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Example clinical scenario

A 42-year-old woman with a history of weight loss and jaundice comes to clinic following a CT scan, which reveals an 18cm liver lesion and circumferential sigmoid thickening. Sigmoid biopsy reveals a moderately differentiated adenocarcinoma. There is no significant family history of cancer. You wish to undertake genomic testing and are considering what constitutional (germline) and somatic (tumour) genetic testing is available and appropriate for her.

When to consider genomic testing

Constitutional (germline) testing

  • If the tumour demonstrates microsatellite instability (MSI), or if MSH2, MSH6 or PMS2 IHC results are abnormal, or the MLH1 IHC is abnormal and MLH1 promoter hypermethylation test is negative, the patient should be tested for Lynch syndrome by genetic testing of constitutional (germline) DNA.
  • If tumour-based testing identifies a variant in MLH1, MSH2, MSH6, PMS2, or in the exonuclease domains of POLE or POLD1 at a variant allele frequency in excess of 30%, consideration should be given to the possibility that the variant may be of constitutional (germline) origin, necessitating confirmatory site-specific testing of constitutional DNA (usually lymphocyte-derived, from whole blood), after appropriate genetic counselling and patient consent.
  • 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 <40 years;
    • ≥5 adenomatous polyps and colorectal cancer;
    • ≥5 adenomatous polyps <40 years, ≥10 adenomatous polyps <60 years, or ≥20 adenomatous polyps at any age; or
    • ≥5 adenomatous polyps (age <60 years) and first-degree relative with ≥5 adenomatous polyps (<60 years);
    • Serrated polyposis:
      • ≥5 serrated lesions/polyps proximal to the rectum all being at least 5mm in size with two or more being at least 10mm in size; or
      • more than 20 serrated lesions/polyps of any size distributed through the large bowel with at least five being proximal to the rectum.
    • Hamartomatous polyposis syndromes:
      • ≥5 hamartomatous polyps of the colorectum; or
        ≥ 2 hamartomatous polyps throughout the GI tract; or
        ≥ 1 hamartomatous polyp and a first / second degree relative has hamartomatous polyp.
  • 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).

Somatic (tumour) testing

  • All patients with metastatic colorectal cancer are eligible for a multi-target next-generation sequencing (NGS) panel to check for variants in KRAS, NRAS, BRAF, MLH1, MSH2, MSH6, PMS2, POLD1 and POLE.
    • Patients who are KRAS or NRAS wild-type are eligible for anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (cetuximab and panitumumab) in combination with 5-fluoropyrimidine doublet chemotherapy in the metastatic setting.
    • Patients with tumours demonstrating KRAS or NRAS mutations do not usually benefit from anti-EGFR monoclonal antibodies.
    • Patients with tumours demonstrating BRAF V600E mutations may also be eligible for treatment with the BRAF inhibitor encorafenib, in combination with cetuximab, via the Cancer Drugs Fund.
    • Somatic testing for NTRK1, NTRK2 and NTRK3 fusion genes is available for metastatic colorectal cancer patients as an biomarker for treatment with an NTRK inhibitor when all other approved lines of treatment have been exhausted.
    • If variants are identified in MLH1, MSH2, MSH6 or PMS2, further assessment of mismatch repair (MMR) and/or MSI should be undertaken, and the result should be discussed at a Genomic Tumour Advisory Board meeting or with the clinical genetics team to determine if site-specific germline testing is indicated to confirm/rule out germline origin of the variant.
    • Tumours associated with MMR deficiency are typically hypermutated, while tumours associated with polymerase proofreading deficiency are typically ultramutated.
  • All patients with colorectal cancer are eligible for testing of MMR or MSI.
    • In the context of a metastatic colorectal cancer, MMR/MSI status provides important prognostic information and guides potential benefit from immunotherapy.
    • This may be done by immunohistochemistry (IHC) four‑panel test for MLH1, MSH2, MSH6 and PMS2 protein expression, PCR for MSI (a genomic test), or as part of the approved multi-target NGS panel.
    • If the MLH1 IHC result is abnormal, BRAF gene analysis should be undertaken to determine if further germline genetic testing for Lynch syndrome is indicated.
      • Pathogenic somatic variants in BRAF are a surrogate marker of MLH1 promoter hypermethylation – being present in around two-thirds of MLH1 hypermethylated tumours, but being rare in tumours associated with Lynch syndrome. If the BRAF V600E test is negative, an MLH1 promoter hypermethylation test should be performed.
  • In the future, somatic (tumour) testing is likely to be expanded to include larger somatic gene panels. Ultimately, it is likely that paired somatic and constitutional (germline) whole genome sequencing (WGS) will be performed.
  • All patients with solid tumours who have exhausted all standards of care testing and treatment are eligible for WGS in order to explore clinical trial options.

What do you need to do?

  • Consult the National Genomic Test Directory eligibility criteria to ensure your patient is eligible for testing. There is also a spreadsheet of all available tests.
  • For information on the genes that are 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, germline genetic testing options include:
    • R210 Inherited MMR deficiency (Lynch syndrome); and
    • R211 Inherited polyposis and early-onset colorectal cancer.
  • Testing of tumour tissue for MMR or MSI, as well as other somatic testing, are performed on formalin-fixed tumour tissue. These tests 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. Tests available are:
    • M1.1 Multi-target NGS panel – small variant (KRAS, NRAS, BRAF, MLH1, MSH2, MSH6, PMS2, POLD1, POLE, DPYD);
    • M1.2 KRAS hotspot;
    • M1.3 NRAS hotpot;
    • M1.4 MSI testing; and
    • M1.5 MLH1 promoter hypermethylation testing – for tumours demonstrating loss of MLH1/PMS2 deficiency on IHC, without evidence of a pathogenic somatic BRAF variant.
  • NTRK1, NTRK2 and NTRK3 fusion gene analysis can be requested as test M1.6. This consists of NGS structural variant analysis.
  • DPYD testing is undertaken on constitutional (germline) DNA (M1.7).
  • For constitutional testing, 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. No RoD form is required for DPYD mutation testing.
  • For DNA-based tests (germline DPYD, Lynch syndrome or polyposis tests), and EDTA blood sample is required. Please refer to your local Genomics Laboratory Hub (GLH) for details of test request forms and where to send samples.
  • Depending on the details you provide and the test that is chosen, 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):
  • WGS of solid tumours where the patient has exhausted all standards of care testing and treatment is requested as code M232. WGS requires access to a fresh tumour sample and a matched EDTA blood sample for germline testing. An RoD form must be completed for this investigation. Please discuss with your local GLH before submitting samples for WGS to confirm the local test pathway details.


For clinicians

For patients

Tagged: Colorectal cancer

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  • Last reviewed: 03/05/2022
  • Next review due: 03/05/2023
  • Authors: Dr Alison Berner
  • Reviewers: Dr Ellen Copson, Dr Amy Frost, Dr Terri McVeigh