1. Genetic Testing in Sarcoma: Current practice and future perspectives
Sarcoma SSG: 21/06/2016
Chris Wragg, Head of Oncology Genomics, BGL

2. Role of Genetic/Genomic testing
Diagnostic marker;
May aid discrimination between small round cell tumours
Spindle cell sarcoma
Rhabdomyosarcoma subtype
Unusual clinicopathological presentations
Typically exhibited from earliest disease presentation; persist in metastiatic and previously treated lesions
Retained in neoplasms as they become less differentiated/lose immunophenotype
Dedifferentiated liposarcoma with MDM2 amplification
Direct treatment strategy:
Some fusion genes important in directing therapy e.g. dermatifibrosarcoma protruberans, t(17;22), COL1A1-PDGFB
Clear cell sarcoma vs. conventional melanoma
Lipoma vs. Atypical lipomatous tumour/well differentiated liposarcoma

3. Genomic Abnormalities in Sarcoma
Relatively simple genomic aberrations, often as the sole abnormality. Recurrent structural abnormality e.g. translocation or activating mutation
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
More complex genomic imbalance which can be a) reproducible or b) of no specific pattern

4. Cell cycle & DNA Packaging

5. Genomic Rearrangement

6. Genomic Rearrangements- Sarcoma
Pierron et al. Nat Genet. (2012)

7. Molecular Testing in Sarcomas
Use RT-PCR specific for t(11;22)
EWSR1 (Chr 22)
FLI1 (Chr 11)
Control
Fusion

8. Fluorescence in situ hybridisation (FISH)

9. EWSR1

10. FISH substrates Fresh/Cultured material Direct/imprint preparations
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)
Formalin fixed paraffin embedded
Extensive processing required
Time consuming 3-10 days
Retains in situ structure 10

11. Ewing sarcoma Small, round cell tumour
Varying degrees of neuroectodermal differentiation
Recurrent translocations (fusion genes) involving typically EWSR1 and a member of the ETS family of transcription factors

12. EWSR1 Ewing Sarcoma breakpoint region 1 (EWSR1)
Located on chromosome 22q12
Encodes 656-amino acid nuclear protein:
Meiotic cell division
Mitotic spindle formation and stabilisation of microtubules
DNA repair mechanisms
Cellular ageing
Member of TET family of genes; other TET genes can substitute for EWSR1 as a fusion partner e.g. FUS
‘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

13. 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
Further information from karyotype: also +8
EWSR1 rearrangement
EWSR1 probe 13

14. EWSR1

15. MDM2 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

16. MDM2 amplification

17. Sarcoma Genomics – BGL Disease Test http://www.nbt.nhs.uk/genetics
Synovial Sarcoma
SS18 (18q11) rearrangement
Myxoid Liposarcomas
CHOP (12q13) rearrangement
Low grade fibromyxoid sarcoma, angiomatoid fibrous histiocytoma, myxoid liposarcoma
FUS (16p11) rearrangement
Alveolar Rhabdomyosarcoma
FOXO1 (13q14) rearrangement
ATL/WDL de-differentiated lipsarmcoma, osteosarcomas and oesophageal carcinomas.
MDM2 (12q15) amplification
Dermatofibrosarcoma protuberans (DFSP) or giant
cell fibroblastoma (GCF)
COL1A1/PDGFB t(17;22)
Various tumours including Ewings, DSRCT etc (see above)
EWSR1 (22q12) rearrangement
Infantile fibrosarcoma
ETV6 (12p13) rearrangement

18. 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

19. The 100,000 Genomes Project
West of England Genomic Medicine Centre (WEGMC)

20. WEGMC – Cancer Pathway

21. 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

22. Sarcoma SSG
Bristol Genetics Laboratory, Southmead Hospital, BRISTOL BS10 5NB
(0117)