4. Colorectal Cancer - HNPCC, FAP, MUTYH

Question 1

What causes Lynch syndrome?

Answer 1

Germline mutations in mismatch repair genes - MLH1, MSH2, MSH6, PMS2

Followed by secondary somatic loss of remaining copy

Question 2

What cancers are caused by HNPCC?

Answer 2

CRC, endometrial carcinoma, small intestine, pancreatic, gastric, ovariant

Question 3

What’s the contribution of each gene to HNPCC?

Answer 3

40% MLH1
40% MSH2
10% MSH6
7% PMS2
3% deletion of 3’ of EPCAM - creates EPCAM-MSH2 fusion transcript with hypermethylated MSH2 promoter –> reduced expression

Question 3

What is the purpose of IHC?

Answer 3

Test for presence of MLH1, MSH2, MSH6, PMS2 with antibodies

Often shows concurrent loss of MSH2 & MSH6 or MLH1 & PMS2 - occur as heterodimers in MMR pathway

Question 4

What is the purpose of MSI testing? How is it tested?

Answer 4

Most Lynch syndrome cases show length alterations of microsatellites due to MMR

Test 5 mononucleotide markers - 2 or more altered markers = MSI-H

MSI-H = increased risk tumour due to MMR gene defect

Question 4

What tests are carried out to identify sporadic cases of CRC?

Answer 4

1. MLH1 promoter methylation - in high proportion of sporadic cases, rarely in germline. Results in loss of MLH1 on IHC
2. BRAF mutations associated with MLH1 hypermethylation, indicates sporadic

V600E commonly seen in non-MSI-H tumours

Question 5

Why is it necessary to determine the size of EPCAM deletions?

Answer 5

Higher risk of extracolonic cancers if MSH2 coding sequence included (similar to MSH2 mutations)

Lower risk if deletion causes promoter hypermethylation

Question 6

Why do mismatches occur?

Answer 6

Incorporation of mismatched bases and DNA slippage during replication causes insertion/deletion loops

Failure to repair –> missense and frameshift

Question 7

What is the role of the different MMR complexes?

Answer 7

MSH2-MSH6 (MutS-alpha) - repairs single base mismatches and mononucleotide repeats

MSH2-MSH3 (MutS-beta) - repairs dinucleotide repeat errors

MLH1-PMS2 - coordinates the other DNA-repair protein effectors to repair mismatches arising during DNA replication

Question 8

What is the treatment for HNPCC?

Answer 8

Full colectomy, routine colonoscopy as preventative measure, removal of pre-cancerous polyps, chemoprevention

Question 9

What are the considerations for predictive testing in HNPCC?

Answer 9

Risk depends on gene and gender

Highest risk in males with MLH1 and MSH2 variants

Question 10

What are the first tests in the Lynch syndrome pathway?

What if the result if negative/positive?

Answer 10

MSI or IHC

Normal IHC or MSI - no further testing for Lynch, suggests it’s sporadic - do BRAF, RAS

Positive IHC for MSH2, MSH6, PMS2 - do germline panel

Positive IHC for MLH1 - do BRAF V600E

Positive MSI - do BRAF V600E

Question 11

What testing is indicated following BRAF V600E?

Why?

Answer 11

BRAF V600E positive –> no further testing as V600E associated with sporadic CRC

Negative –> MLH1 promoter hypermethylation

Question 12

What testing is indicated following MLH1 promoter hypermethylation?

Answer 12

Positive –> no further testing as MLH1 hypermeth associated with sporadic CRC

Negative –> germline Lynch syndrome testing

Question 13

What is the clinical phenotype of FAP?

Answer 13

Familial adenomatus polyposis

Development of 1000s of adenomatous colonic polyps in 2nd decade

100% risk of CRC if untreated, ~10 years after polyps

Question 14

What gene causes FAP? What is its role?

Answer 14

APC
TSG involved in Wnt signalling pathway
Regulates B-canetin degradation - accumulates in absence of APC –> activates TFs –> constitutive activation of the canonical WNT signaling pathway
Increased proliferation, decreased apoptosis and differentiation

Question 15

What is attenuated FAP and what causes it?

Answer 15

Fewer polyps, due to APC mutations in exon 9 and 5’/3’ UTRs

Question 16

What is MUTYH-associated polyposis?

Answer 16

AR, similar presentation to AFAP

Question 17

What is the role of MUTYH?

Answer 17

Base excision repair gene

Question 18

When is MUTYH testing indicated?

Answer 18

Test alongside APC

Usually patients with 10-100 polyps, no APC mutation + AR family history

Question 19

What is MSI?

Answer 19

Microsatellite instability

Change in length of of microsatellite due to errors in DNA replication that aren’t corrected due to deficient MMR

Question 20

What does MSI testing inform, other than MMR deficiency?

Answer 20

Guides therapy: MSI-H = better response to immunotherapy rather than 5-FU chemo

Question 21

How is MLH1 promoter methylation tested for?

Answer 21

Bisulphate treatment of DNA - converts unmethylated cytosines at CpG islands to uracil by deamination

PCR of promoter region then analyse of degree of methylation at five CpG sites using pyrosequencing

Question 22

When is somatic Lynch syndrome testing indicated?

Answer 22

Tumours with MMR loss on IHC
BRAF codon 600 normal
MLH1 promoter normal
No germline MMR mutations

Question 23

Why is the presence of MSI not enough to diagnose Lynch syndrome?

Answer 23

10-15% of sporadic CRC exhibit MSI