Colorectal cancer - Lynch

Question 1

Provide a brief breakdown of the causes of colorectal cancer

Answer 1

~85% sporadic vs. ~15% = established familial genetic syndrome:

• Lynch syndrome (3-5%)
• FAP, PJS, PTEN (<1%)
• Unspecified familial cancer (~10%)

Question 2

What did Lynch syndrome used to be known as?

Answer 2

Hereditary non-polyposis colorectal cancer (HNPCC)

Question 3

What is the mode of inheritance of Lynch syndrome?

Answer 3

Autosomal dominant

Question 4

What are the basic genetics of Lynch syndrome?

Answer 4

Germline mutation in mismatch repair genes (MLH1, MSH2, MSH6, PM2, EPCAM).

Followed by secondary somatic loss of remaining copy of the gene (LoH).

Question 5

Do mutations in the mismatch repair genes only cause Lynch/colorectal cancer?

Answer 5

No - they predispose to a wider spectrum of cancers

E.g. MLH1 = GI cancers, MSH2 = widest range, MSH6 = bladder/renal

Question 6

What is the mean age of onset for Lynch syndrome?

Answer 6

~45 years (higher in MSH6 cases)

Question 7

Which genes cause a higher percentage of Lynch?

Answer 7

80-90% = MLH1 (chr3) / MSH2 (chr2)

The rest:
7-10% = MSH6 (chr2)
<1% = PMS1/2 (chr7)
3% = EPCAM 3’ dels (chr2)

Additional causes:
Germline methylation of MLH1 promoter
10Mb inversion on chr2p (disrupts MSH2)

Question 8

What are the two sets of clinical criteria that can be applied to select a patient for Lynch syndrome testing?

Answer 8

Amsterdam criteria
- Developed to identify LS for research studies and to distinguish HNPCC vs non-HNPCC

Bethesda guidelines
- Developed to identify patients with CRC who should be tested for LS
- More sensitive but less specific - helps those with smaller families

Question 9

What is the current testing strategy for Lynch syndrome?

Answer 9

Tumour samples first tested for MSI or IHC MMR 4 gene panel (MLH1/MSH2/MSH6/PMS2) to identify DNA MMR-deficient tumours and guide further sequential testing.

Negative MSI / normal IHC > no further testing.

Abnormal MSH2/MSH6/PMS2 > confirm LS by genetic testing of germline DNA.

Positive MSI / abnormal MLH1 > BRAF V600E testing.
- If positive > no further testing
- If negative > MLH1 promoter hypermethylation testing
- If positive > no further testing
- If negative > confirm LS by genetic testing of germline DNA

Question 10

Provide some detail around IHC testing in Lynch syndrome

Answer 10

Tumour blocks/slides are assessed by histopath labs for presence/absence of MLH1/MSH2/MSH6/PMS2 proteins using commercial antibodies.

IHC ~95% sensitive for DNA MMR deficiency.

Often see concurrent loss of MSH2/MSH6 or MLH1/PMS2 as these form heterodimers in MMR pathway.

Question 11

Provide some detail around MSI testing in Lynch syndrome

Answer 11

Genetic instability characterised by length alterations to microsatellites – occurs in majority of LS-associated cancers, plus small proportion of sporadic cancers.

Lab assay commonly tests panel of 5 mononuc markers with 3 outcomes:

• Microsatellite stable (MSS) = not supportive of MMR gene defect.
• 1 unstable marker = insufficient for LS-associated instability but may be significant so further investigation.
• 2 or more altered mononuc markers = high-level MSI (MSI-H) = increased risk tumour due to MMR gene defect.

Note: tissue mosaicism can occur.

Question 12

Provide some detail around BRAF V600E testing in Lynch syndrome

Answer 12

BRAF mutations associated with MLH1 promoter hypermethylation (tumour only, not germline) and indicate sporadic cancer.

V600E mutation commonly occurs in non MSI-high tumours.

Used as screening method to avoid unnecessary MMR gene screening.

Question 13

Provide some detail around MLH1 promoter hypermethylation testing in Lynch syndrome

Answer 13

MLH1 promoter hypermethylation has been shown in high proportion of sporadic cancers (~15%) - results in absence of MLH1 protein on IHC.

Hypermeth = somatic change in tumour, very rarely seen in germline DNA as heritable germline mutation. If available, blood DNA can be tested for germline hypermethylation at same time as the tumour.

MS-MLPA kit available - detects abnormal methylation at 5 sites in the hMLH1 promoter, 4 sites in the MSH2 promoter (used to confirm 3’ EPCAM deletions) and other MMR promoter regions.

Occurs in testing pathway after BRAF V600E testing. If no abnormal methylation then should be tested for a germline MLH1 mutation.

Question 14

Provide some detail around germline MMR gene testing in Lynch syndrome

Answer 14

Gene panel approach used in multiple labs for sequence/dosage analysis of MMR genes.

PMS2 challenging due to highly homologous pseudogene. The 3’ end of gene is non-amenable to NGS analysis and requires long-range nested PCR to amplify PMS2 only.

Large rearrangements can be detected by MLPA, NGS copy number analysis (depending on NGS method/ analysis pipeline) or targeted array.

Question 15

What are the four main treatment categories for CRC/Lynch syndrome?

Answer 15

Treatment of manifestations
- Full colectomy

Prevention of 1ary manifestations
- Prophylactic removal of colon not generally recommended as routine colonoscopy is a good preventative measure.
- Prophylactic removal of uterus and ovaries can be considered after childbearing is complete.

Surveillance
- Colonoscopy with removal of pre-cancerous polyps every 1-2yrs from aged 20-25 (or 10yrs before earliest age of diagnosis in family)

Chemoprevention
- Aspirin is recommended in people with high risk of CRC

Question 16

What is the risk for presymptomatic patients found to carry the causative familial Lynch variant?

Answer 16

The risks of developing cancer depend on the gene involved and the gender of the patient.

MLH1 has the highest risk of developing CRC by 70yrs (78% male, 57% female).

MSH2 has the highest risk of developing a LS-associated cancer by 70yrs (77% both genders).