Fluorescence in-situ hybridization
FISH uses a uorescently labeled probe targeted
towards a specic genetic sequence. The uores-
cent probe can then be assessed in situ using a
uorescence microscope. Dual-color dual-fusion
probes are designed towards a particular gene
fusion target, while dual-color break-apart probes
have greater utility for gene fusions in which the
partner may not be known (e.g. EWSR1). Large
gene amplications and deletions may also be
detected by comparing the ratio of lost target
signals to a control signal (e.g. MDM2).
Reverse-transcriptase polymerase
chain reaction (RT-PCR)
PCR-based tests utilize PCR amplication of
certain primer sequences that can be built to
accommodate a set of gene rearrangements. This
is particularly useful for larger panels that use
known genetic breakpoints, but this method
generally lacks the ability to detect new fusion
Next-generation sequencing (NGS)
NGS has become standard for detection of both
prognostic and therapeutic mutations. In
sarcoma, new RNA-based methods, including
hybrid-capture and anchored multiplex PCR,
offer better detection for gene rearrangements.
These methods have the added benet of detect-
ing new gene partners, which is particularly
useful with promiscuous genes like EWSR1. The
development of various commercial platforms for
NGS fusion testing has signicantly increased the
availability of this test to pathologists. Addition-
ally, tertiary institutions with large sample
volume in bone and soft tissue pathology may
opt to bring these methods in-house to improve
turnaround times.
Methylation testing
Initially developed for usage in the diagnosis of
brain tumors, methylation testing has expanded
to begin to include soft tissue and bone tumors.
Methylation tests use an array chip to detect the
Issue 22 || March 2023
Farres Obeidin
Department of Pathology, Northwestern University
Feinberg School of Medicine, Chicago, IL, USA
Corresponding Author: Farres Obeidin, MD
Department of Pathology, Northwestern University
Feinberg School of Medicine, Chicago, IL, USA
E-mail: farres.obeidin@nm.org
Farres Obeidin
Our understanding of bone and soft tissue tumors
has thoroughly evolved as a consequence of
modern molecular techniques. DNA and RNA
sequencing methods play an important diagnos-
tic and therapeutic role in sarcoma pathology.
Herein, we discuss current guidelines and best
practices for molecular testing in bone and soft
tissue tumors.
While translocation driver events are very rare in
epithelial malignancies, they occur in up to 25%
of sarcomas. As a result, molecular techniques for
identifying recurrent translocations currently play
an important diagnostic role in bone and soft
tissue pathology.
methylation patterns of thousands of CpG islands
to produce a “signature” for a particular tumor.
Through statistical methods, these signatures can
be clustered into groups of similar tumors,
providing a tentative “cell of origin” diagnosis
(Fig. 1). While still in its infancy, methylation
has great potential for future diagnostics in soft
tissue and bone.
The constantly growing number of translocation-
driven soft tissue and bone tumors in the litera-
ture necessitates judicious use of diagnostic
genetic testing. By employing a morphology-
based approach, pathologists can triage mesen-
chymal neoplasms for both diagnostic and
therapeutic testing. The following review takes a
“line of differentiation” approach to decide on
appropriate testing strategies.
MDM2 amplication is the key differentiating
factor between lipoma and well-differentiated/
dedifferentiated liposarcoma (Fig. 2). MDM2
amplication may also rarely be seen in other
high-grade sarcomas; as such, caution is
warranted when interpreting this nding
without the presence of a well-differentiated
liposarcoma component.
RB1 deletion (tested by loss of RB1 on immu-
nostaining or NGS) is pathognomonic for
spindle cell/pleomorphic lipoma and may be
seen in about 50% of atypical spindle cell/
pleomorphic lipomatous tumor.
Myxoid liposarcoma is driven by translocations
involving DDIT3, most commonly with FUS.
These tumors show a distinct myxoid back-
ground, chicken-wire capillary architecture,
and univacuolated lipoblasts. High-grade
variants show round cell features.
Nodular fasciitis and similar disorganized,
Sponsored by an unrestricted grant from USCAP
rearrangement, usually with CREB3L2 or
CREB3L1; cellular spindle cell neoplasm with
mixed collagenous and myxoid features
(low-grade bromyxoid sarcoma) or sclerotic
tumor with bland epithelioid cytology
(sclerosing epithelioid brosarcoma). Addi-
tional fusions involving KMT2A and YAP1
that show similar morphological features to
sclerosing epithelioid brosarcoma have been
- EWSR1::SMAD3-positive broblastic tumor:
EWSR1::SMAD3 fusion; newly described
fascicular, bland spindle cell proliferation with
distinct zonation (hypocellular central region
and increased peripheral cellularity) with ERG
IHC positivity.
- Supercial CD34-positive broblastic tumor:
PRDM10 rearrangements; low-grade fascicu-
lar proliferation of spindle cells with marked
nuclear pleomorphism.
Fusion testing is not required but helpful in the
diagnosis, particularly in the case of the
low-grade malignancies and hemangioendothe-
Epithelioid hemangioma is dened by recur-
rent fusions involving FOS and FOSB. Fusion
distinction relies on a combination of clinical
history and pathologic features.
Translocation-associated broblastic neoplasms
are typically benign to low-grade neoplasms
with monotonous cytology and characteristic
architectural features. An NGS panel can
conrm the diagnosis, though this is often not
required because the morphology is typical and
molecular analogue immunohistochemical
(IHC) stains exist for many of these tumors
with relatively high sensitivity and specicity.
- Solitary brous tumor: NAB2::STAT6 fusion;
prominent collagenous background and
staghorn vasculature.
- Dermatobrosarcoma protuberans:
COL1A1::PDGFB fusion; dermal tumor with
diffuse storiform architecture and honeycomb/
septal inltration into fat. Fibrosarcomatous
transformation can mask the storiform
architecture and gives the tumor metastatic
- Inammatory myobroblastic tumor: ALK
rearrangement; bland myobroblastic prolif-
eration with prominent mixed inammatory
inltrate. Malignant versions exist, often with
more epithelioid morphology.
- Low-grade bromyxoid sarcoma/sclerosing
epithelioid brosarcoma: FUS or EWSR1
myxocollagenous, variably cellular broblastic/
myobroblastic neoplasms with or without
reactive bone formation (including myositis
ossicans, bro-osseous pseudotumor of the
digit, aneurysmal bone cyst, and a subset of
broma of tendon sheath), form a growing
spectrum of tumors that are dened by USP6
gene rearrangements with multiple different
partners (Fig. 3).
As in spindle cell lipoma, RB1 loss is a dening
feature in both cellular angiobroma and
myobroblastoma. These tumors have some
overlapping histologic and immunophenotypic
features in addition to the same genetics, so
Fig. 1. A t-distributed stochastic neighbor embedding (t-SNE) plot is a visualization method used in clinical laboratories to show the aggregate methylation data of a tu-
mor. Each dot represents a single tumor and the closeness of the dots represents similarity in their methylation profiles. From this, a classification scheme can be created.
Each cluster represents a separate tumor type. In this example case, the black dot is the current tumor and is clustering near the category of MYOD1-mutated rhabdo-
Fig. 2. MDM2 amplification, seen here as an increase
in red fluorescent probe signals, is a hallmark of cer
tain cancers. The prototypical example is well-differ-
entiated/dedifferentiated liposarcoma.
testing is not required but can be useful in
more cellular cases with atypical cytologic
Epithelioid hemangioendothelioma:
WWTR1::CAMTA1 fusion or rarely
TFE3::YAP1 fusion; these malignant tumors
show more primitive vascular differentiation,
epithelioid morphology, intracytoplasmic
lumens, and a distinct myxohyaline stroma that
distinguishes them from epithelioid angiosar-
coma. The CAMTA1 fusion or IHC stain may
be used to conrm the diagnosis.
Pseudomyogenic hemangioendothelioma:
FOSB gene fusions, usually with SERPINE1 or
ACTB; histologically, shows a rhabdomyoblas-
tic-like appearance but stains for keratins and
vascular markers.
Angiosarcoma, like other high-grade sarcomas,
shows complex genomic changes. The presence
of MYC gene amplications (tested by IHC,
FISH, or NGS) is seen in most cases of post-
irradiation or lymphedema-associated angiosar-
coma. This nding is very useful in distin-
guishing post-irradiation atypical vascular
lesions from true angiosarcoma.
Pericytic and smooth muscle
While the diagnosis of glomus tumors is gener-
ally based on morphology, examples of deep,
gastrointestinal, or malignant glomus tumors
may confound the diagnosis. Most glomus
tumors (including malignant variants) show
recurrent fusions involving the NOTCH family
of genes, most commonly
Distinct from classical leiomyosarcoma,
inammatory leiomyosarcoma is a low-grade
malignancy that has been shown to have a
recurrent karyotypic pattern that is best seen on
mRNA microarray technology, such as
OncoScan™. These neoplasms show a near-
haploid genotype that is thought to be a relevant
driver of the disease process. Some tumors may
show whole genome duplication afterwards,
resulting in a pseudo-hyperdiploid karyotype
that may signal transformation to a higher-grade
malignancy. Recent studies have demonstrated a
number of cases with rhabdomyoblastic IHC
staining, and new terminology (inammatory
rhabdomyoblastic tumor) has been suggested.
Skeletal muscle
Molecular testing in rhabdomyoblastic tumors
is best utilized in the differentiation of embryo-
nal and alveolar rhabdomyosarcoma. Both
tumors may have a solid small round blue cell
morphology and skeletal muscle staining with
IHC. Myogenin IHC tends to be more diffuse
in alveolar rhabdomyosarcoma; however,
denitive diagnosis requires molecular detec-
tion of the typical FOXO1 fusion with either
PAX3 or PAX7 in alveolar rhabdomyosarcoma.
Embryonal rhabdomyosarcoma may show
recurrent mutations in the RAS family of genes
or DICER1 in some syndromic patients. The
distinction between the alveolar and embryonal
subtypes is necessary because of the variation in
prognosis and treatment.
Spindle cell rhabdomyosarcoma falls into three
distinct molecular groupings. Congenital and
infantile tumors are most often translocation-
driven, with fusions involving VGLL2, SRF,
READ1, NCOA2, and CITED2. Another
subset of tumors in adolescents and young
adults shows mutations in the MYOD1 gene.
The third category does not have recurrent
genetic abnormalities. Additionally, some intra-
osseous variants may show EWSR1, FUS, or
MEIS1::NCOA2 rearrangements.
Gastrointestinal stromal tumor (GIST)
All GISTs should be tested for mutational
status, as these mutations predict response to
treatment and prognosis.
Mutations most commonly occur in KIT (exons
9, 11, 13, 14, or 17) and second most com-
monly in PDGFRA (exons 12, 14, and 18).
Immunostaining for cKIT (CD117) does not
imply the presence of a KIT mutation.
SDH-decient GISTs are negative for KIT and
PDGFRA mutations and show mutations in
mutations are typically screened by assessing
for loss of SDHB IHC staining, which picks up
mutations in any of the four genes. SDH-
decient GISTs are more common in younger
patients in the stomach.
Some GISTs will instead show mutations in
RAS family genes.
Fig. 3. New NGS methods are highly sensitive and specific for translocations and can also be used to detect new fusions with previously undescribed partners. This ex-
ample shows the readout from the NGS software demonstrating a fusion of the USP6 gene with the SERPINF1 gene in a case of nodular fasciitis.
A small proportion of GISTs may be negative
for all four of these mutations, called quadruple
wild-type GIST.
Uncertain differentiation and round cell
Undifferentiated round to spindle cell tumors
and those with monomorphic cytology should
be considered for large panel NGS fusion
testing. IHC has been found to show much
overlap in this category of tumors, and NGS
testing offers the ability to pick up non-classical
examples as well as discover new fusion
Currently, Ewing and Ewing-like round cell
sarcomas can be split into six overall categories
(Table 1). New fusion partners continue to be
discovered, and the particular fusion may affect
treatment and prognosis.
Certain neoplasms show bi-immunophenotypic
staining patterns that may lead to confusion.
Molecular testing can be conrmatory.
- Angiomatoid brous histiocytoma:
EWSR1::ATF1, FUS::ATF1, or
EWSR1::CREB1; a low-grade malignancy
with EMA and desmin co-positivity and
prominent lymphoid cufng.
- Ossifying bromyxoid tumor: Most commonly
PHF1 rearrangements (50% of cases), rarely
rearrangements in BCOR or SUZ12, suggest-
ing a genetic overlap with endometrial stromal
sarcoma; low-grade malignancy with promi-
nent peritumoral metaplastic bone formation
and often co-positivity for keratins, S100, or
- Myoepithelial neoplasms of soft tissue: EWSR1
Dr. Obeidin has been an author for
PathologyOutlines since 2018 and part
of the PathologyOutlines editorial board since
January 2022. He is currently an Assistant
Professor of Pathology at Northwestern
University Feinberg School of Medicine.
He obtained his M.D. at the Medical College
of Georgia and then completed his Anatomic
and Clinical Pathology residency at
Northwestern University. He then completed
a fellowship in General Surgical Pathology
and Bone and Soft Tissue Pathology at the
University of California, Los Angeles.
Meet the Author
Table 1. Ewing and Ewing-like round cell sarcomas
Category Molecular abnormalities
Classic Ewing sarcoma and Ewing family tumors EWSR1::FL1
CIC-rearranged sarcoma CIC::DUX4
BCOR-rearranged sarcoma BCOR::CCNB3
BCOR internal tandem duplication