Endometrial cancers

All endometrial cancers referred for detection of small DNA variants will be examined using the Illumina TSO500 gene panel for the following genes only:

• Essential targets: POLE (M215.5 in Table 1)
• Desirable/investigative targets: POLD1, PIK3CA, FGFR2 and FGFR3
• *Please note that we will not currently be using TSO500 to report on either microsatellite instability (MSI) or on copy number variant detection of ERBB2

Reporting for somatic variants in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, PMS2 using the TSO500 panel will only be undertaken where this is specifically requested clinically and in line with NICE guidelines for endometrial testing for Lynch syndrome DG42 (M 215.4 in Table 1).

Testing for MLH1 promoter methylation testing will be undertaken using a methylation specific assay where this is specifically requested, according to NICE Lynch syndrome guidelines DG42 (M 215.2 in Table 1). MLH1 promoter methylation testing is a separate test currently undertaken on those endometrial cancers that have shown evidence of mismatch repair deficiency. A separate report will be issued where you have requested this test. It helps to identify those patients where such mismatch repair efficiency is more likely to be somatic (where MLH1 hypermethylation is identified) or where there is a possibility it may be due to a germline change (where MLH1 is unmethylated). In the latter case, referral to clinical genetics and germline testing of MMR genes is recommended.

Testing for structural variants of NTRK1, NTRK2 and NTRK3 genes (M 215.1 in Table 1) will be undertaken where there is a specific clinical request, using the Illumina TruSight gene fusion panel. Pathogenic fusions of any genes in the panel will be reported where relevant. In time, this testing will also move to the TSO500 panel.

Requests for testing should be made via your local Cellular Pathology laboratory that holds the tumour tissue and samples should initially be routed via your local genomics laboratory who will direct the testing and reporting as appropriate, so that they can act as your single point of call for any of your queries.

Costs from your local genomics laboratory onward, including transport and test costs, are commissioned and paid for by NHSE directly to the GLH, so do not need to be paid for by the referring cellular pathology lab. Tissue preparation and transport costs prior to that are not covered by that commissioning.

Until a single GLH-wide referral form is available, we will accept requests on existing genetics lab forms. However, please ensure that the form:

• Includes email addresses for your Cellular Pathology secretaries and pathologist;
• Clearly specifies which tests are needed;
• Clearly specifies tumour cell nuclei as a percent of nucleated cells (in marked area if sections used).

1. For DNA or RNA extraction only, e.g. TruSight RNA fusion panel or DNA only part of TSO500 panel:

• Sample with >20% neoplastic cells: send one tube (Eppendorf or Universal) containing 5-10 x 10μm FFPE curls
• Sample with lower tumour content: please either;
• a. macro dissect tumour-rich regions and send in a single tube or
• b. send 10 x 5μm slide mounted sections along with marked H&E with tumour rich area(s) marked.

2. For MLH1 methylation testing

• Samples must have >30% tumour nuclei
• Please send 10 x 5μm slide mounted sections along with marked H&E with tumour rich area(s) marked. These slides are in addition to material sent for TSO500

Genomic results will be returned to the referring pathologist and/or any other agreed histopathology email address for receiving results for integration into the histopathology reports.

Please note that where more than one of MLH1 promoter methylation testing, NTRK testing and TSO500 panel are ordered, reports will alert the referrer that a subsequent report of any other tests ordered will follow.

M215.5 POLE

The most significant change the latest version of the test directory brought for endometrial cancers was the inclusion of POLE as an essential target for testing. The recent BAGP guidance on testing POLE in endometrial cancer sets out recommendations for which specific types should have POLE testing undertaken.

Desirable/investigative targets

Whilst desirable/investigative targets are not included in the public-facing version of the test directory, NHSE are keen to encourage genomic labs to report on these where possible. We will continue to classify any variants in these targets (POLD1, FGFR2, FGFR3 and PIK3CA) and report driver variants, but no further interpretation on these will be undertaken

M215.4 Mismatch repair (MMR) genes

The mismatch repair genes MLH1, MSH2, MSH6, PMS2 are also included within M215, however note that the test directory eligibility criteria states that somatic testing should be according to NICE guidelines for Lynch syndrome, in the following scenarios:

1. Proband has colorectal or endometrial cancer with a deficient mismatch repair (dMMR) tumour with normal BRAF and MLH1 hypermethylation analysis AND germline testing did not reveal a pathogenic mutation AND personal/family pattern of disease whereby demonstration of acquired MMR mutations (and therefore exclusion of constitutional MMR abnormality) enables downscaling of surveillance
2. Deceased affected individual with colorectal or endometrial cancer ≤60 years AND tumour featuring high/intermediate MSI or loss of staining of MMR protein(s) on IHC, AND one first degree relative with Lynch-related cancer ≤60 AND no living affected individual is available for genetic testing.

Therefore in most cases this testing will happen AFTER germline testing for Lynch syndrome.

We propose that somatic MMR gene testing is only activated when specifically requested, in patients that meet the eligibility criteria stated above. This should ensure that testing is undertaken according to the test directory eligibility criteria, and we are able to interpret any findings in the context of the germline results.