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Genetic Testing in Sarcoma:
Current practice and future
perspectives
Sarcoma SSG: 21/06/2016
Chris Wragg, Head of Oncology Genomics, BGL
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http://www.nbt.nhs.uk/genetics
Role of Genetic/Genomic testing
1.
Diagnostic marker;
a) May aid discrimination between small round
cell tumours
b) Spindle cell sarcoma
c) Rhabdomyosarcoma subtype
d) Unusual clinicopathological presentations
2. Typically exhibited from earliest disease presentation; persist in
metastiatic and previously treated lesions
3. Retained in neoplasms as they become less differentiated/lose
immunophenotype
a) Dedifferentiated liposarcoma with MDM2 amplification
4. Direct treatment strategy:
a) Some fusion genes important in directing therapy e.g.
dermatifibrosarcoma protruberans, t(17;22), COL1A1-PDGFB
b) Clear cell sarcoma vs. conventional melanoma
c) Lipoma vs. Atypical lipomatous tumour/well differentiated liposarcoma
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Genomic Abnormalities in Sarcoma
1. Relatively simple genomic aberrations, often as the sole
abnormality. Recurrent structural abnormality e.g. translocation or
activating mutation
a. Non-random, reciprocal translocation creates fusion genes
(30/117(26%) WHO soft tissue tumours). Functionally can
encode for:
 Aberrant transcription factors (deregulation) e.g. Ewing
sarcoma, synovial sarcoma, ARMS
 Chimeric tyrosine kinases (deregulated signalling) e.g.
inflammatory myofibroblastic tumour
 Chimeric autocrine growth factors e.g.
dermatofibrosarcoma protruberans
2. More complex genomic imbalance which can be a) reproducible or
b) of no specific pattern
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Cell cycle & DNA Packaging
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Genomic Rearrangement
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Genomic Rearrangements- Sarcoma
Pierron et al. Nat Genet. (2012)
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Molecular Testing in Sarcomas
Use RT-PCR specific for t(11;22)
EWSR1 (Chr 22)
FLI1
Control
Fusion
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(Chr 11)
Fluorescence in situ hybridisation (FISH)
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EWSR1
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FISH substrates
1.
Fresh/Cultured material
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2.
Metaphase and interphase growth
Time consuming (2~4 weeks)
Does not retain in situ structure
Some rearrangement cryptic
Direct/imprint preparations
 No metaphase growth
 Rapid (1-2 days)
 Does not retain in situ structure
3.
Formalin fixed paraffin embedded
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No metaphase growth
Extensive processing required
Time consuming 3-10 days
Retains in situ structure
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Ewing sarcoma
1. Small, round cell tumour
2. Varying degrees of neuroectodermal differentiation
3. Recurrent translocations (fusion genes) involving typically EWSR1
and a member of the ETS family of transcription factors
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EWSR1
1. Ewing Sarcoma breakpoint region 1 (EWSR1)
2. Located on chromosome 22q12
3. Encodes 656-amino acid nuclear protein:
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Meiotic cell division
Mitotic spindle formation and stabilisation of microtubules
DNA repair mechanisms
Cellular ageing
4. Member of TET family of genes; other TET genes can substitute
for EWSR1 as a fusion partner e.g. FUS
5. ‘Promiscuous’ gene; identified as a fusion partner in a wide range
of clinically and pathologically divers tumours:
 Many associated with unique entities
 Some fusions with a single gene can result in morphologically/
behaviourally different neoplasms
 Other translocations may result in genetically different but
phenotypically identical entities
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Ewings Sarcoma
t(11;22)(q24;q12)
• EWSR1-FLI1
• EWSR1 gene on chromosome 22 is split in the
rearrangement
• FISH Fusion signal – normal gene
• Red and green signal – gene split apart
EWSR1 rearrangement
EWSR1 probe
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• Further information from karyotype: also +8
EWSR1
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MDM2
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Murine double minute 2 (MDM2)
Located on chromosome 12q14.3
This gene encodes a nuclear-localized E3 ubiquitin ligase
The encoded protein can promote tumour formation by targeting
tumour suppressor proteins, such as p53, for proteasomal
degradation
 MDM2 amplification arised on supernumerary ring chromosomes
and/or giant rod shaped chromosomes derived from chromsome 12
 MDM2 amplification identified in:
 atypical lipomatous tumour/well differentiated liposarcoma (ALT/WDL)
 Dedifferentiated liposarcoma
 MDM2 amplifiction not reported in:
 Lipoma
 Other, poorly differentiated/dedifferntiated, sarcomas
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MDM2 amplification
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Sarcoma Genomics – BGL
Disease
Test
Synovial Sarcoma
SS18 (18q11) rearrangement
Myxoid Liposarcomas
CHOP (12q13) rearrangement
Low grade fibromyxoid sarcoma, angiomatoid fibrous histiocytoma,
myxoid liposarcoma
Alveolar Rhabdomyosarcoma
FOXO1 (13q14) rearrangement
ATL/WDL de-differentiated lipsarmcoma, osteosarcomas and
oesophageal carcinomas.
Dermatofibrosarcoma protuberans (DFSP) or giant
cell fibroblastoma (GCF)
Various tumours including Ewings, DSRCT etc (see above)
Infantile fibrosarcoma
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FUS (16p11) rearrangement
MDM2 (12q15) amplification
COL1A1/PDGFB t(17;22)
EWSR1 (22q12) rearrangement
ETV6 (12p13) rearrangement
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Sarcoma Genomics – what’s next?
• Studies to further define the genomic landscape
of sarcoma
• Genome, epigenome and transcriptome
• New recurrent mutations identified e.g. TP53
and STAG2 mutations by WGS in ES
• Numerous large scale sequencing projects e.g.
100K genomes project
– Biodiscovery
– NHS Transformation
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The 100,000 Genomes Project
 West of England Genomic Medicine Centre (WEGMC)
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WEGMC – Cancer Pathway
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Conclusions
• Genetic testing can help inform the diagnosis
and management of patients with sarcoma
• Different methodologies available, each with
relative strengths and weaknesses
• Expansion of test portfolio possible, requires
clinical input
• 100K genomes may expand the scope of
actionable genetic lesions and has the potential
to inform transformational change
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Sarcoma SSG
Bristol Genetics Laboratory,
Southmead Hospital, BRISTOL
BS10 5NB
(0117) 4146141
[email protected]
www.nbt.nhs.uk
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