Molecular biology of soft tissue sarcoma

MOLECULAR BIOLOGY OF
SOFT TISSUE SARCOMAS
Dr. Trivikrama Rao M
Med Onco resident
Dharamshila Narayana Hospital
New Delhi

CONTENTS
A. Translocation
associated soft tissue
sarcomas
• Myxoid / Round cell
liposarcoma
• Ewing sarcoma
• Desmoplastic small
round cell tumor
• Synovial sarcoma
• Alveolar
rhabdomyosarcoma
• Alveolar soft-part
sarcoma
• Dermatofibrosarcoma
protuberans
• Extraskeletal myxoid
chondrosarcoma
• Solitary fibrous tumor &
hemangiopericytoma
B. Soft tissue sarcomas of
simple karyotype
associated with mutations
• Desmoid fibromatosis
C. Complex soft tissue
sarcoma types
• Well differentiated &
dedifferentiated
liposarcoma
• Pleomorphic liposarcoma
• Myxofibrosarcoma &
undifferentiated
pleomorphic sarcoma(
malignant fibrous
histiocytoma)
• Myxofibrosarcoma
• Undifferentiated
pleomorphic sarcoma(
malignant fibrous
histiocytoma)
• Leiomyosarcoma
• Malignant peripheral
nerve sheath tumor
• Angiosarcoma
I. Soft tissue sarcomas

Molecular pathways in sarcoma
Quesada J and Amatol R. The Molecular Biology of Soft-Tissue
Sarcomas and CurrentTrends in Therapy. Hindwai publishing 2012

 Comparative genomic hybridization
 Gene expression profiling
 Genomic sequencing

FISH
 This technique uses fluorescently
labeled probes to locate the positions
of specific DNA sequences on
chromosomes

Comparative genomic
hybridization
 This popular technique provides a
means of genome-wide screening
for copy number variations. First
developed to detect copy number
changes in solid tumors, CGH uses two
genomes, a test and a control, which are
differentially labeled and competitively
hybridized to metaphase chromosomes.
The fluorescent signal intensity of the
labeled test DNA relative to that of the
reference DNA can then be linearly
plotted across each chromosome,
allowing the identification of copy
number changes.

 Comparative Genomic Hybridisation
(otherwise known as CGH) is a type of
Fluorescence In Situ Hybridisation
{FISH} technique that compares and
measures differences in copy number
changes between 2 DNA samples

Advantages Of CGH
 Visualisation of deletions and duplications in very
small DNA segments (which is of high
importance as these can occur in birth defect
syndromes and in cancer)
 Searches of the whole genome without prior
knowledge about the chromosomal aberration at
hand
 Analysis without the need for specific probes
 The detection of the presence of amplified genes
in cancer and map their location
 Unlike FISH, CGH is able to;
 1. Identify the chromosome with the aberration 2.
Identify the specific location from which the extra
material originated

DNA sequencing
 is the process of determining the
precise order of nucleotides within
a DNA molecule. It includes any
method or technology that is used to
determine the order of the four
bases—adenine, guanine, cytosine,
and thymine—in a strand of DNA.

• Translocations, inversions or
specific activating mutations
• 30-50 years
• Oncogenesis results from
transcriptional deregulation
induced by fusion genes
Simple
karyotypes &
simple genetic
alterations
• 50-70 years
• Alterations in cell cycle genes
TP53, MDM2, RB1 & INK4a
Aberrant,
highly
complex
genomes
I. SOFT TISSUE SARCOMAS

1. Myxoid / round cell liposarcoma

• Typically arise in the thigh or other deep
soft tissues
• Adults (peak age, 30 to 50 years).
• characteristic morphology: a myxoid matrix,
a plexiform vasculature, and lipoblasts.
• Balanced translocation, t(12;16)(q13;p11)
• Fusing FUS (also known as TLS) with
DDIT3 (aka CHOP, GADD153)
1. Myxoid / round cell liposarcoma
Thway K, Noujaim J, Jones R.L, Fisher C. Advances in the Pathology
and Molecular Biology of Sarcomas and the Impact on Treatment.
Clinical Oncology 2017,1-10.

• Fusion product results is activation of
critical pathways
• related to angiogenesis (interleukin 8 [IL-8]),
• early adipose differentiation (PPARγ),
• growth factor signaling (insulinlike growth factor
[IGF], RET), and
• cell-cycle control (cyclin D, CDK4).
• Another result is repression of miR-486 and IL-
24, which might otherwise act as tumor
suppressors.

 High levels of p53, IGF1R/IGF2, AXL, and RET may be
adverse factors.
 In addition, mutations in PIK3CA, found in 18% of
myxoid/round cell liposarcomas, were associated with a
worse outcome.
 Myxoid liposarcomas have dense microvasculature and high
expression of IL-8 and vascular endothelial growth factor
(VEGF). These characteristics suggest a value for
antiangiogenic therapies and may underlie the observed
sensitivity to radiotherapy and trabectedin.
 Trabectedin may also function by disrupting the binding of
FUS-DDIT3 to target promoters.
 Agents designed to target FUS-DDIT3 are not yet available.

 Most commonly in adolescents and young adults;
 primary sites are most often in bone but can also
be in soft tissues.
 EWSR1, is fused to one of several ETS family
transcription factor genes (usually FLI1).
 The net result is the activation of pathways driving
proliferation and cell survival and the repression of
pathways promoting mesenchymal differentiation.
2. Ewing sarcoma

 Several agents (inhibitors of IGF/mammalian target
of rapamycin [mTOR], histone deacetylase, and
cyclin-dependent kinase) that target translocation-
induced mechanisms and pathways are currently in
clinical trials for Ewing sarcoma, and strategies to
inhibit the oncoprotein itself are in active
development.

3. Desmoplastic small round cell tumor

 EWSR1 involved in Ewing sarcomas are fused to WT1,
a tumor suppressor deleted in Wilms tumor.
 Despite some similarities to Ewing sarcoma family
tumors, desmoplastic small round cell tumors are rarely
cured with aggressive conventional chemotherapy
combined with surgical debulking; prognosis is dismal,
so new therapies are needed.
 Several targets of EWSR1-WT1 have been identified.
EWSR1-WT1 directly induces PDGFA expression,
which explains the desmoplastic background and, along
with VEGFA and VEGFR2 overexpression, accounts for
the observed partial responses to sunitinib.
 IL2RB is also induced, and its downstream JAK/STAT
and AKT/mTOR signaling pathways appear active,
representing potential targets for novel treatment
approaches.
3. Desmoplastic small round cell tumor

 Typical synovial sarcomas demonstrate classical
biphasic morphology, with prominent paler
glandular-like structures interspersed within
fascicles and sheets of monomorphic spindle cells
that have a more darkly stained appearance on
haematoxylin and eosin staining.
 Defining translocation, t(X;18)(p11;q11).
 Encode epigenetic regulators, not transcription factors that
bind DNA directly.
4. Synovial sarcoma

 Fusion of SS18 (aka SYT) gene with an SSX gene.
 The net result is Polycomb-mediated epigenetic
repression of target genes, including the tumor
suppressors EGR1 and CDKN2A.
 Key genes and oncogenic pathways that become
activated, directly or indirectly, in synovial sarcoma
include histone deacetylases, SOX2, Wnt/β-
catenin, TWIST1, FGFR2, BCL2, and the
Akt/mTOR pathway via IGF2.
 Therefore, these represent candidates for targeted
therapy approaches in the absence of known drugs
that inhibit SS18-SSX directly.

 An aggressive cancer of older children and
adolescents.
 FOXO1 from 13q14 is fused to the DNA-
binding domain of paired box transcription
factor PAX3 (2q35) or PAX7 (1p36).
 Translocations involving PAX3 may be
associated with a worse prognosis than
those involving PAX7.
5. Alveolar rhabdomyosarcoma

 Thus, to optimize patient care, the
diagnosis should be confirmed by FISH
and/or RT-PCR.
 Direct targets of PAX3-FOXO1 include P-
cadherin (CDH3), GREM1, DAPK1, and
MYOD1, ALK as well as PDGFRA.
 Inhibitors of PDGFRA are effective in
mouse models.

 ASPSCR1 (i.e., ASPL) gene on 17q25 is
fused to exon 3 or 4 of TFE3 on Xp11.
 Although alveolar soft-part sarcoma has
distinctive histology, two useful diagnostic
adjuncts are the
◦ detection of TFE3 rearrangements by FISH and
◦ detection of translocations by RT-PCR or by
immunohistochemistry for TFE3.
6. Alveolar soft part sarcoma

 Antiangiogenic therapy is effective in
xenograft models.
 A single-arm phase II study of the VEGFR
inhibitor cediranib in metastatic alveolar
soft-part sarcoma showed a high rate of
disease control in association with
downregulation of angiogenic genes
supporting advanced trials of
antiangiogenic agents.

7. Dermatofibrosarcoma
protuberans

 This is the most common dermal sarcoma, and
comprises characteristic fascicles of uniform, bland
elongated spindle cells in a prominent storiform
pattern.
 Immunohistochemically, diffusely and strongly
positive for CD34, expressed by subsets of
fibroblasts.
7. Dermatofibrosarcoma
protuberans

 A hallmark of dermatofibrosarcoma protuberans
(DFSP) is supernumerary ring chromosomes that
contain material from chromosomes 17 and 22, or
less commonly, an unbalanced t(17;22)(q21-
23;q13).
 The molecular consequence of both types of
aberration is the overexpression of the PDGF beta
(PDGFB) gene on chromosome 22, through fusion
with the collagen gene COL1A1 on chromosome
17.

 FISH and comparative genomic hybridization
(CGH) studies indicate increased COL1A1–
PDGFB copy number.
 The COL1A1-PDGFB fusion product signals
through the PDGF receptor in an autocrine loop.
 This signaling can be blocked using tyrosine-
kinase inhibitors acting at PDGFR, such as
imatinib.
 A number of clinical studies have shown a high
response rate to imatinib therapy in both locally
advanced and metastatic DFSP.

8. Extra-skeletal Myxoid
chondrosarcoma

 Most extraskeletal myxoid
chondrosarcomas show reciprocal
translocations that fuse NR4A3 in 9q22-
q31.1 with one of four partners:
 EWSR1 in 22q12 (the most common),
 TAF15 in 17q11,
 TCF12 in 15q21, or
 TFG in 3q12.
 FISH is used for diagnosis.
8. Extra-skeletal Myxoid
chondrosarcoma

9. Solitary fibrous tumor and
Hemangiopericytoma

 Solitary fibrous tumor (SFT) and
hemangiopericytoma share similar
histopathologic features and are now classified
as a single biologic entity.
 A recurrent NAB2-STAT6 fusion was identified
in virtually all SFTs, regardless of anatomic
location (pleura, meninges, or soft tissue).
 SFTs showed a high-level expression of both
EGR1 target genes, including NAB2, NAB1,
IGF2, FGF2, and PDGFD, and receptor
tyrosine kinases, such as FGFR1 and NTRK1.
9. Solitary fibrous tumor and
Hemangiopericytoma

 IGF2 is uniformly overexpressed in SFT, regardless
of anatomic location.
 IGF2 signaling occurs through the insulin receptor
A pathway.
 Overexpression of IGF2 and consequent activation
of the insulin receptor results in hypoglycemia.
 This syndrome, known as Doege-Potter syndrome,
has been associated with large tumor size and
aggressive clinical behavior and is resolved by
surgical resection of the lesion.

B. SOFT TISSUE SARCOMAS OF
SIMPLE KARYOTYPE ASSOCIATED
WITH MUTATIONS

 Desmoid-type fibromatoses are locally
infiltrative, clonal fibroblastic proliferations
that arise in the deep soft tissues and never
metastasize.
 70% - APC or CTNNB1,
 pregnancy, trauma, and prior surgery.
 Two groups:
 sporadic desmoids
 heterozygous germ-line mutation in
the APC gene (chromosome 5q).
Desmoid fibromatosis

 Among sporadic desmoids, a majority
(52% to 85%) have an activating point
mutation in the β-catenin gene, CTNNB1.
 β-Catenin, a mediator of Wnt signaling, is
negatively regulated by APC, so both APC
inactivation and CTNNB1 activating
mutations result in the upregulation of the
Wnt pathway.

 β-catenin antagonists would be useful.
 An inhibitor of matrix metalloproteinase, a
downstream target of β-catenin,
substantially reduced tumor volume and
tumor invasion in a transgenic mouse
model of aggressive fibromatosis.
 Hedgehog signaling is activated in human
and murine desmoid tumors.
 Hedgehog antagonists are a promising
therapy for desmoid patients.

 Patients with desmoids were found to have
elevated levels of PDGF-AA and PDGF-BB,
leading to a trial of the tyrosine-kinase
inhibitor Imatinib in patients with advanced
disease.
 Sorafenib, a multitargeted tyrosine-kinase
inhibitor, results in tumor shrinkage in 25%
of desmoid patients and stable disease in
70%, along with symptom relief in 70% of
patients.

C. COMPLEX SOFT TISSUE
SARCOMAS TYPES

1. Well differentiated and Dedifferentiated
Liposarcoma

 Most common biologic group of liposarcoma.
 Characterized by amplification of 12q.
 The amplified region generally includes the
oncogenes MDM2, HMGA2, and CDK4.
 Nutlin-3a, a selective MDM2 antagonist may be
useful.
 PD0332991, a selective CDK4/CDK6 inhibitor,
inhibits proliferation by inducing G1 cell-cycle
arrest is under trial.
1. Well differentiated and Dedifferentiated
Liposarcoma

 Aside from 12q aberrations, dedifferentiated
liposarcomas contain significant amplifications of
1p, 1q, 5p, 6q, and 20q.
 Amplification of JUN (on 1p32) - block in adipocyte
differentiation in
 C/EBPα (19q), is a tumour supressor gene, which
results in 50% decrease in proliferation, a G2/M
arrest, apoptosis, and restoration of the ability to
induce early adipogenesis markers.
 C/EBPα is under expressed (24% cases) in
sarcomas – poor prognosis.

 8.3% had somatic mutations in the histone
deacetylase HDAC1.
 Treating with histone deacetylase inhibitor
vorinostat decreases proliferation, induces
apoptosis, and reduces xenograft tumor
growth by 50% to 70%.

 Least common subtype (5%).
 Most common alteration - del3q14.2-q14.3,
including the RB1 tumor suppressor in 60%
cases.
 Second most common alteration - loss of
17p13.1, including TP53.
 In TP53-mutant cells, antagonism of MDM2
by nutlin-3a enhances chemosensitivity.
2. Pleomorphic liposarcoma

 A third genetic alteration – del 17q11.2,
including the tumor suppressor NF1.
 Loss of NF1 function activate the RAS and
mTOR pathways
 MEK or mTOR inhibitors may have clinical
utility.

 Myxofibrosarcoma,
 Also known as myxoid variant of malignant
fibrous histiocytoma,
 Karyotypes tend to be highly complex, with
chromosome numbers in the triploid or
tetraploid range.
 55% harbores chromosome 5p
amplification.
3. Myxofibrosarcoma

 This region contains RICTOR (a binding
partner of mTOR), CDH9, and LIFR.
 Myxofibrosarcoma also harbores deletions
of tumor suppressors, including
CDKN2A/CDKN2B, RB1, TP53, NF1, and
PTEN.

4. Undifferentiated pleomorphic sarcoma

 Histologically pleomorphic and high grade.
 The karyotypes are highly complex.
 Most have chromosome numbers in the
triploid or tetraploid range.
 Mutations and/or deletions of TP53, RB1, and
INK4a have been suggested to be drivers of
oncogenesis.
4. Undifferentiated pleomorphic sarcoma

 Recent work show activation of both the
Hedgehog and Notch pathways in UPS.
 Targeting signaling pathways may be a
promising approach for these
undifferentiated tumors.

 Leiomyosarcoma - malignant tumor with
smooth muscle differentiation.
 Karyotypes tend to be complex.
 Frequently observed aberrations include:
◦ losses of 1p12-pter, 2p, 13q14-q21
(RB1),10q ( PTEN), and 16q and
◦ gains of 17p, 8q, and 1q21-31;
5. Leiomyosarcoma

 The myocardin (MYOCD) gene on 17p is
significantly amplified and overexpressed in
retroperitoneal tumors.
 Knockdown of MYOCD in leiomyosarcoma
cell lines harboring this amplification
decreases smooth muscle differentiation
and inhibits cell migration.
 In addition to PTEN inactivation,
homozygous deletions in MTOR were
identified.

 Because PTEN is a repressor of Akt, both
these events suggest a critical role for
aberrant Akt-mTOR signaling in
leiomyosarcoma.
 mTOR inhibitors such as everolimus and
temsirolimus have shown some efficacy in
patients with leiomyosarcoma in clinical
trials.
 RB1 deletion is common in
leiomyosarcomas, with 70% harboring
heterozygous deletions.
 There is high incidence of leiomyosarcoma

6. Malignant peripheral nerve
sheath tumor

 Malignant peripheral nerve sheath tumors
(MPNST) - highly aggressive soft tissue
sarcomas
 The NF1 gene (17q del) is implicated in
sporadic as well as NF1-associated
MPNST.
 NF1 encodes neurofibromin, a protein that
accelerates Ras–GTP hydrolysis and thus
negatively regulates Ras.
 Display complex karyotypes and clonal
chromosomal aberrations.
6. Malignant peripheral nerve
sheath tumor

 NF1-deficient Schwann cells show an
activation of mTOR,
 They are highly sensitive to the mTOR
inhibitor rapamycin.
 Chemokine receptor CXCR4 is highly
expressed in NF1-associated MPNST and
that CXCR4, along with its ligand CXCL12,
promotes MPNST growth by stimulating
cyclin D1 expression
 CXCR4 antagonist AMD3100 may
represent a promising therapy for MPNST.

 The inactivation of NF1 and the consequent
activation of the Ras/Raf/MAPK pathway in
the majority of MPNSTs supported targeting
B-Raf with the B-Raf tyrosine-kinase
inhibitor Sorafenib.

7. Angiosarcoma
 Angiosarcomas are rare vascular
malignancies of endothelial cell
differentiation that arise either de novo or
secondary to radiation therapy or chronic
lymphedema.
 Upregulation of vascular-specific receptor
tyrosine kinases, including TIE1, KDR
(VEGFR2), TEK (TIE2), and FLT1
(VEGFR1).

 The most frequent recurrent abnormalities
in CGH were high-level amplifications on
chromosome 8q24.21 (50%), followed by
amplification on 10p12.33 (33%) and
5q35.3 (11%).
 A high-level amplification of MYC on
8q24.21 was confirmed by FISH in most
angiosarcomas associated with radiation
and chronic lymphedema.

COMMERCIALLY AVALIABLE PROBES USEFUL
FOR FLOURESCENCE IN SITU HYBRIDIZATION IN
SARCOMAS
Tanas MR, Goldblum JR. Fluorescence in situ hybridization in the
diagnosis of soft tissue neoplasm: a review. Adv Anat Pathol.

Quesada J and Amatol R. The Molecular Biology of Soft-Tissue
Sarcomas and CurrentTrends in Therapy. Hindwai publishing 2012

Molecular biology of soft tissue sarcoma

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