This document discusses the utility of integrating molecular genetics with soft tissue pathology. Key points include: 1) Molecular genetics can help more accurately define disease entities, improve diagnostic accuracy, and identify prognostic and therapeutic targets. 2) Techniques like FISH and PCR are useful for distinguishing subtypes of sarcomas and supporting diagnoses, especially in non-canonical cases. 3) Specific gene fusions and mutations have been identified that are diagnostic for entities like Ewing sarcoma, synovial sarcoma, and GIST. 4) While molecular findings cannot replace morphology, genetics provides an important adjunct for diagnosis, especially in challenging cases. The integration of both disciplines has enhanced understanding and classification of soft

1.  Special thanks to my resident Dr Babar Yasin for preparing the
presentation
 ADAPTED FROM THE ABOVE ARTICLE FROM Seminar in diagnostics
Pathology 30 (2013) 375-381
 Angelo P. Dei Tos, MD
 Department of Pathology, Treviso General
Hospital, Piazza Ospedale,1 31100 Treviso, Italy

2. The marriage of Molecular
Genetics and soft tissue
Pathology.

3.  More accurate definition of disease entities
and validation of classification schemes.
 Improved diagnostic accuracy.
 Identification of molecular predictive and
prognostic markers.
 Discovery and validation of therapeutic
molecular targets.

4.  Nucleic acids (RNA and DNA), either via
hybridization on a slide(i.e., fluorescent in
situ hybridization—FISH)
OR
 On isolated DNA or RNA via polymerase chain
reaction (PCR) techniques (i.e.,reverse
transcriptase PCR and quantitative PCR)It is MANDAOTORY that the results get
interpreted in context with morphology.
 Genetic assessement is an
important ADJUNCT not a
replacement to
conventional
morphological tools.

5. Soft tissue tumors are heterogeneous group of
neoplasm which can be :
 Benign
 Boderline
 Malignant
Challenge for Pathologists
Diagnostic inaccuracy affects the
treatment and development of new
drugs as clinical trials depend upon
tumor classification.

6.  Rare group of diseases (< 2% of all cancers)
 Extremely heterogenous. Over 100 subtypes
are being described.
 Usual features of malignancy (?) are not always
applicable to these.
Histologically worrisome leisons may
actually be benign e.g. Nodular fascitis
Cytollogically innocent neoplasm may
behave aggressively e.g. Low-Grade
Fibromyxoid Sarcoma.

7.  Distinguishing specific subtypes of
sarcomas.
 Supporting diagnosis in non-canonical
clinical presentations.
 Distinguishing sarcomas from benign
mimickers.

8.  Molecular genetics has proved diagnostically
useful in two relatively large groups of
mesenchymal malignancies: round cell
sarcomas and pleomorphic sarcomas.
Round cell sarcomas include :
 Ewing sarcoma
 Desmo-plastic small round cell tumor
 Alveolar rhabdomyo-sarcoma
 Poorly differentiated round cell synovial
sarcoma
 Mesenchymal chondrosarcoma
 Minority of cases of round cell liposarcoma
Distinction is
crucial because
the therapeutic
approach
differs.

12.  The demonstration by FISH of
EWSR1,SS18, and FOXO1 rearrangements in
EWS, PDSS, and
ARMS, respectively, or, alternatively, of
specific chimerical transcriptsby PCR-based
techniques is of great help for achieving
acorrect diagnosis.
EWSR1 FISH results need to be
interpreted in context with
morphology and IHC findings.

13.  Sub classification is very important.
 myogenic differentiation in pleomorphic
sarcomas is associated with a less favorable
outcome.
 Another important point is the
distinction, among retroper-itoneal
sarcomas, of dedifferentiated
liposarcoma (DDLPS) from other
pleomorphic sarcomas, most often
pleomorphic leiomyosarcoma.

15.  The recognition of DDLPS is based on the
identification of a well-differentiated
lipogenic component associated with a high-
grade, most often non-lipogenic, sarcoma.
 Core biopsies usedfor diagnostic purposes
may leave the lipogenic component
unsampled.
 DDLPS exhibit better out- comes when
compared to other pleomorphic
sarcomas, and its accurate recognition may
lead to adopt a more aggressive surgical
strategy. (Locally aggressive)

16.  Detection of MDM2 amplification by FISH or
quantitative RT-PCR certainly represents a useful
diagnostic adjunct.
 The MDM2 gene (as well as CDK4 and HMGA2) maps at
the 13q12–15 chromosome region and is amplified in
both well-differentiated and dedifferentiated
liposarcomas.
 MDM2 testing is also potentially useful in
distinguishing between myxoid liposar-coma (MDM2
negative) and WD/DDPLS with myxoid change.
 The separation of the two conditions again allows
adoption of proper treatment in consideration of the
high sensitivity of myxoid liposarcoma to the marine-
derived alkaloid named trabectedin.

18. Tumor Gene Mutation Gene Involved
Alveolar rhabdomyosarcoma t(2;13)(q35;q14)
t(1;13)(p36;q14)
PAX3–FOXO1A
PAX7–FOXO1A
Alveolar soft part sarcoma (X;17)(p11.2;q25) ASPL–TFE3
Angiomatoid fibrous
histiocytoma
t(12;16)(q13;p11)
t(12;22)(q13;q12)
t(2;22)(q34;q12)
FUS–ATF1
ATF1–EWSR1
CREB1–EWSR1
Aneurysmal bone cyst t16;17)(q22;p13) CDH11–USP6
Atypical lipomatous
tumour/dedifferentiated
liposarcoma
Amplification MDM2, CDK4, and HMGA2
Central/periosteal
osteosarcoma
Point mutation IDH1/IDH2
Clear cell sarcoma t(12;22)(q13;q12)
t(2;22)(q34;q12)
ATF1–EWSR1
CREB1–EWSR1
Dermatofibrosarcoma
protuberans
t(17;22)(q22;q13) COL1A1–PDGFB
Desmoid-type fibromatosis Activating mutation BCTN1

19. Desmoplastic round cell
tumor
t(11;22)(p13;q12) WT1–EWSR1
Endometrial stromal
sarcoma
t(7;17)(p15;q21)
t(6;7)(p21;p15)
t(6;10)(p21;p11)
t(10;17)(q22;p13)
JAZF1–JJAZ1
PHF1–JAZF1
PHF1–EPC1
YWHAE–FAM22
Ewing sarcoma/PNET t(11;22)(q24;q12)
t(21;22)(q22;q12)
t(7;22)(p22;q12)
t(17;22)(q12;q12)
t(16;21) (q13;q22)
t(2;22)(q33;q12)
EWSR1–FLI1
EWSR1–ERG
ETV1–EWS
EIAF–EWS
FUS–ERG
FEV–EWS
Extraskeletal Myxoid
Chondrosarcoma
t(9;22)(q22;q12)
t(9;15)(q22;q21)
EWSR1–NR4A3
TCF12–NR4A3
TFG–NR4A3
GIST Activating mutation KIT and PDGFRA
Intramuscular myxoma Activating mutation GNAS1

20. Pericytoma with t(7;12)
t(7;12)(p22;q13)
ACTB–GLI
Pigmented
villonodular synovitis
t(1;2)(p13;q37) COL6A–CSF1
Soft tissue
myoepithelioma
t(1;22)(q23;q12)
t(19;22)(q13;q12)
EWSR1–PBX1
EWSR1–ZNF444
Synovial sarcoma t(X;18)(p11;q11) SS18–SSX1
SS18–SSX2
SS18–SSX4

21.  The combination of morphological criteria
and genetics validates the recognition of rare
diseases even when arising at non-canonical
anatomic locations.
 This is particularly true for referral centers
wherein challenging cases tend inevitably to
concentrate.
 Molecular genetics has greatly
 Contributed to the identification of primary
Ewing sarcoma of the skin, kidney, and dura
mater, as well as of viscerally located
synovial sarcomas.

22.  Morphological appearance of mesenchymal
lesions does not always reflect the clinical
behavior.
 The distinction of sarcomas from benign
mimics most often relies on morphology.
 In a minority of cases molecular genetics may
also prove diagnostically helpful.

23. Fibroid and myxoid areas.
Swirling whorled growth pattern.
Low to moderate cellularity.
Bland cells.
Minimal nuclear pleomorphism.

24.  Deceptively bland-looking spindle cell
mesenchymal malignancY with an aggressive
clinical behavior.
 The differential diagnosis of LGFMS includes
benign lesions such as
perineurioma, neurofibroma, cellular
myxoma, and nodular fasciitis, as well locally
aggressive neoplasms such as desmoid
fibromatosis.
MUC4 expression is
regarded as key
diagnostic feature.
Identification of FUS
rearrangement via FISH
or identification of
FUS-CREB3L2 transcript
via PCR is very useful.
B- Catenin

25. Several attempts have been made to
determine the prognostic value of
molecular genetic findings.
Focused on Ewing sarcoma, alveolar
rhabdomyosarcoma, and synovial
sarcoma.
 No meaningful molecular prognostic
stratification can be foreseen for
now.

26. A notable exception is represented by
a molecular signature named
CINSARC, which allows better
separation of grade 2 sarcomas. This
attempt is based on the use of a
complex technique (CGH-array) and
requires availability of fresh
material, which hampers a large scale
clinical application of CINSARC.

27.  Type of mutations involving both the KIT and
PDGFRA genes are associated with distinctive
outcomes.
 Deletions occurring at the exon 11 of the KIT
gene are associated with more aggressive
disease, whereas mutations of exon 18 of the
PDGFRA gene generally identify a more
indolent clinical course.

28.  Distinct mutation types in GIST reflect
different objective response rates (greater
for KIT exon 11 mutation and much lower for
so-called wild-type GIST).
 presence of specific mutations in the exon 18
of the PDGFRA gene (D842V) predict primary
resistance to tyrosine kinase inhibitors.
Molecular assessement in GIST
assumes a central role in clinical
decision making.

29. The identification of the two specific
fusion products of CHOP/DDIT3 gene
with FUS and more rarely with
EWSR1 is extremely helpful in
distinguishing challenging examples
of myxoid liposarcoma from other
myxoid sarcomas and therefore to
apply the adequate therapeutic
regimen.

30.  Another examples is use of crizotinib in
inflammatory myofibroblastic tumors wherein
assessment of the ALK gene may represent an
important diagnostic confirmatory finding as
well as a key biomarker of prediction.
Molecular Genetics represents the
most valuable tool to identify and
validate new therapeutic targets.

31. Good examples are represented
by MDM2, amplified in
dedifferentiated liposarcoma
and potentially targetable by
Nutlin-A3, the mTOR pathway in
malignant PEComa and
lymphangioleiomyomatosis, PDG
FB in DFSP, and KDR in
angiosarcoma.

32. Promiscuity

33.  Molecular pathology/genetics does not
represent an alternative but a complement
to surgical diagnostic pathology.
 Considering the degree of molecular
promiscuity of EWSR1 gene aberrations, the
results of FISH analysis need to be
mandatorily evaluated in context with
morphology as EWSR1 aberrations are
described in a variety of unrelated entities.

34. ETV6–NTRK3/t(12;15) Infantile fibrosarcoma
Acute myeloid leukemia
Secretory breast carcinoma
ALK gene fusions Inflammatory
myofibroblastic tumor
Anaplastic large cell
lymphoma
Subsets of lung
adenocarcinoma
FUS–ERG/t(16;21) Ewing sarcoma
Acute myeloid leukemia
ASPL–TFE3/t(X;17) Alveolar soft part sarcoma
Subset of pediatric renal
EWS–ATF1/t(12;22) and EWS-
CREB1/t(2;22)
Clear cell sarcoma
Angiomatoid fibrous
histiocytoma

35.  Genetics has certainly played a key role in
allowing a better understanding of many
lesions.
 The unification of myxoid and round cell
liposarcoma within a single tumor entity
represents one of the best examples.
 Genetics has greatly helped in recognizing the
close relationships between several tumors
such as giant cell fibroblastoma and DFSP,
LGFMS and epithelioid sclerosing
fibrosarcoma, and hemosiderotic
fibrolipomatous tumor and myxoinflammatory
fibroblastic sarcoma.

36.  The definition of new entities has been
strongly supported by genetics.
 The identification of t(7;19)(q22;q13)
translocation in pseudomyogenic (epithelioid
sarcoma-like) hemangioendothelioma.
 The future classifications will include more
genetic observation.
 Genetic aberrations will also contribute to the
definition of the specific tumor entities.

37. The marriage between
Pathology and Genetics
not only proved to be
fruitful but also to be
stable.