Sarcoma and soft tissue tumour genetic studies
Soft tissue tumours are a diverse group of neoplasms affecting tissues which connect, support, or surround other structures and organs of the body. Classification is traditionally based on histology, although tumour location, size, growth pattern, recurrence risk, patient age, and distribution of metastases are also important. Most soft tissue tumours are either benign or malignant (i.e. sarcomas) but some are intermediate, ie locally aggressive with a potential to metastasise.
Although histopathology remains the gold standard for diagnosis, advances in genetic technology are reshaping approaches to diagnosis and disease management. The majority of soft tissue tumours contain acquired cytogenetic changes – many demonstrate disease-specific chromosome translocations, whilst some contain other molecular events such as oncogene amplifications. Cytogenetic and molecular testing plays an increasingly important role in aiding pathology diagnosis and providing information important to disease progression and outlook.
Cytogenetic testing and molecular genetic testing is performed by the Leeds Genetics/Histopathology Molecular Oncology team for an expanding number of soft tissue tumour types using a variety of techniques such as G-banding, FISH, and Sanger sequencing.
FISH showing PAX-FOXO1 amplification in alveolar rhabdomyosarcoma
FISH showing 13q14 deletion in spindle cell lipoma
FISH showing EWSR1 rearrangement in Ewing’s sarcoma
G-banding showing large marker chromosome containing MDM2 amplification in well-differentiated liposarcoma.
Sanger sequencing showing KIT exon 9 mutation in a GIST
Sample Requirements – G-Banding
Whilst the great majority of genetic testing in soft tissue tumours involves the use of DNA-based tests, a cell culture and karyotyping service is available on fresh tissue for exceptional cases where this may be of clinical benefit.
Cell culture requires living cells. Please ensure that the sample reaches us as quickly as possible (within 24 hours). First class post is satisfactory.
Samples should be placed in sterile containers, containing sterile, fresh (i.e. less than one month since opening) culture medium – this should ideally be Ham’s F10, but any balanced salt solution is acceptable. Transport medium is available from the laboratory on request. On no account should samples be transported in formalin.
Samples from within St James’s should be sent in sealed plastic bags, with the request card protected from the sample.
Samples from other hospitals should be placed in taped absorbent boxes containing appropriate padding and packing.
Request cards should be protected from samples.
Where delays over 24 hours are unavoidable, e.g. for biopsies taken on Saturday afternoon or Sunday, samples should be refrigerated and sent to the laboratory to arrive on the next working day.
Open biopsies from the tumour itself are preferred.
Sample size is important, and the chances of obtaining a successful result increase with biopsies above 5mm in diameter.
Trucut biopsies and fine needle aspirates will also be accepted if no open biopsy is available, although smaller sized samples can result in a higher failure rate.
Where consent for research is available, surplus tumour tissue from paediatric patients will be stored for possible future research or development purposes, in line with the agreed St James’s Hospital Paediatric Oncology tumour storage pathway.
Where appropriate, other fluids where tumour infiltration is known or suspected may be sent. These include pleural effusions, cerebro-spinal fluid, bone marrow, blood and cystic fluid.
Cytogenetic analysis of blood or bone marrow may also be undertaken by the molecular oncology team where infiltration into blood or bone marrow is known or suspected.
Sample Requirements – FISH
Fresh tumour biopsies for short-term (overnight) culture for interphase FISH studies should be sent to the lab as ‘urgent’ in transport medium, sterile saline or dry.
All Formalin fixed and paraffin wax embedded (FFPE) samples should be sent to the Leeds histopathology laboratory for entry onto the CoPath LIMS. Samples can either be sent as cut sections or as a block. Cut sections should be of 4µ thickness on positively charged coated superfrost slides, with an accompanying marked H&E stained slide with the tumour area marked on it to guide FISH analysis, along with the % tumour nuclei present. If sending paraffin blocks, these will be cut and marked by the Leeds Histopathology team prior to genetic analysis. Blocks should contain >20% tumour, and will be returned once the analysis is complete.
Sample requirements – GISTs
See Molecular Oncology section on GIST tumour testing for more details.
Sample labelling and referral forms
All referrals must be sent with an appropriate referral form. Please use clear patient identifiers on the sample containers, slides and referral form. All patient samples must be labelled with at least three patient identifiers.
Sample transport
When sending samples by post a secure container should be used to conform to current postal regulations, i.e. P650 and UN3373 as applicable. See packaging regulations for further information.
For the laboratory address and contact details, see the laboratory contacts page.
FISH analysis
FISH tests are available for a wide variety of soft tissue tumour types, mainly using validated commercially available probe sets:
Tumour Type:
Alveolar rhabdomyosarcoma
Test type and method:
FOXO1 gene rearrangement – break apart FISH
FOXO1-PAX gene fusion – single fusion FISH
MYCN amplification – single copy FISH
PAX7 & PAX3 rearrangements – break apart FISH
Tumour Type:
Ewing’s sarcoma tumours
Test type and method:
EWSR1 gene rearrangement – break apart FISH
EWSR1-FLI1 gene fusion – dual fusion FISH
EWSR1-ERG1 gene fusion – dual fusion FISH
Tumour Type:
Desmoplastic small round cell tumours (DSRCT)
Test type and method:
EWSR1 gene rearrangement – break apart FISH
WT1 gene rearrangement – break apart FISH
Tumour Type:
Clear cell sarcoma
Test type and method:
EWSR1 gene rearrangement – break apart FISH
Tumour Type:
Angiomatoid fibrous histiocytoma
Test type and method:
EWSR1 gene rearrangement –break apart FISH
Tumour Type:
Extraskeletal myxoid chondrosarcoma
Test type and method:
EWSR1 gene rearrangement – break apart FISH
NR4A3 gene rearrangement – break apart FISH
Tumour Type:
Other tumours with EWSR1 rearrangements –
Myoepitheloid tumour, occasional myxoid liposarcoma, slerosing epitheloid fibrosarcoma
Test type and method:
EWSR1 gene rearrangement – break apart FISH
Tumour Type:
Nodular fasciitis, giant cell granulomas, aneurysmal bone cysts, and myositis ossificans
Test type and method:
USP6 – break apart FISH
Tumour Type:
Endometrial stromal sarcoma
Test type and method:
JAZF1 rearrangement (low grade tumours) – break apart FISH
YWHAE rearrangement (high grade tumours) – break apart FISH
PHF1 rearrangement – break apart FISH
SMARCB1 (INI1) deletion – single copy FISH
Tumour Type:
**Synovial sarcoma
Test type and method:
SS18 gene rearrangement – break apart FISH
Tumour Type:
Well-differentiated and dedifferentiated liposarcoma
Test type and method:
MDM2 amplification – single copy FISH
Tumour Type:
Myxoid liposarcoma
Test type and method:
FUS-DDIT3 gene rearrangement – break apart FISH
Tumour Type:
Spindle cell lipoma and well differentiated spindle cell liposarcoma
Test type and method:
13q deletion – single copy FISH
Tumour Type:
Cellular angiofibroma and mammary-type myofibroblastoma
Test type and method:
13q deletion – single copy FISH
Tumour Type:
Alveolar soft part tumour
Test type and method:
TFE3 gene rearrangement – break apart FISH
Tumour Type:
DFSP/giant cell fibroblastoma
Test type and method:
COLIA1-PDGFB rearrangement – single fusion FISH
Tumour Type:
Low grade fibromyxoid sarcoma and sclerosing epitheloid sarcoma
Test type and method:
FUS gene rearrangement – break apart FISH
Tumour Type:
Inflammatory myofibroblastic tumour
Test type and method:
ALK gene rearrangement – break apart FISH
Tumour Type:
Rhabdoid tumours
Test type and method:
SMARCB1 (INI1) deletion – single copy FISH
Tumour Type:
Mesoblastic nephroma/congenital fibrosarcoma
Test type and method:
ETV6 gene rearrangement – break apart FISH
Tumour Type:
Lipoblastoma
Test type and method:
PLAG1 gene rearrangement – break apart FISH
Tumour Type:
Undifferentiated sarcoma/small round cell sarcoma
Test type and method:
CIC rearrangement – break apart FISH
Tumour Type:
Spitzoid tumours
Test type and method:
Break apart FISH for rearrangements of either ALK, ROS1, NTRK1 or NTRK3
Tumour Type:
Ossifying fibromyoid tumours
Test type and method:
PHF1 rearrangement – break apart FISH
Tumour Type:
Myxoinflammatory fibroblastic sarcomas
Test type and method:
TGFBR3 rearrangement – break apart FISH
** RNA based analysis using reverse transcriptase PCR (RT-PCR) is also available for detection of SSX2-SS18 rearrangements in FFPE tissue in synovial sarcoma.
Additional FISH probes are available for a number of carcinomas of soft tissues. These include:
- TFE3 break apart probe for TFE3 associated renal cell carcinoma
- MAML2 break apart probe for mucoepidermoid carcinoma, hidradenoma
- ETV6 break apart probe for ETV6 rearrangements in mammary analogue secretory carcinoma
- NUTM1 break apart probe for NUT midline carcinoma
FISH reports, turnaround times and prices
Reports can be e-mailed if you supply the Genetics laboratory with an nhs.net address. A paper copy will only be posted if specifically requested.
The expected turnaround time for pre-labelled FFPE slides is 3 working days. This increases to 5 working days if just blocks are sent.
Prices are available on contact to [email protected]
Further information on the Leeds Sarcoma Service is also available on the Sacroma Service page.
Future developments, including RT-PCR
The laboratory is keen to introduce new tests as necessary. Newly available FISH probes can be ordered and validated where there is a clinical utility. We are also keen to expand our repertoire of RNA based testing for translocations.