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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 Ä¢¹½tvÊÓƵ 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 Ä¢¹½tvÊÓƵ 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 Ä¢¹½tvÊÓƵ 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:

** 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 Ä¢¹½tvÊÓƵ 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.