Fluorescence in-situ hybridization

(FISH)

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

partners.

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

WHAT’S NEW IN BONE

AND SOFT TISSUE

PATHOLOGY 2023:

GUIDELINES FOR

MOLECULAR TESTING

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

ORCID

Farres Obeidin

https://orcid.org/0000-0001-8461-9238

Abstract

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.

COMMON MOLECULAR

METHODS

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.

TESTING GUIDELINES FOR

SPECIFIC TUMOR CLASSES

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.

Adipocytic

• 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.

Fibroblastic/myoﬁbroblastic

• Nodular fasciitis and similar disorganized,

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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

found.

- 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.

Vascular

• Fusion testing is not required but helpful in the

diagnosis, particularly in the case of the

low-grade malignancies and hemangioendothe-

liomas.

• 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

potential.

- 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-

myosarcoma.

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

features.

• 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

MIR143::NOTCH1/2/3.

• 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

SDHA, SDHB, SDHC, or SDHD. These

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

tumors

• 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

partners.

• 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

desmin.

- 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

EWSR1::ERG

EWSR1::FEV

EWSR1::ETV1

ESWR1::ETV4

FUS::ERG

FUS::FEV

CIC-rearranged sarcoma CIC::DUX4

CIC::FOXO4

CIC::LEUTX

CIC::NUTM1

CIC::NUTM2B

BCOR-rearranged sarcoma BCOR::CCNB3

BCOR internal tandem duplication

BCOR::MAML3

GLI1-altered sarcoma GLI1::MALAT1

GLI1::ACTB

GLI1 ampliﬁcations or other rearrangements

Non-Ewing family gene fusions EWSR1::PATZ1

EWSR1::NFATC2

EWSR1::SP3

EWSR1::SMARCA5

Unclassiﬁed round cell sarcoma Fusion negative or unknown

or FUS rearrangements with several different

partners; keratin and S100 co-positivity with

myxoid to myxocollagenous background and

bland round to spindled cells. INI1 is lost in a

subset. These tumors show different genetics

to salivary gland myoepithelial neoplasms,

which are often governed by PLAG1 rear-

rangements.

- Extraskeletal myxoid chondrosarcoma: NR4A3

rearrangement, most commonly with EWSR1.

Keratin, S100, neuroendocrine, or myoepithe-

lial markers may be nonspecically positive.

Morphologic and immunophenotypic overlap

with myoepithelial neoplasms may cause

diagnostic difculty, and because of the

presence of EWSR1 as a partner in both, NGS

testing is recommended to assess the partner

gene to distinguish these two.

• Phosphaturic mesenchymal tumor: in patients

with clinical evidence of hypophosphatemia

and/or osteomalacia, serum testing may be

performed for increased FGF23 secretion. Most

of these tumors show fusions involving

FN1::FGFR1 or FN1::FGF1.

• NTRK gene rearrangements have been seen in

an increasing spectrum of mesenchymal

tumors. The prototypical infantile brosarcoma

is dened by ETV6::NTRK3. However, NTRK

fusions have now been seen in cellular meso-

blastic nephroma as well as various myxoid soft

tissue tumors with bland cytology and possible

co-positivity for S100 and CD34, including

lipobromatosis-like neural tumor. Some

uterine and soft tissue neoplasms with brosar-

coma-like morphology also dene a new subset

of NTRK-rearranged sarcomas.

• A subset of PEComa is driven by rearrangements

in TFE3; often not required for the diagnosis,

as the combination of myogenic and melano-

cytic markers is specic enough in most

instances to diagnose PEComa.

• Recent studies have shown that the majority of

true intimal sarcomas show amplications in

MDM2, similar to well/dedifferentiated

liposarcoma. Intimal sarcoma may have variable

differentiation and immunostaining. The

presence of a luminal mass in the pulmonary or

cardiac vasculature should prompt FISH testing

for MDM2 to conrm the diagnosis.

Bone and cartilage

• The diagnosis of primary bone lesions is still

based most heavily on the morphology coupled

with the radiological imaging.

• Some distinct exceptions where molecular

testing can be diagnostically useful:

- Aneurysmal bone cyst: USP6 gene rearrange-

ments; cystic, giant cell-rich neoplasm with

reactive woven bone formation.

- Low-grade central osteosarcoma/parosteal

osteosarcoma: MDM2 amplications; low-

grade osteoblastic tumors that, similar to

well-differentiated liposarcoma, have potential

to dedifferentiate. MDM2 testing by FISH can

help to distinguish from reactive or benign

bone-forming tumors.

- Giant cell tumor of bone/chondroblastoma:

both show unique and specic mutations in

H3F3A or H3F3B. IHC testing is useful as a

molecular adjunct.

- Conventional chondrosarcoma: IDH1 or IDH2

mutations in a subset of chondrosarcoma; can

be diagnostically useful in small biopsies and

dedifferentiated examples.

- Mesenchymal chondrosarcoma:

HEY1::NCOA2 fusion; small round blue cell

sarcoma with cartilaginous maturation.