Whole genome sequencing

Whole genome sequencing (WGS) for cancer involves two parallel processes of sequencing:

• Tumour WGS, where the entire genome of the tumour itself is sequenced. This will identify both somatic (acquired) and germline (inherited/heritable) alterations, but will not reliably determine whether a given variant is somatic or germline.

• Germline WGS, where the entire genome of the individual (not tumour) is sequenced. This will identify only germline alterations and will therefore allow alterations detected in the tumour to be classified as either somatic or germline.

WGS is the most comprehensive way of assessing the genome. In theory, it should identify any genomic alteration within the genome of the tumour and the individual.

What do I need for WGS?

It is essential that the possibility of WGS is considered well in advance of tissue sampling. This is because there are two important differences from non-WGS genomic testing:

• Explicit consent is required with completion of a record of discussion.

• Fresh tumour tissue is required; formalin-fixed tissue cannot be used.

Planning is therefore required. It is important that tissue acquisition is planned in such a way as to allow fresh tissue to be sampled and frozen.

How do I request WGS?

They key steps are:

1. The patient (or family) and clinician complete the record of discussion →

2. The clinician arranges acquisition of a germline sample, usually in the form of a blood sample, which is sent to the GLH for DNA extraction →

3. The clinician takes a tumour sample and the pathologist samples tissue →

4. The pathologist assesses the sample →

5. The tumour sample is sent to the GLH for DNA extraction →

6. Extracted DNA from the germline and tumour samples is sent to Genomics England for WGS →

7. WGS data is returned to the GLH, which analyses the significance of the findings →

8. The WGS findings are discussed at a regional Genomic Tumour Advisory Board (GTAB) →

COMPLETE THE RECORD OF DISCUSSION

Explicit consent is required from the patient (or family) prior to WGS. A record of discussion form must be completed by the clinician and patient (or family). This should be included in the patient’s notes, and copies will need to be sent alongside samples.

Acquire a Germline Sample

A germline sample must be obtained to establish whether any alterations detected could be germline. This sample is usually blood, and should be collected in an EDTA tube. This should be sent to the local GLH together with a completed request form and a copy of the record of discussion.

Sample the tumour

It is essential that tumour sampling is planned in advance of the procedure:

• The pathology laboratory must be informed well in advance that a WGS sample is being taken to ensure that staff are available and ready to handle it.

• Whoever is taking the sample must be aware that it must not be immersed in formalin.

• Once taken, the sample must be transported immediately to the pathology laboratory.

• The sample should be transported with a copy of the record of discussion (or this should be available to the pathology department through the electronic patient record).

• Staff receiving the sample in the pathology must be aware that it is WGS sample, and therefore that it must not be immersed in formalin, and that it must be dealt with immediately.

The exact method of sampling depends on the specimen type and whether the diagnosis is already known:

• There may be a strong clinical suspicion that the patient will be eligible for WGS before a histological diagnosis has been made, for example, in a patient with a soft tissue tumour or a patient aged under 25 years with suspected cancer. Here, the tumour biopsy may be sent to the pathology laboratory both for diagnosis and WGS.

• A patient may already have a known histological diagnosis, and a new sample is being taken purely for the purpose of WGS. In these situations, the histology should be checked to make sure there are no surprises, but the tissue should be used for WGS.

• A patient may already have a known histological diagnosis and is having their tumour resected. In these situations, some of the tumour should be sampled for WGS, but then the remainder of the specimen used to establish staging, completeness of resection, etc.

Sampling from resection specimens is generally more straightforward:

Use of biopsies is more complex, and differs depending on whether the diagnosis is already known or not:

Assess the sample

The histopathologist assesses the quality of the tumour sample in terms of neoplastic cell percentage and percentage area necrosis.

Send the tumour sample to the GLH

The sampled, frozen tumour should be sent to the local GLH. This must be accompanied by a completed request form and a copy of the record of discussion. It is worth informing the GLH in advance to ensure that they are ready to receive it.

GLH extracts DNA which is sent to Genomics England

The GLH extracts DNA from both the germline and tumour sample. This is then forwarded to Genomics England. Genomics England undertakes WGS at its laboratories at the Wellcome Genome Campus near Cambridge.

WGS data is returned to the GLH

Results of WGS are returned to the GLH. The GLH then establishes the significance of the findings.

Findings are discussed at GTAB

The findings of WGS are discussed at a regional Genomic Tumour Advisory Board (GTAB). This is effectively a genomic multidisciplinary team (MDT) meeting. They are usually attended by clinical scientists from the GLH, as well as local oncologists, pathologists and clinical geneticists. It is helpful for clinicians and pathologists involved in the patient’s care to attend.

The results of WGS will usually be compared to the results of standard of care testing. Sometimes, further testing may be required to confirm findings from WGS.

The GTAB puts together a summary of important clinical findings and recommendations which are sent to the patient’s clinical team.