1. Dr Luke Farmery Luke.farmery@nhs.net @LFarmery
TERA
Repairing the mismatched diagnostic pathway for colorectal Lynch Syndrome
Dr Luke Farmery
@LFarmery

2. Contents Introduction What is Lynch Syndrome?
How does Lynch Syndrome cause cancer?
Why do we need to know?
What problems face us knowing?
Further problems…
How are we currently doing?
How should we move forwards?

3. Introduction
Colorectal cancer (CRC) is the 4th most common cancer in the U.K.
colorectal resections a year, per department
Lynch Syndrome: 3-5% (estimate: 15 people/year)
Current diagnostic yield: 0.16% / year. Or 0.74% over
We must be missing patients
Figure reference:

4. But what is Lynch Syndrome?

5. Background (1) Lynch Syndrome (LS) - autosomal dominant
Predisposes to early onset cancer with an 80% lifetime risk of coloretctal cancer (CRC)
Most common form of inherited (CRC)
Pathogenic variations in DNA mismatch repair (MMR) genes.
Causing microsatellite instability (MSI).
Table 1: The MMR genes, their protein products and how commonly each gene shows pathogenic variation in LS.
Gene ID
Protein name
Comments
% of LS cases (1, 2)
MLH1
MutL homolog 1
Forms a heterodimer with protein PMS2
42%
PMS2
Mismatch repair endonuclease
Forms a heterodimer with protein MLH1. 7%
MSH2
MutS homolog 2
Forms a heterodimer with protein MSH6. Pathogenic variation in EPCAM can cause epigenetic inactivation of MSH2.
33% (3% subset due to EPCAM)
MSH6
MutS homolog 6
Forms a heterodimer with protein MSH2
18%
Lynch Syndrome (LS) is an autosomal dominant condition caused by germline pathogenic
variations in the deoxyribonucleic acid (DNA) mismatch repair (MMR) genes or their upstream
regulators, such as EPCAM (Epithelial Cell Adhesion Molecule). The four MMR proteins
function in two pairs to correct errors in DNA replication (Table 1). Defective MMR (dMMR)
proteins will allow errors to accumulate in the form of di- or trinucleotide repeats, leading to
a state of hypermutability know as microsatellite instability (MSI).

6. Lynch Syndrome Cancers Urinary Tract / Bladder Cancer
Background (2)
LS predisposes to other types of cancer too.
This table displays just some of them (it’s a work in progress)
Table 2: The Lynch Syndrome cancers and their lifetime risk and mean age at diagnosis.(5)
Lynch Syndrome Cancers
Lifetime risk
Mean age at diagnosis
Colorectal Cancer
30-90%
44-61
Endometrial Cancer
25-60% (females)
48-62
Gastric Cancer
6-13% 56
Ovarian Cancer
4-12% (females)
42.5
Urinary Tract / Bladder Cancer
Low (increased vs. general popn)
Unavailable
Small bowel Cancer
Glioblastoma

7. How exactly does this happen?

8. Molecular Background (1): Mismatch Repair

9. Molecular Background (2): Where dMMR becomes a problem (MSI)
During DNA replication, slippage can occur.
Short tandem repeats, known as microsatellites are highly repetitive and therefore difficult to replicate accurately.
They accumulate mismatch errors, which are not repaired and lead to genomic instability.

10. Molecular Background (3): Two Hit Hypothesis
One allele is lost, and a second hit is acquired to “trigger” disease. (LOH)
Biallelic mutations lead to a distinctive syndrome: childhood cancer syndrome

11. But why is this important to us?

12. The importance of diagnosing:
Relatively common
Guides management decisions
Affects young people
Early detection enables cost-effective colonscopy programs, improving patient outcomes
Avoids expensive surgery and chemotherapy if detected early
Inherited; for every proband identified, there could be an affected family member
Sporadic; could influence chemotherapy
NICE Concordance (which I will come on to)

13. So what is the problem?

14. Mismatch Repair & Microsatellites are not specific
DNA sequencing, the “gold standard” of diagnosing LS is expensive.
IHC or MSI testing can be used as surrogate molecular markers, but are not specific, but are cost-effective.
Figure reference:

15. NICE recommended diagnostic pathway (Feb 2017)
Test all patients with CRC when first diagnosed. Use IHC or MSI on either tissue biopsy, resected tumour or polyp
Do an MSI test
Do a four panel IHC test (MLH1, MSH2, MSH6, PMS2)
No further testing
BRAF V600E test
Do an MLH1 promoter methylation test
Counsel the patient
Confirm Lynch Syndrome by genetic testing of germline DNA
(MSS)
MSI-L or MSH-H
MLH1 abnormal
All normal
BRAF mutation +ve
BRAF mutation -ve
MSH2, MSH6 or PMS2 abnormal
Test positive
Test negative

16. Don’t forget ethics:
Test all patients with CRC when first diagnosed. Use IHC or MSI on either tissue biopsy, resected tumour or polyp
Do an MSI test
Do a four panel IHC test (MLH1, MSH2, MSH6, PMS2)
No further testing
BRAF V600E test
Do an MLH1 promoter methylation test
Counsel the patient
Confirm Lynch Syndrome by genetic testing of germline DNA
(MSS)
MSI-L or MSH-H
MLH1 abnormal
All normal
BRAF mutation +ve
BRAF mutation -ve
MSH2, MSH6 or PMS2 abnormal
Test positive
Test negative

17. Further Problems Up front cost
Debate over long-term cost effectiveness
Availability of testing
Possible patient reluctance
Referral influx on already strained systems

18. So how are we doing?

19. Audit
The current practice of selective screening for microsatellite instability / mismatch repair status.
Possible Lynch Syndrome
Choice of Chemotherapy
Jan 2013 – Aug 2017
All cases of CRC, excluding patient duplicates

20. Method
Data for workload was acquired from John Siewruk in Pathology Systems.
Cases sent for molecular testing were identified using “Send Away” spread sheet.
Cases with molecular testing had available results reviewed.
Calculations were performed in Microsoft Excel

21. Audit *All Figures subject to coding bias
21 cases a month, 252 cases a year.
*All Figures subject to coding bias

22. Current Diagnostic pathway (Jan 2015 – Aug 2017)
Jan 2015 – Aug 2017 CRC cases (n≈672)
Current Diagnostic pathway (Jan 2015 – Aug 2017)
No MMR testing
MMR testing (n=89, 13%)
dMMR CRC (n=19)
No dMMR (n=67)
MMR results unavailable
(n =3)
Loss of MLH1 & PMS2 (n=14)
Isolated loss of PMS2 (n=1)
Loss of MSH6 & MSH2 (n=3)
Loss of PMS2 & MSH6 (n=1)
BRAF Mutation -ve
BRAF Testing
Only 13% cancers were tested, versus 100% recommended by recent NICE guidance. This figure is a guideline only and its accuracy is reduced by the number of CRC cases not taking into account metastasis or recurrence.
Only 2.9% of CRC cancers were detected as dMMR, versus the 20% reported in the literature.
MMR results were unavailable in three cases. MSI instead of MMR IHC, which was returned as MSI-L (n = 1) No addendum on system (n =1) Tissue block sent was inadequate (n = 1)
Loss of MLH1 & PMS2 is most common, in keeping with the literature. The paired relationship is to be expected based on their molecular relationship.
Most cases of MLH1 & PMS2 loss will be epigenetic (sporadic) and should be tested for BRAF mutation. All cases were requested (100%). One case was unavailable (no addendum available on system). 7 cases were positive, 6 cases were negative.
NICE guidance recommends negative BRAF tumours be tested with MLH1 methylation testing. All cases were requested (100%). Testing failed in 2 cases. Addendum’s were unavailable in 3 cases. MLH1 methylation was negative in one case, and further germline sequencing was recommended.
Overall, 7 cases lacked molecular report addendums. 5 cases had evidence raising the possibility of Lynch Syndrome. No germline sequencing data is available.
U/A (n=1)
+ve (n=7)
-ve (n=6)
Partial MLH1 methylation
MLH1 Methylation +ve
(n=0)
MLH1 Methylation
–ve
(n=1)
Methylation testing unavailable (n=3)
Testing failed
(n=2)
Possible Lynch Syndrome (n=5)

23. Audit 89 cases were screened, 67 were not dMMR, 19 dMMR
Our rate of insufficient results is too high.

24. Under diagnosis
Confirmed dMMR cases / all CRC cases = 19 / 1183 = 1.6% (expected 20%)
Possible Lynch Syndrome cases / all CRC cases = 5 / 1183 = 0.4% (expected 3-5%)

25. What tumour characteristics were common?
Low powered, but a bit of fun.

26. Characteristics (1)

27. Characteristics (2)

28. Characteristics (3)

29. Characteristics (4)

30. Problems with the audit
Coding error bias
Sendaway bias
Unable to complete the loop due to no access to “gold standard” sequencing results
Low statistical power

31. Problems identified by audit
We are missing cases of Lynch Syndrome
Incidence: 3-5%
Possible cases identified : 0.74%
We do not get access to all molecular results
This could impact MDT decisions

32. Proposal: repairing the mismatched diagnostic pathway for colorectal Lynch Syndrome
A service evaluation project to determine whether screening all initial colorectal cancer resections for dMMR can increase our detection rate of possible Lynch Syndrome.
Also, informing cost-effectiveness at a regional level.

33. References
1. FC DAS, Wernhoff P, Dominguez-Barrera C, Dominguez-Valentin M. Update on Hereditary Colorectal Cancer. Anticancer Res. 2016;36(9): Richman S. Deficient mismatch repair: Read all about it (Review). Int J Oncol. 2015;47(4): UK CR. Bowel cancer statistics 2017 [Available from: type/bowel-cancer - heading-Five. 4. Colling R, Church DN, Carmichael J, Murphy L, East J, Risby P, et al. Screening for Lynch syndrome and referral to clinical genetics by selective mismatch repair protein immunohistochemistry testing: an audit and cost analysis. J Clin Pathol. 2015;68(12): Kohlmann W GS. Lynch Syndrome. . In: In: Pagon RA AM, Ardinger HH, et al., editors., editor. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; Feb 5 [Updated 2014 May 22] Excellence NIoC. Molecular testing strategies for Lynch syndrome in people with colorectal cancer [Available from: 7. Snowsill T, Huxley N, Hoyle M, Jones-Hughes T, Coelho H, Cooper C, et al. A systematic review and economic evaluation of diagnostic strategies for Lynch syndrome. Health Technol Assess. 2014;18(58): Warrier SK, Yeung JM, Lynch AC, Heriot AG. Managing young colorectal cancer: a UK and Irish perspective. World J Surg. 2014;38(7): Alex AK, Siqueira S, Coudry R, Santos J, Alves M, Hoff PM, et al. Response to Chemotherapy and Prognosis in Metastatic Colorectal Cancer With DNA Deficient Mismatch Repair. Clin Colorectal Cancer Brennan B, Hemmings CT, Clark I, Yip D, Fadia M, Taupin DR. Universal molecular screening does not effectively detect Lynch syndrome in clinical practice. Therapeutic Advances in Gastroenterology. 2017;10(4): Bombard Y, Rozmovits L, Sorvari A, Daly C, Carroll JC, Kennedy E, et al. Universal tumor screening for Lynch syndrome: health-care providers' perspectives. Genet Med. 19. United States2017. p