Presentation: Patient with localised colorectal cancer

A 72-year-old male is diagnosed with a 3cm colorectal tumour at the right hepatic flexure. He undergoes a right hemicolectomy and is found to have a poorly differentiated adenocarcinoma, pT3N0M0, with clear margins and no lymphovascular invasion. There is no significant family history of cancer. You wish to undertake genomic testing and are considering what constitutional (germline) and somatic (tumour) genomic testing is available and appropriate for him.

Somatic (tumour) testing:

• All patients with colorectal cancer are eligible for testing of MMR or MSI of the tumour.
  • In the context of an intermediate-risk stage II colorectal cancer, MMR/MSI status provides important prognostic information and predicts likely benefit from adjuvant chemotherapy.
  • MMR/MSI status may be assessed by an immunohistochemistry (IHC) 4‑panel test for MLH1, MSH2, MSH6 and PMS2 proteins (not a genomic test), or PCR for MSI (a genomic test).
  • If tumour IHC results demonstrate loss of MLH1/PMS2, BRAF analysis should be undertaken. Pathogenic BRAF variants (most commonly BRAF V600E) are common in sporadic MMR-deficient colorectal cancers, but rare in tumours associated with Lynch syndrome.
    • If BRAF testing does not identify a pathogenic variant in an MLH1/PMS2-deficient colorectal cancer, MLH1 promoter hypermethylation testing should be performed.
  • If IHC/ MSI/ MLH1 methylation analysis has indicated that Lynch syndrome testing is indicated and a germline sample is not available (for example, in the case of deceased index case) or a constitutional (germline) MMR variant has not been detected, then genomic analysis of somatic MMR genes can be requested. This should be coordinated by the clinical genetics team.

Constitutional (germline) testing

• If microsatellite instability (MSI) testing demonstrates instability, if the MSH2, MSH6 or PMS2 IHC results are abnormal, or if the MLH1 +/- PMS2 results are abnormal and the MLH1 promoter hypermethylation test is negative, the patient should be tested for Lynch syndrome by genomic testing of constitutional (germline) DNA.
• Consideration should be given to broader panel testing in patients with bowel polyps. Patients with bowel cancer are eligible for testing of the following genes: APC, BMPR1A, GREM1, EPCAM (3’ CNV analysis only), MLH1, MSH2, MSH6, MUTYH, NTHL1, PMS2, POLD1*, POLE*, (*exonuclease domains only) PTEN, RNF43, SMAD4 and STK11 if they meet one of the following criteria:
  • any colorectal cancer diagnosis before age 40 years; or
  • five or more adenomatous polyps and colorectal cancer; or
  • five or more adenomatous polyps before age 40 years, 10 or more adenomatous polyps before age 60 years, or 20 or more adenomatous polyps at any age; or
  • five or more adenomatous polyps before age 60 years and one or more first-degree relatives with five or more adenomatous polyps or colorectal cancer diagnosed before age 60 years; or
  • a clinical diagnosis of serrated polyposis syndrome; if:
    • diagnosed before age 50; or
    • if the patient has a family history of one or more affected FDR with SPS; or
    • if there is evidence of dysplasia within any polyp.
  • hamartomatous polyposis syndromes:
    • five or more hamartomatous polyps of the colorectum; or
    • two or more hamartomatous polyps throughout the GI tract; or
    • one or more hamartomatous polyps and one or more first-/ second-degree relative also has one or more hamartomatous polyps.
• This patient would be eligible for fluoropyrimidine chemotherapy and would therefore require testing for constitutional (germline) dihydropyrimidine dehydrogenase (DPD) deficiency.
  • Testing for select recurrent DPYD variants is performed on constitutional (germline) DNA (indication M1.7).

• Consult the National Genomic Test Directory to ensure your patient is eligible for testing. You can also access a spreadsheet containing details of all available tests.
  • For information about how to arrange testing in Wales, Scotland or Northern Ireland, see our dedicated Knowledge Hub resource.
• For information on the genes included on different gene panels for constitutional (germline) testing, see the NHS Genomic Medicine Service signed-off panels resource.
• Decide which tests best suit the needs of your patient. For patients affected with colorectal cancer, options include the following.

Somatic testing:

• M1.1 Multi-target NGS panel for patients with colorectal cancer (CRC) eligible for anti-EGFR therapy and/or BRAF status required as part of pathway for Lynch testing (KRAS, NRAS, BRAF);
• M1.2 KRAS hotspot testing in CRC patients eligible for anti-EGFR therapy where NGS panel has failed;
• M1.3 NRAS hotspot testing in CRC patients eligible for anti-EGFR therapy where NGS panel has failed;
• M1.4 MSI testing testing in CRC patients where MMR testing has failed;
• M1.5 MLH1 promoter hypermethylation in CRC patients, where indicated as per the Lynch Syndrome testing algorithm. This is best done alongside constitutional (germline) DNA to facilitate the detection of constitutional hypermethylation of the MLH1 promoter
• M1.6 NTRK rearrangement panel for CRC patients who may be clinically eligible for NTRK inhibitor (advanced disease only).
• M1.9 Multi-target NGS panel for the analysis of MMR genes (MLH1, MSH2, MSH6, PMS2, POLE, POLD1) where constitutional (germline) testing is uninformative but IHC/MSI results suggestive of a diagnosis of Lynch Syndrome, or where constitutional sample is not available for testing.
• For constitutional (germline) testing for Lynch syndrome, a record of discussion (RoD) form is required. If you have not completed an RoD form before and/or do not have access to one, please review this Knowledge Hub article on how to complete an RoD form.
• All of the above somatic tests can be performed on formalin-fixed tumour tissue. Testing of tumour tissue for MMR deficiency or MSI, as well as subsequent somatic testing, may be done reflexively by your histopathology department, but you may need to follow this up within the multidisciplinary team meeting and clarify patient eligibility.
• Depending on the details you provide and the chosen test, a range of different genomic investigation techniques will be applied to your patient’s/their family’s (if appropriate) DNA. These include (but are not restricted to):
  • Single gene sequencing
  • Gene panel sequencing
  • MLPA
  • Methylation testing

Constitutional (germline) testing:

• R210 Inherited MMR deficiency (Lynch syndrome) after working through the tumour-based MSI/MMR IHC pathway; and
• R211 Inherited polyposis and early onset colorectal cancer, for patients with polyps fulfilling eligibility.

Information about patient eligibility and test indications was correct at the time of writing. When requesting a test, please refer to the National Genomic Test Directory to confirm the right test for your patient.

For clinicians

• ESMO: Clinical practice guidelines – localised colon cancer
• NHS England: National Genomic Test Directory and eligibility criteria (note that somatic (tumour) tests are listed in the directory for cancer, while constitutional (germline) tests are listed in the directory for rare and inherited disease. See this article for more information)
• NICE: Molecular testing strategies for Lynch syndrome in people with colorectal cancer

For patients

• Macmillan: Lynch syndrome
• Royal Marsden NHS Foundation Trust: Beginner’s guide to Lynch syndrome (PDF, 64 pages)