Lynch Syndrome

Question 1

What is Lynch Syndrome?

Answer 1

• previously known as hereditary non-polyposis colorectal cancer (HNPCC)
• caused by a germline mutation in one of several Mismatch Repair Genes
• results in the patient having an increased risk of developing cancer over their lifetime with a quotable risk for colorectal cancer of 80%!
• for endometrial cancer in women it is close to 50%.

Question 2

What do mismatch repair genes do?
What happens when they don’t work?
Which MMR genes are involved in Lynch?

Answer 2

• recognises and repairs errors in our DNA such as those that occur during replication
• errors go unrecognised and are free to replicate, as these errors accumulate over time this is what causes cancer
• MLH1, MSH2, MSH6 and PMS2

Question 3

Which genes account for the majority of Lynch Syndrome at the moment?
What is the disease process?

Answer 3

MLH1 and MSH2

‘First hit’ - germline mutation followed by
‘second hit’ - somatic loss of second copy

Question 4

What is an important hallmark of MMR deficient tumour such as one you would see in Lynch syndrome?
What else is it seen in?

Answer 4

Microsatellite instability.

Seen in 15% of sporadic colorectal cancer (but in this case the underlying cause is biallelic hypermethylation of MLH1)

Question 6

According to the most recent BPG for Lynch Syndrome which are the main genes that should be tested?

Answer 6

MLH1
MSH2
MSH6
PMS2

Testing of other genes not currently recommended.

Question 7

What are the Lynch related cancers?

Answer 7

Colorectal
Endometrial 
Small intestine
Gastric 
Ovarian
Bladder
Prostate
Breast
Brain

Question 8

What is the likelihood of a colorectal cancer with MSI being Lynch Syn?

Answer 8

Age dependant!
If very young, chances are high e.g. 92% at 35yrs.
Much lower as you get older e.g. only 23% at age 70yrs.

Question 9

Before the new guidelines were released, what did clinicians use to identify Lynch patients?

Answer 9

The Amsterdam and Bethesda guidelines.
Primarily designed for research but didn’t work well in clinical setting. 
3-2-1 criteria:
>3 family members with cancer
>2 successive generations
>1 diagnosed under 50yrs

Question 10

Why is immunohistochemistry (IHC) useful in Lynch Syndrome? Why is it better than MSI?

Answer 10

• the gene which has a germline mutation will usually show loss of staining
• e.g. MLH1 loss by IHC would be consistent with Lynch
• again, issue is that loss of MLH1 would also be seen in a sporadic colorectal cancer
• as the proteins heterodimerise you can see loss of MLH1 and PMS2

Question 11

What is the basic concept on IHC?

Answer 11

• tumour specimen can be investigated by IHC
• fluorescently labelled antibodies are applied to tumour which bind to their respective antigen
• in this case the antigen is the MLH1 protein
• the antibody is specific for the MLH1 antigen
• absence of binding suggests an issue with MLH1 and will often be accompanied by loss of PMS2

Question 12

Why is microsatellite instability (MSI) testing useful in Lynch?

Answer 12

Microsatellite = sequence of repetitive DNA (2-6 base repeats) spaced throughout the genome

• MSI commonly seen in Lynch Syn
• these regions are prone to accumulation of mutations, mainly due to DNA polymerase slippage,
• the correction of these errors is the responsibility of the MMR genes so when a defect is present in an MMR gene these errors accumulate and instability of the microsetellite is the result

Question 13

Why is IHC preferred over MSI with respect to our Lynch service?

Answer 13

MSI will tell you if there is any instability in the tumour but can’t direct testing as to which gene.
IHC will give you a clue as to which gene is involved and potentially avoid an unnecessary test e.g. if MLH1 loss then would send for MLH1 hypermethylation test however if MSH2 or MSH6 lost there wouldn’t be any point!

Question 14

How did you know which region of the promoter to interrogate?

Answer 14

• paper by Deng et al
• they demonstrated an association between methylation of ‘region C’ of the MLH1 promoter and silencing of the MLH1 gene
• this paper is mentioned in BPG

Question 15

Are mutations of BRAF associated with LS? Is BRAF testing compulsory?

Answer 15

LS is usually negative for BRAF V600E but rare cases of BRAF mutation have been reported.
BPG don’t say that BRAF testing is essential.

Question 16

What tissue should be tested for MLH1 hypermeth? Why?

Answer 16

Both tumour tissue (minimum 20% neoplastic cell content) and normal tissue should be tested.

There are rare cases of constitutional hypermthylation of MLH1 referenced in the literature so BPG state that either normal tissue or a blood sample should be tested for completeness.

Question 17

What is the cause of 15% of sporadic colorectal cancer?

Answer 17

Biallelic hypermethylation of MLH1.

Question 18

How would you report:

1. a hypermethylated tumour sample with ‘normal’ blood?
2. Hypemethylated tumour and hypermethylated blood sample
3. Normal/unmethylated tumour.

Answer 18

1. Tumour more likely to be sporadic in origin.
2. Evidence of constitutional hypermethylation of MLH1, rare reports in the literature and quote paper. Refer to clinical where they can discuss family testing.
3. This result makes it more likely than this patient tumour could have arisen due to underlying Lynch Syndrome, refer to clinical genetics for further testing.

Question 19

What is the purpose of the MLH1 pilot?

Answer 19

Allow us to find our feet without the pressure of 10 day turn around.

Figure out how to slot this into our workload in the labs and allow staff to get trained without too much pressure.

Question 20

How many people are estimated in the UK to have Lynch Syndrome?

Answer 20

Approx 175,000 and a large proportion are unaware that they have it.

Question 21

Why is it important we identify these patients?

Answer 21

As it is inherited and is dominant, identifying one patient with Lynch Syndrome could unlock a diagnosis for an entire family very quickly!

A diagnosis of Lynch Syndrome will mean a family has access to relevant screening programmes such as bowel screening which could ultimately save their life!

Lynch syndrome is approved for PGD and therefore giving a family a diagnosis can give a family the power to make decisions about their reproductive options.

Question 22

What was important with regards to consent of patient samples for this project?

Answer 22

Informed consent;
- assumed as they have been recruited to testing through Clinical Genetics

Consent for DNA storage;

• checked all referral cards on LIMS that they had given their consent.
• furthermore, checked that there were no contact logs as if a patient had withdrawn their consent at any point after having the sample taken their would be a note on LIMS.

Anonymised;
- all sample data was anonymised and just Lab No. were used.

Samples;
- ensured I didn’t completely use up any patient samples incase any testing was required. However all samples were sections cut from a histology block which is likely still held by the pathology lab. Furthermore, all samples were from several years ago as samples now go straight to Manchester rather than coming via the laboratory.