do not
express CD99 may also have gene fusions, which may or may not
involve the EWS gene. EWS has been described as a translocation
partner with non-ETS genes such as SP3, ZNF278, and
POU5F1.23,24 Two cases of Ewings-like tumours have also been
described with CICeDUX4 fusions.24 All told, variant fusions (i.e.
neither EWSeFLI1 nor EWSeERG) are estimated to account for
4e9% of cases histologically defined as ES.24
From a practical standpoint, it may be impractible to test for
all possible fusion combinations by RT-PCR. If paraffin
embedded tissue is tested, this becomes even more problematic,
as degradation from processing precludes amplification of large
RNA segments. FISH using break-apart probes for EWS can
partially circumvent this, as described previously in this review.
Any fusion involving EWS should be picked up by FISH, and
similar break-apart probes for FUS can be applied to cases that
lack the EWS fusion.28 Positive break-apart FISH for EWS must
be interpreted cautiously and in the context of other clinical and
pathologic data, as splitting can also be seen in other tumours,
such as desmoplastic small round cell tumour, clear cell
sarcoma, extraskeletal myxoid chondrosarcoma, polyphenotypic
round cell sarcoma of bone29, myxoid liposarcoma, and angiomatoid fibrous histiocytoma.

years despite extensive multimodality therapy.31 In its classic

presentation, this tumour usually does not present diagnostic
difficulties. However, cases occasionally arise outside of the
abdomen and at a younger age; the fibrous stroma may be poorly
represented in small biopsies, and sometimes immunohistochemistry may be inconclusive.32
A defining molecular feature of DSRCT is the reciprocal t
(11;22)(p13;q12) translocation, which rearranges the EWS gene
to the WT-1 gene on chromosome 11, producing a chimeric
transcript. In most cases, the first seven exons of EWS are fused
to the last three exons of WT-1. As this is the C-terminal region
of WT-1, immunohistochemistry for the carboxy terminus of
WT-1 is usually positive (nuclear) in DSRCT, whereas immunohistochemistry for the N-terminus is usually negative.33
Variants have been reported with alternative EWS breakpoints
containing additional EWS exons and conservation of the WT-1
portion, as well as rare variants with non-typical transcripts
from both EWS and WT-1.34 These rare transcripts may be
associated with aberrant (negative) staining for the C-terminus
end of WT-1. Given the small number of cases, it is unclear if
these more uncommon translocation variants carry any prognostic implications. Interestingly, these variant fusions appear
more common in DSRCTs that arising in unusual sites.34 Also,
the EWSeWT-1 fusion is not entirely specific, as there is
a recent report of two cases of EWSeWT-1 positive paediatric
tumours with spindle cell morphology, expression of smooth
muscle antigens and a favourable clinical course, quite unlike
DSRCT.35

Infantile fibrosarcoma
Infantile fibrosarcoma is a tumour of very young childhood, and
is the most common soft tissue sarcoma seen during the first
year of life, with half of the cases presenting as congenital
tumours. Although characterized by rapid growth, it seldom
metastasizes. It most commonly occurs in the extremities, but
may also be seen in axial locations. Histologically, infantile
fibrosarcoma is similar to adult fibrosarcoma, composed of
fascicles of variably mitotically active spindled cells, often in
a herringbone-type pattern (Figure 5b). However, unlike adult
fibrosarcoma, infantile fibrosarcoma is often infiltrated by
inflammatory cells, and tends to have less pleomorphism. A
prominent hemangiopericytoma-like vascular pattern is
a common feature. Immunohistochemistry tends to be nonspecific, with near universal reactivity for vimentin, and variable positivity for neuron specific enolase, smooth muscle actin
and muscle specific actin.4
Most infantile fibrosarcomas carry a t(12;15)(p13;q26) translocation which fuses the genes ETV6(TEL) and NTRK3.36 This
translocation is identical to that seen in cellular mesoblastic
nephroma, a rare kidney tumour of infants with a similar
histology. Importantly, this fusion has not been described in adult
fibrosarcomas, which have complex karyotypes and carry a graver
prognosis. The fusion may be difficult to recognize with standard
cytogenetic banding techniques, and either FISH or RT-PCR performed on frozen or paraffin embedded tissue are recommended
techniques to evaluate for it.31 The fusion protein leads to activation of the P13-Akt pathway which mediates cell survival, most
likely through the intermediate activation of c-Src.37 Trisomies for

Desmoplastic small round cell tumour

Desmoplastic small round cell tumour (DSRCT) is a highly
aggressive and lethal tumour which mainly afflicts young adults
and adolescents, and has a strong male predilection. First
described 20 years ago,30 this tumour typically presents as an
intrabdominal mass with peritoneal seeding, and in its classic
histologic form is composed of sheets of undifferentiated small
blue cells interrupted by broad swaths of paucicellular fibrosis
(Figure 5a). It displays a characteristic polyphenotypic immunohistochemical profile, co-expressing neural, muscular and
epidermal markers, including desmin, cytokeratins, epithelial
membrane antigen, vimentin, and neuron specific enolase.4 The
overall prognosis is dismal, with most patients dying within 2

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a Desmoplastic small round cell tumour. b Infantile fibrosarcoma. c Clear cell sarcoma. d Low-grade fibromyxoid sarcoma, with large fibrous
rosette (all haematoxylin & eosin).
Figure 5

chromosomes 11, 20, 17 and 8 are also typical for infantile fibrosarcoma, and have been described in over 90% of cases.38

nucleoli, and multinucleated giant cells may be seen. Mitotic

activity is usually low and pleomorphism can be minimal; in
these cases distinction from benign tumours such as cellular blue
nevi can be challenging. Immunohistochemically, CCS will be
positive at least focally for melanoma related antigens such as S100, Melan-A and HMB-45 in almost all cases.4
Typically, CCS is associated with a t(12;22)(q13;q12) translocation, in which EWS is fused with the ATF1 gene on chromosome
12.44 Using FISH, rearrangements of the EWSR1 locus can be
demonstrated in greater than 90% of CCS, and it has been suggested
that the cases that lack such a EWSR1 rearrangement may have been
true melanomas.45 To date, four variants of the EWSR1eATF1
chimeric transcript have been documented, with the most common
being fusions of the EWSR1 exon 8 with ATF1 exon 4 (type 1 fusion)
and EWSR1 exon 7 and ATF1 exon 5 (type 2 fusion). There is no
apparent prognostic significance imparted by the fusion type.46
Other EWSR1eATF1 fusion types have been described, but are
quite rare.45 In addition to the EWSR1eATF1 fusion, Antonescu et al
described a variant fusion EWSeCREB1 in three cases of CCS.47
These cases were unusual in that they had a lack of melanocytic
differentiation, and all were located within the abdomen. Subsequently, three additional cases of CCS with the EWSeCREB1 were
described, all of which were soft tissue based.42,45
Molecular diagnosis of CCS can be accomplished by break-apart
FISH for EWSR1 rearrangements, as well as RT-PCR assay using

Clear cell sarcoma of soft tissue

This tumour is another example of a translocation associated
sarcoma involving the EWS gene. Previously referred to as
malignant melanoma of soft parts, it has morphologic and
immunophenotypic similarity to malignant melanoma. However,
clear cell sarcoma of soft tissue (CCS) is a distinctive clinical and
biologic entity, as melanoma does not carry the translocation, and
CCS almost always arises in deep soft tissues, closely related to
aponeuroses and tendons. CCS also lacks melanoma-associated
BRAF mutations.39 CCS is primarily a tumour of adolescents and
young adults, and is unusual in children younger than 10. It is the
most common malignancy of the ankle and foot in adolescents,
with up to 75% arising in the lower extremity. It is aggressive,
with 5- year survival rates ranging from 47 to 63%. 40e43 Early
diagnosis with complete wide excision offers the best chance of
cure.
Histologically, the tumour typically displays a nested architecture, with groups of tumour cells subdivided by fibrous septae
of variable thickness (Figure 5c). Tumour cells range from oval
to spindled, with either clear or pale cytoplasm, imparted by
a high glycogen content. Nuclei are vesicular, with prominent

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both CREB1 and ATF1 primers. Both methods have been shown to
be highly sensitive in paraffin embedded tissue, with a recent study
showing detection of the EWS rearrangement by FISH in all 15 cases
tested, and detection of a CCS related fusion transcript by RT-PCR in
13/13 cases in which sufficient RNA was available.45
Curiously, the two fusions associated with CCS are not
specific for that entity, as both have also been associated with
another soft tissue tumour, angiomatoid fibrous histiocytoma
(AFH), which has a distinct morphology, clinical presentation,
and most importantly a much more favourable prognosis than
CCS.48 AFH is a rare tumour of soft tissues that predominantly
affects children and young adults. Previously referred to as
malignant angiomatoid fibrous histiocytoma based on malignant behaviour in some of the earliest described cases, the word
malignant was subsequently dropped, as larger series have
shown an overall excellent prognosis, with less than 1%
metastasizing.49 In addition to the EWSReCREB1 and
EWSR1eATF1 fusions, cases of AFH have also been shown to
harbour a third fusion; FUSeATF1. As it is only of low malignant
potential, AFH will not be further discussed here.

Synovial sarcoma
Despite its name, synovial sarcoma (SS) is not a tumour of
synovial origin, and indeed few examples occur within joints.
The most common locations are in the soft tissues of the
extremities, especially around the knee, but synovial sarcomas
have been reported in a wide variety of anatomic sites, including
visceral organs and the head and neck. In the paediatric age
group, the tumour most commonly affects adolescents. In its
classic histologic form, synovial sarcoma displays a biphasic
pattern of spindled cells and epithelial gland components; which
is very useful diagnostically (Figure 6). However, the more
common form in children is monophasic, without gland formation. Documentation of biphenotypic expression of both mesenchymal and epithelial markers by immunohistochemistry is
useful in these cases.59 The majority of SS express cytokeratins
and epithelial membrane antigen, as well as vimentin. Approximately one-third express S-100, and more than half will express

Low-grade fibromyxoid sarcoma

Low-grade fibromyxoid sarcoma (LGFMS) is a neoplasm that
displays a deceptively bland microscopic appearance, and yet
carries a significant risk of metastases, with a propensity for a long
latent period before metastases develop. First described by Evans
in 1987,50 it has multiple histologic appearances, and cytogenetic
and molecular data have played a key role in reconciling its
diverse histologies into a single diagnostic entity. Although a very
high rate of metastases was documented in initial reports (up to
68%), subsequent studies showed lower rates (4e27%).51,52 This
may reflect the adoption of more aggressive surgery as the biologic
behaviour of this tumour became better understood. However
given the long latency seen in some metastatic cases, the true
incidence of metastatic disease has yet to be determined. It occurs
primarily in young adults, but 19% of cases are seen in children.51
Histologically, the tumour typically shows a mixture of myxoid
zones and hypocellular fibrous areas, often with an abrupt transition between zones. Branching, curvilinear small blood vessels
are characteristic. Cytology is generally bland, and mitotic activity
is low. In up to 40% of tumours, strikingly large fibrous rosettes are
present (Figure 5d). Originally designated as hyalinizing spindle
cell tumour with giant collagen rosettes tumours with this
histology are now subsumed into the category of LGFMS.53
Immunohistochemically, these tumours have a fibroblastic
immunophenotype and may express smooth muscle actin in
addition to vimentin; other antigens being only rarely expressed.
LGFMS is associated with a t(7;16)(q34;p11) translocation,
which is seen in examples of the tumour with and without giant
collagen rosettes.54 This translocation fuses the FUS gene to
CREB3L2 in 95% of cases, with a small subset displaying a t
(11;16)(p11;p11) fusion of FUS to CREB3L1.55,56 These fusions
have not been described in any other neoplasms, with the
exception of a small number of sclerosing epithelioid fibrosarcomas which were shown to harbour FUSeCREB3L2 fusions,
raising the possibility that at least some of those tumours may be
closely related to LGFMS.52 The fusions can be demonstrated by
FISH and RT-PCR in paraffin embedded tissues.57,58

DIAGNOSTIC HISTOPATHOLOGY 17:1

Synovial sarcoma. a Biphasic pattern with glands and spindled

cells (haematoxylin & eosin). b Immunohistochemistry for INI1 in
synovial sarcoma, with endothelial cells serving as positive
internal controls. Tumour cells demonstrate weak to absent
nuclear expression of INI1.
Figure 6

31

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REVIEW

CD99. Less differentiated forms of SS may assume a small round

cell appearance or a predominantly epithelioid appearance,
creating diagnostic challenges, particularly with malignant
peripheral nerve sheath tumours and Ewing sarcomas, which
may be difficult to resolve by immunohistochemistry, but
amenable to clarification by cytogenetic and molecular
techniques.
In at least 95% of SS, a characteristic t(X;18)(p11.2;q11.2)
translocation is demonstrable, in which the SS18 gene (previously known as SYT ) on chromosome 18 is juxtaposed with one
of the SSX genes on chromosome X. Of the nine SSX gene transcripts, the majority of fusions involve SSX1 and SSX2, with rare
examples of fusions involving SSX4.60 This translocation appears
specific, and has not been associated with other tumour
types.59,61 Demonstration of the fusion by either cytogenetic
analysis, FISH or RT-PCR is considered to be the diagnostic gold
standard for SS. The latter two modalities can be applied to
formalin fixed paraffin embedded tissue successfully, with one
study showing sensitivities of 96% for quantitative RT-PCR, 92%
for conventional RT-PCR, and 86% for FISH.62 The resulting
fusion protein is believed to dysregulate transcriptional activity
and through epigenetic mechanisms alters tumour suppressor
gene expression. One of the pathways implicated in synovial
sarcoma development is the Wnt/b-catenin pathway, and
a component of that pathway, TLE (transducer-like enhancer of
split 1), has recently become a focus of interest as a possible
additional immunohistochemical marker. TLE1 nuclear expression has been found to be a sensitive marker for SS, present in
over 90e100% of cases.63e66 The specificity of TLE1 for synovial
sarcoma is still a matter of debate. In the study of Kosemehmetoglu et al, 37% of non-synovial sarcoma cases stained with
TLE1, including 30% of their malignant peripheral nerve sheath
tumours. However, Jagdis et al found that TLE1 was very
specific, with rare to absent staining in 73 various non-SS
tumours tested, and Knosel et al also felt that TLE1 was relatively
specific in the proper clinical context, with 83% of their non-SS
controls displaying no staining at all, with the remainder displaying only weak (1) staining.
Recently it has been shown that SS tumours display abnormal
expression of SMARCB1/INI1, a ubiquitous protein involved in posttranscriptional chromatin remodelling. Hoot et al first demonstrated
variable INI1 staining in three cases of synovial sarcoma, and
subsequent studies have demonstrated partial to complete loss of
INI1, without evidence of reduced INI1 RNA.67e69 These studies
suggest that the reduced INI1 expression is an epigenetic finding
involving interaction of the fusion protein and INI1.

eosinophilic cytoplasm and globular pink cytoplasmic inclusions,

which correspond to whorled intermediate filaments ultrastructurally (Figure 7).74 They have a polyphenotypic immunohistochemical profile, with almost all being positive for vimentin, and
most expressing EMA, CD99, and cytokeratin, with many
expressing neural markers as well. Of interest, desmin and CD34 are
not expressed.75
Through molecular genetic investigations, a characteristic loss or
mutation of the INI1 (SNF5, SMARCB1, BAF47) gene was found to be
characteristic of MRT, detectable by FISH and sequence analysis in
at least 95% of tumours. This gene is located in chromosome band
22q11.2.76,77, and is part of the SWI/SNF chromatin remodelling
complex. SWI/SNF acts as a classic tumour suppressor gene in
patients with MRT, as germline mutations or deletions of INI1
predispose patients to the development of rhabdoid tumours, with
inactivation of both alleles leading to malignancy.77 Deletion and/or
mutation of both copies of the INI1 gene results in loss of expression
of the INI1 protein, which can be evaluated by immunohistochemistry. INI1 immunohistochemistry is sensitive and specific for the

Malignant rhabdoid tumour

Malignant rhabdoid tumour (MRT) is a rare neoplasm originally
described in the kidneys and central nervous system but subsequently identified in other locations, including soft tissues.70e72
Most tumours occur in infants and young children, and behave
aggressively, demonstrating a poor response to treatment and
rapid fatal outcome.73
Histologically, these tumours tend to grow in sheets entirely or
partially composed of rhabdoid cells. These are large cells with
eccentrically placed nuclei, vesicular chromatin and prominent
eosinophilic nucleoli. They characteristically have abundant

DIAGNOSTIC HISTOPATHOLOGY 17:1

Malignant rhabdoid tumour. a Typical rhabdoid cells with eccentric

eosinophilic globular cytoplasmic inclusions, vesicular nuclei and
prominent nucleoli (haematoxylin & eosin). b Immunohistochemistry for INI1, showing absence of nuclear staining in tumour cells.
Figure 7

32

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REVIEW

5 Cessna MH, Zhou H, Perkins SL, et al. Are myogenin and myoD1
expression specific for rhabdomyosarcoma? A study of 150 cases, with
emphasis on spindle cell mimics. Am J Surg Pathol 2001; 25: 1150e7.
6 Dias P, Chen B, Dilday B, et al. Strong immunostaining for myogenin
in rhabdomyosarcoma is significantly associated with tumors of the
alveolar subclass. Am J Pathol 2000; 156: 399e408.
7 Kumar S, Perlman E, Harris CA, Raffeld M, Tsokos M. Myogenin is
a specific marker for rhabdomyosarcoma: an immunohistochemical
study in paraffin-embedded tissues. Mod Pathol 2000; 13: 988e93.
8 Hostein I, Andraud-Fregeville M, Guillou L, et al. Rhabdomyosarcoma:
value of myogenin expression analysis and molecular testing in
diagnosing the alveolar subtype: an analysis of 109 paraffinembedded specimens. Cancer 2004; 101: 2817e24.
9 Wachtel M, Runge T, Leuschner I, et al. Subtype and prognostic
classification of rhabdomyosarcoma by immunohistochemistry. J Clin
Oncol 2006; 24: 816e22.
10 Morotti RA, Nicol KK, Parham DM, et al. An immunohistochemical
algorithm to facilitate diagnosis and subtyping of rhabdomyosarcoma: the Childrens Oncology Group experience. Am J Surg Pathol
2006; 30: 962e8.
11 Morgenstern DA, Rees H, Sebire NJ, Shipley J, Anderson J. Rhabdomyosarcoma subtyping by immunohistochemical assessment of
myogenin: tissue array study and review of the literature. Pathol
Oncol Res. 2008; 14: 233e8.
12 Heerema-McKenney A, Wijnaendts LC, Pulliam JF, et al. Diffuse myogenin expression by immunohistochemistry is an independent
marker of poor survival in pediatric rhabdomyosarcoma: a tissue
microarray study of 71 primary tumors including correlation with
molecular phenotype. Am J Surg Pathol 2008; 32: 1513e22.
13 Sullivan LM, Atkins KA, LeGallo RD. PAX immunoreactivity identifies
alveolar rhabdomyosarcoma. Am J Surg Pathol 2009; 33: 775e80.
14 Barr FG. Gene fusions involving PAX and FOX family members in
alveolar rhabdomyosarcoma. Oncogene 2001; 20: 5736e46.
15 Sumegi J, Streblow R, Frayer RW, et al. Recurrent t(2;2) and t(2;8)
translocations in rhabdomyosarcoma without the canonical PAXFOXO1 fuse PAX3 to members of the nuclear receptor transcriptional coactivator family. Genes Chromosomes Cancer 2010; 49:
224e36.
16 Sorensen PH, Lynch JC, Qualman SJ, et al. PAX3-FKHR and PAX7-FKHR
gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the childrens oncology group. J Clin Oncol 2002;
20: 2672e9.
17 Barr FG, Qualman SJ, Macris MH, et al. Genetic heterogeneity in the
alveolar rhabdomyosarcoma subset without typical gene fusions.
Cancer Res. 2002; 62: 4704e10.
18 Davicioni E, Anderson MJ, Finckenstein FG, et al. Molecular classification of rhabdomyosarcomaegenotypic and phenotypic determinants of diagnosis: a report from the Childrens Oncology Group. Am J
Pathol 2009; 174: 550e64.
19 Downs-Kelly E, Shehata BM, Lopez-Terrada D, et al. The utility of
FOXO1 fluorescence in situ hybridization (FISH) in formalin-fixed
paraffin-embedded specimens in the diagnosis of alveolar rhabdomyosarcoma. Diagn Mol Pathol 2009; 18: 138e43.
20 Riggi N, Suva ML, Stamenkovic I. Ewings sarcoma origin: from duel
to duality. Expert Rev Anticancer Ther 2009; 9: 1025e30.
21 Kovar H. Downstream EWS/FLI1-upstream Ewings sarcoma. Genome
Med 2010; 2: 8.
22 Zagar TM, Triche TJ, Kinsella TJ. Extraosseous Ewings sarcoma: 25
years later. J Clin Oncol 2008; 26: 4230e2.

diagnosis of malignant rhabdoid tumour, and INI1 protein loss

appears to be quite rare in other tumours.69,78,79 In addition to MRT,
tumours that display complete or partial loss of INI1 protein
expression include the very rare renal medullary carcinoma,
epithelioid sarcomas, synovial sarcomas, and a subset of paediatric
undifferentiated sarcomas. To date, there is no convincing evidence
that INI1 gene mutations or deletions underlie the loss of INI1
protein expression in non-rhabdoid tumours, and the possibility
exists that some of the undifferentiated sarcomas and epithelioid
sarcomas with INI1 loss may actually be examples of MRT that lack
classic morphologic and/or immunohistochemical features.

Epithelioid sarcoma
Epithelioid sarcoma is a rare soft tissue tumour that primarily affects
young adults and adolescents. It most commonly occurs as
a superficial lesion in the distal extremities, particularly in the hands,
and although typically indolent in behaviour, carries a high risk of
recurrence and a high risk of late metastases. Prognosis of epithelioid
sarcoma is poor; in a recent study of 106 patients, 54% developed
metastases and 31% succumbed to the disease.80 The classic form of
the tumour is characterized histologically by a nodular growth
pattern, and commonly displays granuloma like features and central
necrosis. The tumour cells are epithelioid and spindled and tend to
grow in an infiltrative pattern along fascial planes. Akin to synovial
sarcoma, this is another example of a tumour with a biphenotypic
immunohistochemical profile, typically expressing epithelial
markers such as EMA and cytokeratins in addition to vimentin. More
recently, Guillou et al described a deep-seated proximal form of
epithelioid sarcoma, which was axially located, with a propensity for
occurrence in the perineum, pelvis and genitourinary tract. This
proximal form of epithelioid sarcoma shares some histological and
immunohistochemical features with MRT, and is more aggressive
than the classic form of epithelioid sarcoma.81
Cytogenetically, epithelioid sarcomas have shown nonspecific chromosomal gains and deletions. In 2009, three studies
independently described lack of INI1 protein expression in both
the majority (85e93%) of both proximal and classic forms of the
tumour.80,82,83 Although still a matter of some debate, it appears
that loss of INI1 expression in epithelioid sarcomas (particularly
the classic forms) may be an acquired change associated with
complex karyotypic abnormalities, and distinct from the inactivation secondary to INI1 deletion or mutation. Several recent
studies have shown that INI1 alterations at the DNA level are rare
in epithelioid sarcoma, and more likely to be found in proximaltype tumorus.82,84 These findings raise the question as to
whether proximal-type epithelioid sarcomas bear a closer kinship
to MRT than to classic epithelioid sarcoma.
A

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DIAGNOSTIC HISTOPATHOLOGY 17:1

Practice points
C

35

Although it is optimal practice to allocate fresh or frozen tissue

for molecular/cytogenetic studies, some molecular studies,
most notably FISH, can be successfully applied to formalin
fixed paraffin embedded tissue.
Translocation associated sarcomas tend to have simple
karyotypes and often a relatively uniform histologic appearance; whereas those without translocations tend to have more
complex karyotypes and more pleomorphism.
Break-apart FISH and RT-PCR are complimentary; although
break-apart FISH can demonstrate an interruption of a gene
locus, RT-PCR with specific primers can identify the specific
translocation partner.
Immunohistochemistry for myogenin and PAX-5 can be helpful
in distinguishing alveolar rhabdomyosarcoma from embryonal
rhabdomyosarcoma.
In addition to its lack of expression in malignant rhabdoid
tumour, INI1 expression is also altered in epithelioid sarcoma
and synovial sarcoma.

2010 Elsevier Ltd. All rights reserved.