Advances in genetic technologies result in improved diagnosis of mismatch repair deficiency in colorectal and endometrial cancers

Abstract

Background
Testing cancers for mismatch repair deficiency (dMMR) by immunohistochemistry (IHC) is a quick and inexpensive means of triaging individuals for germline Lynch syndrome testing. The aim of this study was to evaluate tumour dMMR and the prevalence of Lynch syndrome in patients referred to the Manchester Centre for Genomic Medicine, which serves a population of 5.6 million.

Methods
Tumour testing used IHC for MMR proteins with targeted BRAF and MLH1 promotor methylation testing followed by germline mutation and somatic testing as appropriate.

Results
In total, 3694 index tumours were tested by IHC (2204 colorectal cancers (CRCs), 739 endometrial cancers (ECs) and 761 other), of which 672/3694 (18.2%) had protein loss, including 348 (9.4%) with MLH1 loss. MLH1 loss was significantly higher for 739 ECs (15%) vs 2204 CRCs (10%) (p=0.0003) and was explained entirely by higher rates of somatic MLH1 promoter hypermethylation (87% vs 41%, p<0.0001). Overall, 65/134 (48.5%) patients with MLH1 loss and no MLH1 hypermethylation or BRAF c.1799T>A had constitutional MLH1 pathogenic variants. Of 456 patients with tumours showing loss of MSH2/MSH6, 216 (47.3%) had germline pathogenic variants in either gene. Isolated PMS2 loss was most suggestive of a germline MMR variant in 19/26 (73%). Of those with no germline pathogenic variant, somatic testing identified likely causal variants in 34/48 (71%) with MLH1 loss and in MSH2/MSH6 in 40/47 (85%) with MSH2/MSH6 loss.

Conclusions
Reflex testing of EC/CRC leads to uncertain diagnoses in many individuals with dMMR following IHC but without germline pathogenic variants or MLH1 hypermethylation. Tumour mutation testing is effective at decreasing this by identifying somatic dMMR in >75% of cases.

Full text

1
Evans DG, etal. J Med Genet 2021;0:1–7. doi:10.1136/jmedgenet-2020-107542
Original research
Advances in genetic technologies result in improved
diagnosis of mismatch repair deficiency in colorectal
and endometrialcancers
D Gareth Evans ,1,2 Fiona Lalloo,2 Neil AJ Ryan,3,4 Naomi Bowers,2 Kate Green,2
Emma R Woodward ,1,2 Tara Clancy,2 James Bolton,5 Rhona J McVey,5
Andrew J Wallace,2 Katy Newton,6 James Hill,6 Raymond McMahon,5
Emma J Crosbie 3,4
Cancer genetics
To cite: Evans DG, Lalloo F,
Ryan NAJ, etal. J Med Genet
Epub ahead of print: [please
include Day Month Year].
doi:10.1136/
jmedgenet-2020-107542
►Additional material is
published online only. To view,
please visit the journal online
(http:// dx. doi. org/ 10. 1136/
jmedgenet- 2020- 107542).
For numbered affiliations see
end of article.
Correspondence to
Professor Emma J Crosbie,
Division of Cancer Sciences,
The University of Manchester,
Manchester M13 9WL, UK;
emma. crosbie@ manchester.
ac. uk
Received 25 October 2020
Revised 17 December 2020
Accepted 23 December 2020
© Author(s) (or their
employer(s)) 2021. Re- use
permitted under CC BY.
Published by BMJ.
ABSTRACT
Background Testing cancers for mismatch repair
deficiency (dMMR) by immunohistochemistry (IHC) is a
quick and inexpensive means of triaging individuals for
germline Lynch syndrome testing. The aim of this study
was to evaluate tumour dMMR and the prevalence of
Lynch syndrome in patients referred to the Manchester
Centre for Genomic Medicine, which serves a population
of 5.6 million.
Methods Tumour testing used IHC for MMR proteins
with targeted BRAF and MLH1 promotor methylation
testing followed by germline mutation and somatic
testing as appropriate.
Results In total, 3694 index tumours were tested by
IHC (2204 colorectal cancers (CRCs), 739 endometrial
cancers (ECs) and 761 other), of which 672/3694
(18.2%) had protein loss, including 348 (9.4%) with
MLH1 loss. MLH1 loss was significantly higher for 739
ECs (15%) vs 2204 CRCs (10%) (p=0.0003) and was
explained entirely by higher rates of somatic MLH1
promoter hypermethylation (87% vs 41%, p<0.0001).
Overall, 65/134 (48.5%) patients with MLH1 loss and
no MLH1 hypermethylation or BRAF c.1799T>A had
constitutional MLH1 pathogenic variants. Of 456 patients
with tumours showing loss of MSH2/MSH6, 216 (47.3%)
had germline pathogenic variants in either gene. Isolated
PMS2 loss was most suggestive of a germline MMR
variant in 19/26 (73%). Of those with no germline
pathogenic variant, somatic testing identified likely
causal variants in 34/48 (71%) with MLH1 loss and in
MSH2/MSH6 in 40/47 (85%) with MSH2/MSH6 loss.
Conclusions Reflex testing of EC/CRC leads to
uncertain diagnoses in many individuals with dMMR
following IHC but without germline pathogenic variants
or MLH1 hypermethylation. Tumour mutation testing is
effective at decreasing this by identifying somatic dMMR
in >75% of cases.
INTRODUCTION
Colorectal cancer (CRC) and endometrial cancer
(EC) are two of the most common malignancies in
humans. They are both characterised by having a
relatively high rate of mismatch repair deficiency
(dMMR) and similar germline rates (3%) of
pathogenic variants in MMR genes.1 CRC is the
third most common cancer in men and women.2
EC is the most common gynaecological cancer in
high- income countries, and its incidence is rising
rapidly.3 Although environmental causes such
as diet (CRC) and obesity (particularly EC)4 and
decreased parity (EC) are major contributors to
incidence, a significant minority of both cancers
(3%) are caused by Lynch syndrome (LS).1 5 LS is
an inherited susceptibility to malignancies associ-
ated with dMMR. Around 1 in 280 of the general
population is heterozygous for a pathogenic variant
in an MMR gene, MLH1, MSH2 (including dele-
tions of EPCAM), MSH6 or PMS2 (path_MMR),
the vast majority of whom are undiagnosed.5–8
Path_MMR heterozygotes have an averaged risk to
age 70 years of EC, CRC and ovarian cancer (OC)
of 35%, 46% and 11%, respectively,9 although
these vary by gene with lower risks of PMS2. These
likelihoods are substantially higher than those of
the general population for EC (3%), CRC (4%)
and OC (1%).10
Since the discovery of the MMR genes in 1993–
1994, germline testing has been targeted towards
those most likely to have an inherited pathogenic
variant. The Amsterdam criteria were developed
in 1991,11 primarily to select high- risk families,
therefore requiring a substantial family history of
CRC. While 45%–60% of index cases in families
fulfilling criteria are path_MMR variant carriers,12
the criteria have low sensitivity.13 14 The addition
of other characteristic tumours of LS, such as EC,
OC and urothelial cancers,15 add little to either the
detection rate12 or sensitivity.13 14 The less restric-
tive Bethesda guidelines were developed in 1997,16
which improved sensitivity but resulted in many
more samples being tested without detection of
all path_MMR variants.13 14 17 18 More recently,
the concept of universal testing of CRC has gained
ground18–22 and is now recommended national
guidance in a number of countries for CRC.20 21
This is also gaining traction for EC23 and is now
recommended by the National Institute for Health
and Care Excellence in the UK.24
We have evaluated our prescreening strategy
with immunohistochemistry (IHC) in Lynch-
related cancers from 2000 to 2020 and, more
latterly, the impact of somatic next- generation
sequencing (NGS) of tumours in individuals
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2Evans DG, etal. J Med Genet 2021;0:1–7. doi:10.1136/jmedgenet-2020-107542
Cancer genetics
with protein loss on IHC in tumours but without a germline
path_MMR.
METHODS
Participants
Individuals referred to the regional genetics department in
Manchester with an LS- related cancer and concerns about the
possibility of LS provided consent for tumour and, if necessary,
germline analysis. The great majority of evaluated patients had
CRC or EC and were selected based on early age at diagnosis
or fulfilling Bethesda guidelines or Amsterdam criteria. Occa-
sional cases were tested as deceased first- degree relatives of clin-
ically unaffected index patients. In addition, 500 women from
the Proportion of Endometrial Tumours Associated with Lynch
Syndrome (PETALS) study with sequential EC were also included
(15/NW/0733).25 Generally, individuals fulfilling Amsterdam
criteria did not undergo prescreening and went straight to germ-
line path_MMR analysis.
The standard pathway for non- Amsterdam criteria tumours
was an initial test for dMMR using IHC of the MMR proteins.
If there was loss of MLH1, the samples were tested for
MLH1 promoter hypermethylation and the BRAF c.1799T>A
(p.Val600Glu) pathogenic variant. Positive results for either of
these are indicative of a somatic mutation of MLH1. All individ-
uals with MLH1 loss, samples and wild type for BRAF and nega-
tive for promoter hypermethylation, as well as all sole PMS2
or MSH2/MSH6 loss, underwent germline lymphocyte testing
where this was possible. BRAF c.1799T>A (p.Val600Glu) was
suspended for EC once it was known this screen was not sensi-
tive (0/23 tested were positive for c.1799T>A (p.Val600Glu).26
Immunohistochemistry
IHC for the four MMR proteins was performed in the MFT
clinical pathology laboratory using the automated Ventana
BenchMark ULTRA IHC⁄ISH staining module and the OptiView,
3′diaminobenzidine V.5 detection system (Ventana Co, USA)
according to standard clinical protocols.25 The proportion of
stained tumour epithelial component/intensity of staining was
scored by two expert independent observers using tumour
stroma as internal control and as described elsewhere.27 28 Only
tumours with complete loss of protein expression were reported
as dMMR (not those with patchy loss).
Methylation analysis
Reflex MLH1 methylation testing was performed on tumours
showing loss of MLH1 protein on IHC. Purified DNA was
amplified with bisulfite- specific primers in triplicate. A region of
the MLH1 promoter containing four CpG dinucleotides whose
methylation status is strongly correlated with MLH1 expression
was sequenced using a pyrosequencer (PSQ 96MA). Two inde-
pendent scientists interpreted the pyrograms. ‘Hypermethyla-
tion’ described >10% mean methylation across the four CpG
dinucleotides on two of three replicate analyses. A proportion
of MLH1 hypermethylation cases underwent reference standard
germline MMR sequencing to exclude coexisting path_MLH1
variants, usually when they had a significant family history. In
addition to methylation, analysis testing was carried out for the
BRAF c.1799T>A (p.Val600Glu) variant.
Germline analysis
DNA was extracted from 2 to 5 mL lymphocyte blood
(EDTA anticoagulant) using Chemagic DNA blood chemistry
(CMG-1097- D) on an automated PerkinElmer Chemagic 360
Magnetic Separation Module and a JANUS Integrator four- tip
Automated Liquid handling platform. DNA was eluted into 400
μL buffer. Extracted DNA samples were measured for DNA
yield, concentration and quality using a Nanodrop ND-8000
spectrophotometer. MMR genes MLH1, MSH2 and MSH6 were
amplified using long- range PCR followed by NGS using Illumina
SBS V.2 2×150 bp and Illumina MiSeq to analyse the coding
region, flanking sequences to ±15 bp and known splicing vari-
ants (minimum 100× coverage depth) of MLH1, MSH2 and
MSH6.25 Variant identification and calling was via an in- house
bioinformatic pipeline. Reported sequence changes and regions
with <100× coverage were retested via Sanger sequencing using
BigDye V.3.1. Copy number analysis to detect large genomic
rearrangements affecting the MMR genes was performed using
MLPA MRC- Holland probe mixes: P003- D1 MLH1/MSH2
and P072- C1 MSH6. Variant nomenclature followed Human
Genome Variation Society guidelines (http://www. hgvs. org/
vamomen) using reference sequences: LRG_216, t1(MLH1);
LRG_218, t1(MSH2); LRG_219, t1(MSH6). Exons were
numbered consecutively starting from exon 1 as the first trans-
lated exon for each probe mix. Cases with PMS2 protein loss,
normal MLH1 methylation and no path_MLH1/MSH2/MSH6
variant underwent path_PMS2 analysis at the regional specialist
Yorkshire and North East Genomic Laboratory which included
MLPA.
Figure 1 Study flowchart diagram. Note: germline testing was done
for MLH1, MSH2 and MSH6 in cases and PMS2 in select cases. Were a
path_MMR was detected in a gene not consistent with the IHC loss, this
is shown in brackets below the result. *The majority of these samples
were Amsterdam criteria II positive. #One sample had constitutional MLH1
hypermethylation. EC, endometrial cancer; CRC, colorectal cancer; IHC,
immunohistochemistry; MMR, mismatch repair; path_, pathogenic variant.
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Cancer genetics
Somatic tumour analysis
Tumour specimens were assessed by specialist pathologists. All
tissues were formalin- fixed and paraffin- embedded according to
local clinical protocols. Tissue blocks with the greatest tumour
content (>70%) were chosen for DNA extraction. Tumour
was either microdissected from 5×10 µm unstained sections or
cored from tissue blocks, depending on tumour content. Non-
malignant adjacent tissue was selected for comparative consti-
tutional microsatellite instability (MSI) analysis. MMR genes
MLH1, MSH2 and MSH6 were analysed as components of a
somatic panel including PTEN, TP53, APC, POLD1 and POLE
using a custom NGS approach based on a Qiagen GeneRead
amplicon based enrichment. PMS2 was not assessed due to the
difficulties with pseudogenes and high copy number variant rate.
Formal loss of heterozygosity (LOH) analysis was not part of the
initial panel but was introduced with microsatellite repeats after
12 months, but did not include LOH for PMS2.
Statistics
Differences between values were tested by a two- tailed Fisher’s
χ2 test.
RESULTS
A total of 3694 index cases aged 8–91 years at diagnosis had
a tumour (figure 1 and table 1) prescreened with tumour IHC
of MMR proteins with 672 (18%) showing loss of at least one
protein (table 2). CRC (n=2204 mean age 50.8 years) and EC
(n=739 mean age=61 years) were by far the most frequently
tested, and further analysis was largely confined to those two
tumour types (online supplemental figure 1A,B). However,
we also tested 761 other cancers and benign tumours (mean
age=50, table 1).
A total of 211 patients with CRC underwent germline MMR
testing without an IHC prescreen with 123 (58%) demonstrating
Table 1 Tumour samples tested, age at diagnosis, IHC loss and path_MMR rate
IHC (n) Age range (median) IHC loss % IHC loss Path_MMR with loss %
Colorectal cancer 2204 14.5–91 (50.8) 422 19.15 155 7.03
Colorectal polyps 244 8.4–82 (54) 8 3.28 3 1.23
Endometrial cancer 739 183 24.76 44 5.95
Genetics service 239 16–79 (51) 28 11.7
PETALS 500 (65) 16 3.2
Gastric cancer 58 17–79 (48) 5 8.62 0 0.00
Ovarian cancer 261 16–89 (49) 27 10.34 8 3.07
TCC/kidney 13 32–61 (45) 3 23.08 1 7.69
Non- melanoma skin cancer 29 37–75 (57) 12 41.38 3 10.34
Cholangiocarcinoma 20 32–76 (50) 5 25.00 0 0.00
Pancreas 13 37–71 (53) 1 7.69 0 0.00
Brain 12 9–84 (48) 4 33.33 1 8.33
Breast 15 33–74 (49) 0 0.00 0 0.00
Small bowel including ampulla 21 29–72 (48) 1 4.76 0 0.00
Unknown primary 19 26–71 (40) 0 0.00 0 0.00
Oesophagus 16 21–61 (51) 1 6.25 0 0.00
Other 30 0 0.00 0 0.00
Total 3694 672 215
TCC urinary tract; others include cervix (n=4), prostate (n=4), sarcoma (n=3), melanoma (n=3), thyroid (n=2) and lung (n=2).
IHC, immunohistochemistry; MMR, mismatch repair; path_, pathogenic variant; PETALS, Proportion of Endometrial Tumours Associated with Lynch Syndrome; TCC, transitional
cell carcinoma.
Table 2 IHC loss and germline path_MMR detection rates in all index samples tested
All Tested (n) IHC loss % Tested germline (n) Germline PV % Germline
MLH1 loss 3694 348 9.42 191 66* 34.55 63 MLH1, 3 PMS2
PMS2 loss alone 3694 33 0.89 26 19 73.08 19 PMS2
MSH2 loss 3694 198 5.36 166 90 54.22 79 MSH2, 11 MSH6
MSH6 loss 3694 215 5.82 176 102 57.95 51 MSH6, 51 MSH2
Either MSH2 or MSH6 3694 291 7.88 239 130 54.39 79 MSH2, 51 MSH6
Any loss 3694 672 18.19 456 215 47.15
MSH6 loss alone 3694 53 1.43 73 38 52.05 38 MSH6
Tested (n) Positive (n)
MLH1 loss hypermethylation 268 167† 62.3 57 1 1.75 MLH1
MLH1 loss BRAF c.1799T>A 181 45 24.86 11 3 27.3
No loss 3022 0 0 329 19 5.78 5 MLH1, 7 MSH2, 3
MSH6, 4 PMS2
*This rose to 65/134 (48.5%) unmethylated samples.
†One patient with colorectal cancer had germline MLH1 methylation.
EC, endometrial cancer; IHC, immunohistochemistry; PV, pathogenic variant.
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4Evans DG, etal. J Med Genet 2021;0:1–7. doi:10.1136/jmedgenet-2020-107542
Cancer genetics
a pathogenic variant (56 MLH1, 59 MSH2 and 8 MSH6). Simi-
larly, 24 women with EC fulfilling Amsterdam criteria went
direct to germline testing, of whom 19 (79%) had a path_MMR
(4 MLH1, 10 MSH2, 4 MSH6 and 1 PMS2).
Of the 672 tumours with dMMR IHC loss in prescreened
samples (table 2), loss of MLH1 was most common (9.4%)
with 7.9% having loss of either MSH2 or MSH6 or both. There
were 215 path_MMR present in 456 lymphocyte samples tested
(47.4%–63 MLH1, 79 MSH2, 51 MSH6 and 22 PMS2). The
relatively low detection rate of only 34.5% for those with MLH1
loss is partially explained by screening of 57 samples showing
MLH1 methylation of which only one had a path_MLH1 germ-
line variant (26 samples from PETALS and 31 clinical samples
were tested). The patient with a germline MLH1 had a caecal
tumour aged 45 and met Amsterdam criteria. Thus, the true
rate of MLH1 promoter methylation- negative samples in this
group was 65/134 (48.5%). The highest detection rates of path_
MMR were for those with PMS2 loss alone (73%) and MSH6
loss alone (58%). Overall 12/22 (54.5%) with a PMS2 germline
path_MMR had a large rearrangement.
Table 3 shows the dMMR tumours and the pathogenic vari-
ants detected for CRC and EC, respectively. Overall, 2204 index
CRCs underwent IHC and 422 (19.1%) showed dMMR with the
highest proportion demonstrating MLH1 loss (10%). For EC,
183/739 (24.8%) samples were dMMR with 15% demonstrating
MLH1 loss. Both overall dMMR rates (p=0.001) and MLH1
loss rates (p=0.0003) were significantly higher in EC, although
the difference is entirely driven by MLH1 loss. MLH1 promoter
hypermethylation rates were much higher in EC at 87% (95/109)
compared with only 41% (67/163) in CRC (p<0.0001). BRAF
c.1799T>A was identified in only 26.5% (45/170) dMMR CRC
samples, compared with 41% (67/163) with MLH1 promoter
hypermethylation (p=0.005). BRAF testing was of no value in
EC and was abandoned for the PETALS study.25 The difference
in path_MMR variant rates between CRC and EC was most
striking for MSH6 with pathogenic variants identified in 24/44
(54.5%) cases of EC compared with 21/155 (13.5%) for CRC
(p<0.0001). Equally, MLH1 variants were more common in
CRC with 61/155 (39%) pathogenic variants compared with
2/44 (0.5%) in EC (p<0.0001).
A subset of samples with EC and CRC still underwent full
germline testing despite no IHC loss. This was in general because
of a strong family history, and it remains routine practice to
test all Amsterdam criteria cases regardless of IHC loss.27 For
CRC, 13/74 (17.6%) Amsterdam criteria and 5/102 (4.9%) non-
Amsterdam criteria cases tested positive for a path_MMR despite
normal IHC. However, for EC, only 1/120 (0.83%) tested posi-
tive for a path_MSH6 and nil in other MMR genes. The higher
figures for CRC may be because these are more common, and a
sporadic tumour without IHC loss could explain at least some of
these false negative results. Sensitivity for IHC loss in CRC was
155/173 (89.6%) and that for EC was 44/45 (97.8%), although
these figures might drop further if all samples with retained IHC
staining were tested.
The results of somatic analysis are shown in table 4 for
CRC and EC, respectively. In seven cases with IHC loss, the
Table 3 IHC loss and germline path_MMR detection rates in CRC and EC index samples tested
Colorectal Number tested IHC loss %
Number tested
germline
Germline path_
MMR % Germline path_MMR
MLH1 and PMS2 loss 2204 171 7.8 104 40‡ 38.5% 37 MLH1, 3 PMS2
MLH1 loss alone 2204 51 2.3 33 24‡ 72.7% 24 MLH1
PMS2 loss alone 2204 25 1.1 21 14 66.7% 14 PMS2
MSH2 and MSH6 loss 2204 81 3.7 76 54 71.0% 48 MSH2, 6 MSH6
MSH2 loss alone 2204 45 2.0 32 8 25.0% 8 MSH2
MSH6 loss alone 2204 41 1.9 30 15 50.0% 15 MSH6
Either MSH2 or MSH6 2204 175 7.9 140 77 55.0% 56 MSH2, 21MSH6
Any loss 2204 422 19.1 298 155 52.0%
Tested (n) Positive (n)
MLH1 loss hypermethylation 163 67* 41.1 23 1 4.3% MLH1
MLH1 loss BRAF c.1799T>A 170 45 26.5 11 3 27.3%
No loss 1782 0 0.0 176 18† 10.2% 4 MLH1, 7 MSH2, 3
MSH6, 4 PMS2
EndometrialC
MLH1 and PMS2 loss 739 108 14.6% 43 2† 4.6% 2 MLH1
MLH1 loss alone 739 4 0.5 4 0 0%
PMS2 loss alone 739 7 0.95 5 5 100.0% 5 PMS2
MSH2 and MSH6 loss 739 32 4.3 29 16 55.2% 12 MSH2, 6 MSH6
MSH2 loss alone 739 2 0.3 2 1 50% 1 MSH2
MSH6 loss alone 739 30 4.1 30 18 60.0% 18 MSH6
Either MSH2 or MSH6 739 64 8.7 61 37 60.7% 24 MSH6, 13 MSH2
Any loss 739 183 24.8 113 44 38.9%
Tested (n) Positive (n)
MLH1 loss hypermethylation 109 95 87.2 32 0 0.0%
MLH1 loss BRAF c.1799T>A Not systematically
tested
No loss 556 – 0.0 120 1 0.8% 1 MSH6
*One patient with CRC had germline MLH1 methylation, #13/74 (17.6%) Amsterdam criteria, 5/102 (4.9%) non- Amsterdam.
†This rose to 2/15 unmethylated samples.
‡This rose to 63/114 (55.3%) of unmethylated samples.
CRC, colorectal cancer; EC, endometrial cancer; IHC, immunohistochemistry; MMR, mismatch repair; path_, pathogenic variant.
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Cancer genetics
individuals were deceased and a germline path_MMR was found
on somatic testing and confirmed in normal tissue material.
Initial somatic MMR testing in the first 40 CRC samples did not
include an analysis of LOH, and thus many variants were only
monoallelic. Overall, 15/20 monoallelic CRC samples did not
have a formal LOH analysis. However, in EC, 17/18 tumours
showed at least one somatic variant with 13/18 (72%) biallelic.
Four tumours had a single variant at low allele frequency (MSH2
c.2458+1G>A (5.04%), MSH2 c.1003dupA (9.5%), MSH6
c.3261delC (10.1%) and MSH6 c.718C>T (13.2%)), which
precluded a sensitive LOH analysis. Of the 183/739 (24.7%)
EC samples showing IHC loss, 95 (52%) were explained by
MLH1 promoter methylation; 44 (24%) had a germline path_
MMR; and 17 (9.3%) showed evidence of somatic involvement
of the relevant gene. This leaves only 27/183 (14.7%), but six
samples did not undergo germline testing and three did not have
promoter methylation with MLH1 loss. Most of the remainder,
bar 1, did not have tumour somatic testing. Assuming the same
detection rates for somatic testing as in the 18 EC samples, this
would leave no more than 3 of 183 (1.6%) unexplained and
only 3/739 (0.4%) of the whole IHC prescreened cohort. Of
the 422/2204 (19.15%) CRC samples showing IHC loss, 67
(15.9%) were explained by MLH1 promotor methylation; 155
(36.7%) had a germline path_MMR; and 65 (15.4%) showed
evidence of somatic involvement of the relevant gene. This
leaves 135/422 (32%), but 73 samples did not undergo germline
testing as patients were deceased and 17 did not have promoter
methylation with MLH1 loss.
DISCUSSION
We have reported IHC tumour prescreening in 3694 tumour
samples, which, to our knowledge, is the largest such series in the
literature. Although IHC does not have 100% sensitivity,12–14 it
has the advantage of identifying the relevant likely genes involved
and allows targeted MLH1 promotor methylation in a lower
number of samples than MSI testing. We have previously shown
that for EC, in particular with the higher rates of involvement of
MSH6 (55% in the present study), MSI is significantly less sensi-
tive (58%) with IHC detecting 100% of 16 path_MMR.25 The
high proportion of MSH6 in EC is confirmed in other studies
with 5/9 (55.5%) in a US universal testing study.29 An Austra-
lian study limiting testing to ECs<60 found 10/22 (45.4%) of
those with path_MMR had a path_MSH6.30 The study confirms
the utility of MLH1 promotor methylation particularly for EC
with 87% of MLH1 loss being explained. This is similar to the
86.3% in a meta- analysis of 29 studies with 1159 showing loss of
MLH1.31 Although MLH1 promoter methylation is less useful in
CRC, it is still superior to BRAF testing. The significantly higher
rates of promoter methylation in EC seem to account entirely for
the higher rates of MLH1 loss. As methylation is a mechanism
that is used to coordinate menstruation in the endometrium,32
we propose that there is increased opportunity for regions in
the DNA to be erroneously methylated, which may explain
increased promoter methylation of MLH1 in EC.
The current study has confirmed the high predictive value of
isolated loss of PMS2 and, to a lesser extent, MSH6,33 although
isolated MLH1 loss was also quite specific with 73% being caused
by a germline path_MMR. We have also shown the importance
of somatic MMR testing in cases with IHC loss unexplained by
either MLH1 promotor methylation or a germline path_MMR.
Somatic bilalleic path_MMRs are found in a high proportion of
these cases. Of the 284 patients with non- methylated MMR loss
in a joint Ohio and Icelandic cohort, 157 had a germline path_
MMR, (55%) and 92 (32.4%) had probable biallelic (double)
somatic variants.33 They concluded that 19 (6.7%) were unex-
plained and 17 had incorrect IHC. While we demonstrated this
well in EC, it was less well shown in CRC. This may be due to
low neoplastic cell counts that preclude a sensitive assessment
of LOH. Recutting tumour FFPE sections for higher neoplastic
content may well overcome this issue. Furthermore, some IHC
loss may be spurious (an overcall) and reanalysis or assessment
of MSI in those that still remains with unexplained IHC loss
may resolve the issue. For EC we have shown that <1% of cases
undergoing IHC are left with an unresolved diagnosis. In reality,
‘Lynch’ like syndrome, which was thought to be due primarily
to missed path_MMR or another inherited mechanism, appears
to be a relatively uncommon situation once somatic testing has
been performed especially in EC.
Although there was a low rate of path_MMR in patients with
tumours with MLH1 loss on IHC and promoter methylation as
a prescreen, we have previously demonstrated that 4/71 (5.6%)
individuals with CRC and germline pathogenic variants in
MLH1 had evidence of promoter methylation.27 Three of these
four fulfilled Amsterdam criteria did not have an IHC prescreen
(they were tested after path_MMR was found); therefore,
overall MLH1 promotor methylation still left a >10% chance of
a germline path_MMR. Similarly, those with Amsterdam criteria
who had proficient MMR tumour on IHC also had a path_
MMR rate above 10%. As such, we would still recommend that
those with CRC fulfilling Amsterdam criteria undergo germline
Table 4 NGS somatic analysis on CRC and EC with IHC loss
IHC loss Number Hypermethylation Germline from tumour Germline negative blood Somatic No cause found Cause of IHC loss found
Colorectal somatic testing
MLH1/PMS2 47 0/46 4 MLH1* 43 30 MLH1 13 34/47 (72%)
10/34 monoallelic
MSH2/MSH6 38 nt 4
2 MSH6
2 MSH2
34 27
8 MSH6
19 MSH2
7 31/38 (82%)
10/38 monoallelic
Endometrial somatic testing
MLH1/PMS2 5 0/5 0 5 4 MLH1 1 4/5 (80%)
3/5 double somatic†
MSH2/MSH6 13 nt 0 13 7 MSH6
6 MSH2
0 13/13 (100%)
10/13 double somatic
For CRC: 13 MLH1 loss no cause found 2/3 MSH−1 double somatic PTEN, 1 POLD1.
7 MSH2 loss no cause found 4/5 MSS? Overcall: 1 MSH double somatic PTEN.
*One mosaic low level 16% VAF missed on germline testing found after tumour somatic c.1975C>T p.(Arg659Ter) MLH1.
†Most samples with monoallelic variants had allele frequencies of <10%, which precludes LOH analysis.
CRC, colorectal cancer; EC, endometrial cancer; IHC, immunohistochemistry; LOH, loss of heterozygosity; NGS, next- generation sequencing; VAF, variant allele frequency.
on January 16, 2021 by guest. Protected by copyright.http://jmg.bmj.com/J Med Genet: first published as 10.1136/jmedgenet-2020-107542 on 15 January 2021. Downloaded from

6Evans DG, etal. J Med Genet 2021;0:1–7. doi:10.1136/jmedgenet-2020-107542
Cancer genetics
path_MMR testing irrespective of the IHC or MLH1 promoter
methylation result. The same may not be true for EC with the
much higher rates of MLH1 promoter methylation and low rate
of pathogenic germline variants in those tested with proficient
MMR on IHC.
Our results show the value of a combined tumour somatic
and germline test after IHC loss. This can especially be seen
in the population- based PETALS study where the 3.2% detec-
tion rate for germline path_MMR in EC is similar to that seen
in unselected CRC. By combining a tumour somatic approach
with germline testing in 500 ECs, this comprehensive testing
left just 1.9% (2/106) MMR deficient tumours unexplained by
a path_MMR variant/epigenetic silencing.25 As such, only 2/500
(0.4%) were left still in the Lynch- like category after testing. A
similar mainstreaming approach for CRC as well as EC would
leave far fewer with an uncertain diagnosis, and only those
with a path_MMR or unexplained IHC would need referral to
genetics. Unfortunately, we cannot be certain the results would
be as good in CRC based on our analysis as this did not involve
LOH analysis for many samples, but others have found a high
rate of double somatic events in CRC.33 While families can be
reassured when double somatic events account for IHC loss this
will still leave some where the age of the patient or family history
requires ongoing management as Lynch- like. Testing of benign
colorectal polyps is quite specific but does not have a high yield,
although it can detect germline path_MMR in individuals with
strong family histories suggestive of LS.
There are some limitations to the present study. We did not
perform MSI testing on all the samples with IHC and cannot
therefore make a direct comparison, although for EC, we have
previously shown reduced sensitivity of MSI.25 The selection
criteria for testing for CRC was stronger than for EC, and
therefore comparisons are likely to overestimate the contribu-
tion of IHC loss in CRC compared with the EC tested in this
study. Nonetheless, this is likely to strengthen further some of
the differences identified between CRC and EC. We also did
not typically prescreen individuals meeting Amsterdam criteria,
meaning that the detection rates for IHC loss and path_MMR
may be underestimated compared with studies that included
individuals meeting Amsterdam criteria. We would still test
patients meeting Amsterdam criteria even if they had hypermeth-
ylation of MLH1 as evidenced by the case presented here. Some
authors now advocate starting analysis with a tumour somatic
approach.33 It is certainly plausible that this will become more
mainstream and may reduce the requirement for a prescreen for
LS testing. However, given the high rate of copy number vari-
ants in LS (11%–46%)34 and especially in this study for PMS2
(54.5%), the sensitivity to detect these in tumour samples needs
to be fully validated first. PMS2 is known to be difficult to screen
in lymphocyte DNA, and therefore testing in stored non- frozen
tissue samples requires a bespoke approach.
In conclusion, we have undertaken prescreening of a very large
series of tumour specimens with IHC for dMMR. Detection
rates for germline path_MMR are similar to previous estimates.
We have shown the superiority of MLH1 promoter hypermeth-
ylation over BRAF testing and the higher utility in EC compared
with CRC. Furthermore, we have shown that somatic MMR
testing with NGS removes most patients from the ‘Lynch’-like
category with previously unexplained IHC loss.
Author affiliations
1Division of Evolution and Genomic Medicine, The University of Manchester,
Manchester, UK
2Clinical Genetics Service, Manchester Centre for Genomic Medicine, North- West
Genomics Laboratory Hub, Manchester University NHS Foundation Trust, Manchester,
UK
3Division of Cancer Sciences, The University of Manchester, Manchester, UK
4Department of Obstetrics and Gynaecology, Manchester University NHS Foundation
Trust, Manchester, UK
5Department of Pathology, Manchester University NHS Foundation Trust, Manchester,
UK
6Department of Surgery, Manchester University NHS Foundation Trust, Manchester,
UK
Twitter Emma R Woodward @ER_Woodward and Emma J Crosbie @
ProfEmmaCrosbie
Contributors DGE was principal investigator for the study and is its guarantor.
DGE and EJC designed the study and supervised its execution. DGE and EJC
wrote the manuscript. All authors contributed to the data collection, analysis and
interpretation; provided critical comment; edited the manuscript; and approved its
final version.
Funding NR was a Doctoral Medical Research Council (MRC) Research Fellow (MR/
M018431/1), DGE a National Institute for Health Research (NIHR) Senior Investigator
(NF- SI-0513-10076), EJC an NIHR Clinician Scientist (NIHR- CS-012-009), and their
work was supported through the NIHR Manchester Biomedical Research Centre (IS-
BRC-1215-20007). This article presents independent research funded by the NIHR
and MRC. The views expressed are those of the authors and not necessarily those of
the MRC, NHS, NIHR or the Department of Health.
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval Informed consent was obtained from all subjects enrolled in the
PETALS study. No identifiable information is provided in the article and the advice of
the ethics committee was that this represented clinical audit/ service evaluation.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available upon reasonable request from
the corresponding author.
Supplemental material This content has been supplied by the author(s). It
has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have
been peer- reviewed. Any opinions or recommendations discussed are solely those
of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and
responsibility arising from any reliance placed on the content. Where the content
includes any translated material, BMJ does not warrant the accuracy and reliability
of the translations (including but not limited to local regulations, clinical guidelines,
terminology, drug names and drug dosages), and is not responsible for any error
and/or omissions arising from translation and adaptation or otherwise.
Open access This is an open access article distributed in accordance with the
Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits
others to copy, redistribute, remix, transform and build upon this work for any
purpose, provided the original work is properly cited, a link to the licence is given,
and indication of whether changes were made. See:https:// creativecommons. org/
licenses/ by/ 4. 0/.
ORCID iDs
D GarethEvans http:// orcid. org/ 0000- 0002- 8482- 5784
Emma RWoodward http:// orcid. org/ 0000- 0002- 6297- 2855
Emma JCrosbie http:// orcid. org/ 0000- 0003- 0284- 8630
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