Loss of MLH1 Expression due
to Promoter Methylation in
Cases Referred for HNPCC
Lorraine Hawkes
Kennedy Galton Centre
Hereditary Non-Polyposis
Colon Cancer


Inherited pre-disposition to colorectal cancer as well as
others - endometrial, ovarian, stomach and small bowel
Mutations in Mismatch Repair genes – commonly MLH1,
MSH2, MSH6, PMS2

Tumours display Microsatellite Instability

Loss of protein seen on Immunohistochemistry (IHC)

Cancer develops through “adenoma carcinoma
sequence”
CpG Island Methylator
Phenotype (CIMP)




Subset of tumours displaying MSI with wide spread
methylation – Toyota et al 1999
Commonly MLH1 expression lost due to promoter
hypermethylation (seen on IHC)
CIMP is seen in ~15% sporadic colorectal cancers
Cancer develops through “serrated pathway” and has a
high association with BRAF V600E mutation
CpG Island Methylator
Phenotype (CIMP)
Categories of tumour:
1.
2.
3.
4.
5.
CIMP
CIMP
CIMP
CIMP
CIMP
high/MSI-H/BRAF V600E
high/MSI-L/BRAF mutation
low/MSS or MSI-L/KRAS mutation
negative/MSS
negative/MSI-H (HNPCC)
Some overlap may be seen
Aims


To set up a routine service to test for
promoter hypermethylation of MLH1 in
tumours with loss of protein
Retrospectively test tumour samples from
HNPCC negative cases with loss of MLH1
and MSI
Testing Strategy
Tumour
sample
IHC
Positive –
Report
Loss of MLH1
Loss of MSH2
or MSH6
Normal
Result
Methylation
studies
Mutation
screen
MSI and
reporting
Negative –
Mutation
screen
Methods

ME001B Tumour Suppressor 1 MS-MLPA Kit
(MRC Holland)
- 41 probes (26 testing for methylation – 2 for MLH1)

ME011 Mismatch Repair kit (released part
way through study)
- 32 probes (21 testing for methylation – 5 for MLH1)
Methylation Specific
MLPA (MS-MLPA)
Example data
Example data
Results


45 tumour samples tested
35 patient blood samples tested (where available)
Tumour Results:
No. of
patients
MLH1 not
methylated
Other genes
methylated,
not MLH1
MLH1 only
methylated
MLH1 and
other genes
methylated
Fail
10
(22%)
3
(7%)
9
(20%)
15
(33%)
8
(18%)
Results



Hypermethylation not found in any patient blood
samples
1 sample with low level methylation
3 of normal patients later found to have variants:
- c.2041G>A, p.Ala681Thr (confirmed pathogenic)
- c.694G>A p.Gly232Arg (unclassified variant)
- c.199G>A. p.Gly67Arg (unclassified variant)

One patient with two different tumours
- colon is normal
- gall bladder tumour hypermethylated
Normal methylation results
and no MLH1 mutation


Mutation in non-coding region of MLH1?
Change of protein/mRNA expression without structural
change to the gene?

HNPCC mutation missed by dHPLC?

Non CIMP, non-HNPCC pathway?
Further Work




Set up testing for BRAF V600E mutation
Investigate promoter methylation in patients with
loss of MSH6 or MSH2 and no mutation
Patients negative for hypermethylation now to have
full gene sequencing
Work on quantifying methylation using Coffalyser
programme
Conclusions


MS-MLPA is a useful pre-screen for tumour samples
referred for HNPCC, with loss of MLH1
 Reliable
 Cost efficient
A significant proportion of samples referred for
HNPCC testing are found have loss of MLH1 due to
promoter methylation
Acknowledgements




Sam Butler
Stewart Payne
Lindsey Sutherland
Marco Novelli (Histopathology, UCL)