Assessment of mismatch repair deficiency in ovarian cancer

Abstract

Background:
Hereditary causes of ovarian cancer include Lynch syndrome, which is due to inherited pathogenic variants affecting one of the four mismatch repair genes involved in DNA repair. The aim of this study was to evaluate tumour mismatch repair deficiency and prevalence of Lynch syndrome in high-risk women referred to the Manchester Centre for Genomic Medicine with ovarian cancer over the past 20 years.

Methods:
Women with ovarian cancer diagnosed before the age of 35 years and/or with a suggestive personal or family history of Lynch syndrome cancers underwent tumour testing with immunohistochemistry for mismatch repair deficiency and, where indicated, MLH1 promoter methylation testing followed by constitutional testing for Lynch syndrome.

Results:
In total, 261 ovarian cancers were tested and 27 (10.3%; 95% CI 6.9% to 14.7%) showed mismatch repair deficiency by immunohistochemistry. Three of 7 with MLH1 loss showed MLH1 promoter hypermethylation, and 18 of the remaining 24 underwent constitutional testing for Lynch syndrome. A further 15 women with mismatch repair proficient tumours underwent constitutional testing because of a strong family history of Lynch syndrome cancers. Pathogenic variants were identified in 9/33 (27%) women who underwent constitutional testing, aged 33-59 years (median 48 years), including one whose tumour was mismatch repair proficient. Most Lynch syndrome tumours were of endometrioid histological subtype.

Conclusions:
Tumour mismatch repair deficiency identified by immunohistochemistry is a useful prescreen for constitutional testing in women with ovarian cancer with personal or family histories suggestive of Lynch syndrome.

Full text

1
Crosbie EJ, etal. J Med Genet 2020;0:1–5. doi:10.1136/jmedgenet-2020-107270
ORIGINAL RESEARCH
Assessment of mismatch repair deficiency in
ovariancancer
Emma J Crosbie ,1,2 Neil A J Ryan,1,2,3 Rhona J McVey,4 Fiona Lalloo,5
Naomi Bowers,5 Kate Green,5 Emma R Woodward ,3,5 Tara Clancy,5 James Bolton,4
Andrew J Wallace,5 Raymond F McMahon,4 D Gareth Evans 3,5
Cancer genetics
To cite: Crosbie EJ,
Ryan NAJ, McVey RJ, etal.
J Med Genet Epub ahead of
print: [please include Day
Month Year]. doi:10.1136/
jmedgenet-2020-107270
1Division of Cancer Sciences,
The University of Manchester,
Manchester M13 9WL, UK
2Department of Obstetrics and
Gynaecology, St Mary’s Hospital,
Manchester University NHS
Foundation Trust, Manchester
Academic Health Science
Centre, Manchester M13 9WL,
UK
3Division of Evolution and
Genomic Medicine, The
University of Manchester,
Manchester M13 9WL, UK
4Department of Pathology,
Manchester University NHS
Foundation Trust, Manchester
Academic Health Science
Centre, Manchester M13 9WL,
UK
5Manchester Centre for
Genomic Medicine, North- West
Genomics Laboratory Hub,
Manchester University NHS
Foundation Trust, Manchester
Academic Health Science
Centre, Manchester M13 9WL,
UK
Correspondence to
Prof Emma J Crosbie, Division of
Cancer Sciences, The University
of Manchester, St Mary’s
Hospital, 5th floor- Research,
Oxford Road, Manchester M13
9WL, UK;
emma. crosbie@ manchester.
ac. uk
Received 15 June 2020
Revised 25 July 2020
Accepted 27 July 2020
© Author(s) (or their
employer(s)) 2020. Re- use
permitted under CC BY.
Published by BMJ.
ABSTRACT
Background Hereditary causes of ovarian cancer
include Lynch syndrome, which is due to inherited
pathogenic variants affecting one of the four mismatch
repair genes involved in DNA repair. The aim of this
study was to evaluate tumour mismatch repair deficiency
and prevalence of Lynch syndrome in high- risk women
referred to the Manchester Centre for Genomic Medicine
with ovarian cancer over the past 20 years.
Methods Women with ovarian cancer diagnosed before
the age of 35 years and/or with a suggestive personal
or family history of Lynch syndrome cancers underwent
tumour testing with immunohistochemistry for mismatch
repair deficiency and, where indicated, MLH1 promoter
methylation testing followed by constitutional testing for
Lynch syndrome.
Results In total, 261 ovarian cancers were tested
and 27 (10.3%; 95% CI 6.9% to 14.7%) showed
mismatch repair deficiency by immunohistochemistry.
Three of 7 with MLH1 loss showed MLH1 promoter
hypermethylation, and 18 of the remaining 24
underwent constitutional testing for Lynch syndrome.
A further 15 women with mismatch repair proficient
tumours underwent constitutional testing because
of a strong family history of Lynch syndrome cancers.
Pathogenic variants were identified in 9/33 (27%)
women who underwent constitutional testing, aged 33–
59 years (median 48 years), including one whose tumour
was mismatch repair proficient. Most Lynch syndrome
tumours were of endometrioid histological subtype.
Conclusions Tumour mismatch repair deficiency
identified by immunohistochemistry is a useful prescreen
for constitutional testing in women with ovarian cancer
with personal or family histories suggestive of Lynch
syndrome.
INTRODUCTION
Ovarian cancer is the seventh most common malig-
nancy worldwide and the most lethal gynaecolog-
ical cancer.1–3 Epithelial ovarian cancer is one of
the most heritable malignancies, frequently due to
pathogenic variants in single high- risk genes. The
heritable component of ovarian cancer is predomi-
nantly due to constitutional pathogenic variants in
BRCA1 and BRCA2 with as many as 22% of women
with high grade serous ovarian cancers (HGSOC)
carrying pathogenic variants in these genes.4 The
other leading heritable cause is Lynch syndrome
(LS), an inherited mismatch repair (MMR) defi-
ciency due to constitutional pathogenic variants
affecting one of the four MMR genes, MSH2,
MLH1, MSH6 and PMS2.5 Around 1:280 of the
general population carries a pathogenic variant
in a MMR gene, the great majority of whom are
undiagnosed.6 Women heterozygous for pathogenic
MMR gene variants have a 3%–17% lifetime risk of
ovarian cancer, and higher risks for colorectal and
endometrial cancers.7 8
Since the discovery of the MMR genes in
1993–1994, clinicians have tried to target consti-
tutional testing for LS to those at highest risk. The
Amsterdam criteria were developed in 1991,9 but
these require a strong family history of colorectal
cancer to be discriminatory. Even adding additional
LS tumours to the criteria, such as endometrial and
ovarian cancer10 has added little to its detection
rate11 or sensitivity.12 13 Testing for LS outside of
the Amsterdam criteria,9 10 where upfront constitu-
tional testing is practised, has largely depended on
a prescreen of the incident tumour using immuno-
histochemistry (IHC) for MMR protein expression
or DNA for microsatellite instability (MSI).12 There
have been very few studies that have looked at the
success of this prescreen in ovarian cancer and most
have included small numbers of tumours and have
concentrated on just one histological subtype of
ovarian cancer (endometrioid).13 14 This ignores
the fact that restricting testing based on histo-
logical subtype misses cases of LS, particularly as
morphology is subjective and can be challenging in
complex cases.15 16
We have evaluated our prescreening strategy
with IHC in women referred to the regional
genetics department with possible LS- associated
ovarian cancer from 2000 to 2020 and assessed the
identification of constitutional MMR pathogenic
variants.
METHODS
Participants
Women referred to the regional genetics department
in Manchester with ovarian cancer and concerns
about the possibility of LS provided consent for
tumour and if indicated constitutional testing.
Most women had a history of another LS- related
cancer in themselves or another family member
(colorectal, endometrial, ovary, biliary tree, urinary
tract, gastric or skin). However, some were selected
based on diagnosis at <35 years of age.
on December 8, 2020 by guest. Protected by copyright.http://jmg.bmj.com/J Med Genet: first published as 10.1136/jmedgenet-2020-107270 on 11 September 2020. Downloaded from

2Crosbie EJ, etal. J Med Genet 2020;0:1–5. doi:10.1136/jmedgenet-2020-107270
Cancer genetics
Immunohistochemistry
IHC for the four MMR proteins was performed in the clinical
pathology laboratory using the automated Ventana BenchMark
ULTRA IHC⁄ISH staining module and the OptiView, 3’diami-
nobenzidine V.5 detection system (Ventana, USA) according to
standard clinical protocols. Tumour epithelial MMR expression
was scored by two expert independent observers using stroma as
internal control and as described previously.17
Methylation analysis
Reflex MLH1 promoter methylation testing was performed
on tumours showing loss of MLH1 on IHC. Extracted DNA
was bisulfite converted and then 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 were sequenced
using a pyrosequencer (PSQ 96MA). Two independent scientists
interpreted the pyrograms. ‘Hypermethylation’ was described
as >10% mean methylation across the four CpG dinucleotides
on a minimum of two of three replicate analyses. In addition to
promoter methylation analysis, testing was carried out for the
BRAF c.1799T>A variant in some cases.
Microsatellite instability analysis
Extracted DNA underwent sodium bisulfite conversion using the
Epitect Plus FFPE kit (Qiagen, UK). The MSI analysis system
V.1.2 (Promega, USA) used fluorescent- labelled primers to
coamplify seven markers, including five mononucleotide- repeat
markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27), and
two control penta- nucleotide- repeat markers (Penta- C/Penta- D).
MSI status was reported as microsatellite stable (MSS) where all
five mononucleotide loci between tumour and matched normal
tissue were identical; MSI- low (MSI- L) where there was discor-
dance in one mononucleotide locus and MSI- high (MSI- H)
where two or more mononucleotide loci were discordant.
Constitutional analysis
DNA was extracted from 2 to 5 mL lymphocyte blood
(EDTA anticoagulant) using Chemagic DNA blood chemistry
(CMG-1097- D) on an automated Perkin Elmer Chemagic 360
Magnetic Separation Module and a JANUS Integrator 4- tip Auto-
mated Liquid handling platform. DNA was eluted into 400 uL
buffer. Extracted DNA samples were measured for DNA yield,
concentration and quality using a Nanodrop ND-8000 spec-
trophotometer. Three MMR genes MLH1, MSH2 and MSH6
were amplified using long range PCR followed by next gener-
ation sequencing using Illumina SBS v2 2×150 bp chemistry on
an Illumina MiSeq. The whole coding region, intronic flanking
sequences to±15 bp and known splicing variants of MLH1,
MSH2 and MSH6 were analysed (minimum 100 x coverage
depth). 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 chemistry. Copy number analysis to detect large
genomic rearrangements affecting the three 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
translated 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.
All women gave written informed consent for tumour and
blood testing except deceased cases, whose tumour was obtained
and tested with a relative’s consent. Advice from our ethics
committee was that the current analysis represented clinical
service evaluation and that no specific ethics application was
required. There is no directly identifiable patient information
presented.
Statistics
Differences between values were tested by a two- tailed Fisher's
χ2 test.
RESULTS
In total, 261 women with ovarian cancer underwent an IHC
prescreen for LS (table 1, figure 1). They were aged between
16 and 89 years (median=49 years). Fifty- one cases were tested
because they were diagnosed at <35 years of age. All histolog-
ical subtypes were tested if indicated, with HGSOC the most
frequently tested. Overall, only 27 (10.3%; 95% CI 6.9% to
14.7%) tumours showed MMR deficiency by IHC with just 7
(2.7%) having loss of MLH1 (table 2). Three of these tumours
showed MLH1 promotor hypermethylation and therefore consti-
tutional LS testing was not performed. Eighteen of the remaining
24 women whose tumours showed MMR deficiency under-
went constitutional testing for MMR pathogenic variants. The
Table 1 Number of ovarian cancers tested for IHC by pathology and proportion of women with MMR pathogenic variants
Pathology Tested (n)
IHC loss
(n)
IHC loss
(%)
BRAF
tested
(n)
C.1799T>A
positive
(n)
C.1799T>A
positive
(%)
Methylation
tested (n)
Hypermethylated
(n)
Hypermethylated
(%)
Tested for
path_MMR
(n)
Lynch
syndrome
(n)
Lynch syndrome
(%)
Endometrioid 43 9 20.9 2 0 0 4 2 50 10 4 40 MSH2,
3 MSH6
Clear cell 19 2 10.5 0 – – 0 – – 4 2 50 MLH1, MSH2
Mucinous 59 6 10.2 1 0 0 1 0 0 10 0 0 –
Low grade serous 10 0 0 0 – – 0 – – 0 0 0 –
High grade serous 79 6 7.6 1 0 0 1 0 0 9 2 22 MSH2, MSH6
Adenocarcinoma
(other)
38 3 7.9 1 0 0 1 1 100 2 0 0 –
Other* 13 1 7.7 0 – – 0 – – 1† 1 100 MSH2
Total 261 27 10.3 5 0 0 7 3 43 36 9 25 4 MSH6, 4
MSH2, 1
MLH1
*Three Mullerian, two granulosa cell, one Sertoli, two secondaries, one mesodermal, one Brenner, three carcinosarcoma.
†Carcinosarcoma of ovary aged 48 years; sister had colorectal cancer aged 34 years.
IHC, immunohistochemistry; MMR, mismatch repair.
on December 8, 2020 by guest. Protected by copyright.http://jmg.bmj.com/J Med Genet: first published as 10.1136/jmedgenet-2020-107270 on 11 September 2020. Downloaded from

3
Crosbie EJ, etal. J Med Genet 2020;0:1–5. doi:10.1136/jmedgenet-2020-107270
Cancer genetics
remaining six did not undergo constitutional analysis because
the ovarian cancer case was deceased and a blood lymphocyte
sample was not available. An additional 15 women underwent
constitutional analysis despite having MMR proficient tumours
due to a strong family history, with eight meeting Amsterdam II
criteria9 (figure 1).
MSI testing was performed for 24/261 cases. Five tumours
were MSI- H, all of which were MMR deficient by IHC and 4/5
women underwent constitutional analysis for MMR pathogenic
variants. Two tumours were MSI- L and MMR deficient, and one
out of two of these women underwent constitutional analysis.
Seventeen tumours were MSS, six of which were MMR deficient
and five of these six women underwent constitutional analysis
for MMR pathogenic variants. Four of six path_MMR carriers
had MSI- H tumours; one was MSI- L (MSH2), one MSS (MSH6)
and the remaining three were not MSI tested (table 3).
MMR pathogenic variants were found in 9/33 (27%) women
who underwent constitutional testing with ages of ovarian
cancer diagnosis of 33–59 years (median 48) (table 3). The
highest rate was for endometrioid ovarian cancer with 10/43
(9.6%) having a constitutional MMR pathogenic variant. The
only LS case whose ovarian tumour was MMR proficient was a
patient with a clear cell carcinoma who also had a constitutional
BRCA1 pathogenic variant. It is therefore likely her ovarian
cancer was not driven by her MLH1 pathogenic variant. There
were four pathogenic variants each in MSH2 and MSH6, the
MSH6 cases only displayed MSH6 IHC loss whereas three of the
MSH2 pathogenic variants had loss of both MSH2 and MSH6.
Selection for ovarian cancer by age <35 years was not effective
as a selection tool as only 4/52 (7.7%) had IHC loss. Only one of
the four women<35 years tested for constitutional pathogenic
variants had a path_MMR variant identified, and that patient
had a parent with four separate bowel primary tumours highly
suggestive of LS (table 3).
DISCUSSION
Here, we describe our 20- year experience of tumour MMR IHC
as a prescreen for constitutional testing women with suspected
LS- associated ovarian cancer. We tested 261 ovarian tumours
for MMR deficiency because women were diagnosed <35 years
of age and/or because they had a suggestive personal or family
history of LS. Those with strong clinical risk factors underwent
constitutional testing even if their tumours were MMR profi-
cient. In total, 27 tumours (10.3%) were MMR deficient and 8
of these had LS. Most were of endometrioid histological subtype.
Figure 1 Study flow diagram. *Includes LS carrier found in a MMR proficient case with a simultaneous constitutional pathogenic variant in BRCA 1. FHx,
family history; IHC, immunohistochemistry; MMR, mismatch repair; LS, Lynch syndrome; OC, ovarian cancer.
Table 2 IHC loss and constitutional MMR pathogenic variant detection rates in all index ovarian cases tested
Tested (n) IHC loss (n) IHC loss (%) Tested for path_MMR (n) Lynch syndrome (n) Lynch syndrome (%)
Any loss 261 27 10.3 18 8 44.4
MLH1 loss 261 7 2.7 3 0 0.0
Either MSH2 or MSH6 261 19 7.3 15 8 53.3
MSH2 loss 261 13 4.9 9 4 44.4
MSH6 loss 261 10 3.8 9 6 66.7
MSH6 loss alone 261 7 2.7 6 4 66.7
PMS2 loss alone 261 0 0.0 0 0 –
No Loss 234 0 0.0 15 1* 6.7
*Ovarian clear cell carcinoma aged 59 years had exon 6–19 deletion of MLH1 with normal IHC –family met Amsterdam II criteria. She also carries a BRCA1 exon 13 duplication.
She developed grade 3 triple negative breast cancer at 71 and sebaceous carcinoma at 67 years.
IHC, immunohistochemistry; MMR, mismatch repair.
on December 8, 2020 by guest. Protected by copyright.http://jmg.bmj.com/J Med Genet: first published as 10.1136/jmedgenet-2020-107270 on 11 September 2020. Downloaded from

4Crosbie EJ, etal. J Med Genet 2020;0:1–5. doi:10.1136/jmedgenet-2020-107270
Cancer genetics
One woman with constitutional path_MMR variant had a MMR
proficient tumour; she also had a constitutional BRCA1 patho-
genic variant. She is unlikely therefore to have developed ovarian
cancer via a MMR driven pathway.
Previous studies examining the MMR status of unselected
endometrioid or clear cell ovarian cancers found similar rates
of MMR deficiency, but overall numbers were very small.13 14 18
Two systematic reviews found that approximately 10% of ovarian
tumours are MMR deficient by IHC, but included studies that
were very limited with respect to their reporting of basic epide-
miological, molecular and clinical features.19 20 There was also
poor reporting of constitutional status. A recent study by Leskela
et al21 examined the MMR status of 502 stage I/II tumours
selected from the GEICO Early Stage Ovarian Cancer Registry.
The authors report MMR deficiency in 18.7% endometrioid
and 2.4% clear cell tumours overall, but do not provide infor-
mation about clinical risk factors for LS in their cohort. It is
perhaps surprising that despite selecting for LS features, such
as early age of cancer onset and indicative personal or family
history, that detection rates were not higher in our study, with
only 10.3% with IHC loss and 3.5% with a path_MMR variant.
Age selection (<35 years) was not an effective triage strategy
for constitutional testing with only 7.7% IHC loss and 1.9%
path_MMR. Furthermore, failure to select for pathology type by
including serous histological subtypes will have further reduced
our detection rates.
There are several strengths to our work. First, we carried out
MMR IHC tumour prescreening for all women referred to the
clinical genetics department whose age and family history were
suggestive of LS- associated ovarian cancer. We did not restrict
testing to any particular histological subtype. This is important
because histological subtyping is subjective, challenging in diffi-
cult cases and has evolved considerably over the past 20 years,
with validated IHC panels increasingly used to assist diagnosis.
Many of our cases pre- dated the now gold standard expert
gynaecological pathology review and confirmation by IHC.16
Restricting testing to endometrioid subtype would deny LS
testing to women with ovarian cancer diagnosed and treated
historically and in non- expert centres. In particular, women
diagnosed with non- endometrioid tumours who have survived
without recurrence from this earlier era may harbour a consti-
tutional MMR pathogenic variant, as survival in LS is known
to be good, and tumours may on review be reclassified with
modern pathology.15 Second, we provide detailed clinical anno-
tation for all proven LS- associated ovarian tumours as well as
comprehensive molecular phenotyping, including MMR, MSI
and, where indicated, MLH-1 promoter methylation status.
Analyses were carried out to quality- assured clinical standards
in specialist pathology and genetics referral laboratories. Data
were collected from our prospective clinical database, ensuring
comprehensive reporting of all cases and minimising issues with
missing data. All non- deceased women with MMR deficient
ovarian tumours unexplained by MLH1 promoter hypermeth-
ylation and 15 others, whose clinical risk factors were particu-
larly suggestive, underwent definitive constitutional LS testing
using blood lymphocyte DNA. This compares favourably with
preceding series where the conversion to constitutional testing
was poor and pathogenic variants were assumed from allele
frequency in adjacent normal tissue.18–21 Third, we tested 15
women with strong clinical risk factors whose ovarian tumours
were MMR proficient, facilitating an assessment of the accuracy
of MMR IHC as a prescreen for LS constitutional testing, which
is poorly reported in the literature. We found only one case of
MMR proficient LS- associated ovarian cancer, in a woman who
also carried a BRCA1 pathogenic variant and whose tumour is
likely to have developed via a non MMR driven pathway.
Limitations of the study include failure to conduct MSI anal-
ysis for all cases, which precludes a direct comparison between
MMR IHC and MSI status as a prescreen for constitutional LS
testing. The single centre nature of this study is another limita-
tion, since we cannot necessarily extrapolate our conclusions to
other healthcare settings where clinical genetics referral criteria
for suspected LS may differ. Our cohort was selected for IHC
testing and downstream analyses based on clinical criteria and
therefore may not reflect the MMR status of unselected ovarian
cancer populations.
The emergence of targeted therapies has led to mainstream
somatic and/or constitutional BRCA1/2 sequencing of women
with ovarian cancer to inform suitability for PARP inhibitor
therapy and clinical trial enrolment.22 23 Given the similar cumu-
lative risk of ovarian cancer in LS to BRCA2, testing premeno-
pausal women with epithelial ovarian cancer for both BRCA1/2
and LS is appropriate, particularly in an era of panel gene testing
where there is little additional cost to add more genes.24 If this
practice becomes widespread, it may reduce the requirement
for a prescreen for LS testing of patient with recently diagnosed
ovarian cancer, although a prescreen would still be useful for
women referred to clinical genetics departments with a previous
history of ovarian cancer, in whom a priori panel gene somatic
testing is unlikely to be indicated.
Table 3 Ovarian cancer cases with Lynch syndrome
Gene
FIGO stage and histological
subtype Age (years)
IHC loss
(4 protein panel) MSI
Type of pathogenic
variant Path_MMR variant
Meets Amsterdam
criteria?
MLH1 Stage 1c clear cell 59 None Not tested Large rearrangement MLH1 exon 6–19 deletion Yes—Amsterdam
modified
MSH2 Stage 1a mixed endometrioid/
clear cell
34 MSH2 loss MSI- H Splice site MSH2 c.1276+2T>C No
MSH2 Stage 3c high grade serous 38 MSH2 and MSH6 loss MSI- L Truncating MSH2 c.528_529delTG No
MSH2 Stage 1a carcinosarcoma 48 MSH2 and MSH6 Loss MSI- H Large rearrangement MSH2 exon 3 deletion No
MSH2 Stage 1c endometrioid 51 MSH2 and MSH6 loss MSI- H Truncating MSH2 c.196delT No
MSH6 Stage 1a endometrioid 47 MSH6 loss Not tested Splice site MSH6 c.3439–1G>T Yes—Amsterdam
modified
MSH6 Stage 1 endometrioid 50 MSH6 loss MSI- H Missense MSH6 c.1346T>C p.Leu449Pro No
MSH6 Stage 2 high grade serous 50 MSH6 loss MSI- S Truncating MSH6 c.3732_3735dupATTT No
MSH6 Stage 3c poorly differentiated
endometrioid with focal
neuroendocrine features
53 MSH6 loss Not tested Truncating MSH6 c.3261delC No
IHC, immunohistochemistry; MMR, mismatch repair.
on December 8, 2020 by guest. Protected by copyright.http://jmg.bmj.com/J Med Genet: first published as 10.1136/jmedgenet-2020-107270 on 11 September 2020. Downloaded from

5
Crosbie EJ, etal. J Med Genet 2020;0:1–5. doi:10.1136/jmedgenet-2020-107270
Cancer genetics
In summary, we report our experience of MMR IHC as a
prescreen for constitutional MMR pathogenic variant testing
in women with clinical risk factors for LS- associated ovarian
cancer. LS is rare if tumours are MMR proficient. While most
LS- associated ovarian tumours are of endometrioid histological
subtype, the subjective and sometimes challenging task of patho-
logical interpretation risks misclassification. Thus, our practice
is to continue to prescreen all ovarian tumours with clinical risk
factors for LS irrespective of tumour histological subtype, espe-
cially if their tumour pre- dates recent multidisciplinary panel
review in an expert centre.
Twitter Emma J Crosbie @ProfEmmaCrosbie, Neil A J Ryan @neilajryan and Emma
R Woodward @ER_Woodward
Contributors DGE is the principal investigator for the study and its guarantor.
EJC and DGE conceived the study. All authors contributed to data acquisition and
interpretation. EJC and DGE wrote the first draft of the manuscript. All authors
reviewed the manuscript and approved its final content.
Funding NAJR 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 a NIHR Clinician Scientist (NIHR- CS-012-009),
and their work was supported through the NIHR Manchester Biomedical Research
Centre (IS- BRC-1215-20007).
Disclaimer 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.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available from the corresponding author
on reasonable request.
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
Emma JCrosbie http:// orcid. org/ 0000- 0003- 0284- 8630
Emma RWoodward http:// orcid. org/ 0000- 0002- 6297- 2855
D GarethEvans http:// orcid. org/ 0000- 0002- 8482- 5784
REFERENCES
1 Zhang Y, Luo G, Li M, Guo P, Xiao Y, Ji H, Hao Y. Global patterns and trends in ovarian
cancer incidence: age, period and birth cohort analysis. BMC Cancer 2019;19:984.
2 Cancer Research UK. Ovarian cancer risk. Available: https://www. cancerresearchuk.
org/ health- professional/ cancer- statistics/ statistics- by- cancer- type/ ovarian- cancer/ risk-
factors [Accessed 9 Apr 2020].
3 Arnold M, Rutherford MJ, Bardot A, Ferlay J, Andersson TM- L, Myklebust Tor Åge,
Tervonen H, Thursfield V, Ransom D, Shack L, Woods RR, Turner D, Leonfellner S, Ryan
S, Saint- Jacques N, De P, McClure C, Ramanakumar AV, Stuart- Panko H, Engholm G,
Walsh PM, Jackson C, Vernon S, Morgan E, Gavin A, Morrison DS, Huws DW, Porter
G, Butler J, Bryant H, Currow DC, Hiom S, Parkin DM, Sasieni P, Lambert PC, Møller B,
Soerjomataram I, Bray F. Progress in cancer survival, mortality, and incidence in seven
high- income countries 1995-2014 (ICBP SURVMARK-2): a population- based study.
Lancet Oncol 2019;20:1493–505.
4 Alsop K, Fereday S, Meldrum C, deFazio A, Emmanuel C, George J, Dobrovic A, Birrer
MJ, Webb PM, Stewart C, Friedlander M, Fox S, Bowtell D, Mitchell G. Brca mutation
frequency and patterns of treatment response in BRCA mutation- positive women with
ovarian cancer: a report from the Australian ovarian cancer Study Group. J Clin Oncol
2012;30:2654–63.
5 Ryan NAJ, Glaire MA, Blake D, Cabrera- Dandy M, Evans DG, Crosbie EJ. The
proportion of endometrial cancers associated with Lynch syndrome: a systematic
review of the literature and meta- analysis. Genet Med 2019;21:2167–80.
6 Win AK, Jenkins MA, Dowty JG, Antoniou AC, Lee A, Giles GG, Buchanan DD,
Clendenning M, Rosty C, Ahnen DJ, Thibodeau SN, Casey G, Gallinger S, Le Marchand
L, Haile RW, Potter JD, Zheng Y, Lindor NM, Newcomb PA, Hopper JL, MacInnis RJ.
Prevalence and penetrance of major genes and polygenes for colorectal cancer.
Cancer Epidemiol Biomarkers Prev 2017;26:404–12.
7 Barrow E, Robinson L, Alduaij W, Shenton A, Clancy T, Lalloo F, Hill J, Evans DG.
Cumulative lifetime incidence of extracolonic cancers in Lynch syndrome: a report of
121 families with proven mutations. Clin Genet 2009;75:141–9.
8 Dominguez- Valentin M, Sampson JR, Seppälä TT, Ten Broeke SW, Plazzer J- P, Nakken
S, Engel C, Aretz S, Jenkins MA, Sunde L, Bernstein I, Capella G, Balaguer F, Thomas H,
Evans DG, Burn J, Greenblatt M, Hovig E, de Vos Tot Nederveen Cappel WH, Sijmons
RH, Bertario L, Tibiletti MG, Cavestro GM, Lindblom A, Della Valle A, Lopez- Köstner
F, Gluck N, Katz LH, Heinimann K, Vaccaro CA, Büttner R, Görgens H, Holinski- Feder
E, Morak M, Holzapfel S, Hüneburg R, Knebel Doeberitz Mvon, Loeffler M, Rahner N,
Schackert HK, Steinke- Lange V, Schmiegel W, Vangala D, Pylvänäinen K, Renkonen-
Sinisalo L, Hopper JL, Win AK, Haile RW, Lindor NM, Gallinger S, Le Marchand L,
Newcomb PA, Figueiredo JC, Thibodeau SN, Wadt K, Therkildsen C, Okkels H, Ketabi Z,
Moreira L, Sánchez A, Serra- Burriel M, Pineda M, Navarro M, Blanco I, Green K, Lalloo
F, Crosbie EJ, Hill J, Denton OG, Frayling IM, Rødland EA, Vasen H, Mints M, Neffa F,
Esperon P, Alvarez K, Kariv R, Rosner G, Pinero TA, Gonzalez ML, Kalfayan P, Tjandra
D, Winship IM, Macrae F, Möslein G, Mecklin J- P, Nielsen M, Møller P. Cancer risks
by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants:
findings from the prospective Lynch syndrome database. Genet Med 2020;22:15–25.
9 Vasen HF, Mecklin JP, Khan PM, Lynch HT. The International Collaborative group
on hereditary non- polyposis colorectal cancer (ICG- HNPCC). Dis Colon Rectum
1991;34:424–5.
10 Vasen HF, Watson P, Mecklin JP, Lynch HT. New clinical criteria for hereditary
nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the
International Collaborative group on HNPCC. Gastroenterology 1999;116:1453–6.
11 Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working
Group. Recommendations from the EGAPP Working group: genetic testing strategies
in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity
and mortality from Lynch syndrome in relatives. Genet Med 2009;11:35–41.
12 Shia J. Evolving approach and clinical significance of detecting DNA mismatch repair
deficiency in colorectal carcinoma. Semin Diagn Pathol 2015;32:352–61.
13 Bennett JA, Pesci A, Morales- Oyarvide V, Da Silva A, Nardi V, Oliva E. Incidence of
mismatch repair protein deficiency and associated clinicopathologic features in a
cohort of 104 ovarian endometrioid carcinomas. Am J Surg Pathol 2019;43:235–43.
14 Aysal A, Karnezis A, Medhi I, Grenert JP, Zaloudek CJ, Rabban JT. Ovarian
endometrioid adenocarcinoma: incidence and clinical significance of the morphologic
and immunohistochemical markers of mismatch repair protein defects and tumor
microsatellite instability. Am J Surg Pathol 2012;36:163–72.
15 Ryan NAJ, Evans DG, Green K, Crosbie EJ. Pathological features and clinical behavior
of Lynch syndrome- associated ovarian cancer. Gynecol Oncol 2017;144:491–5.
16 Ryan NAJ, Bolton J, McVey RJ, Evans DG, Crosbie EJ. BRCA and Lynch syndrome-
associated ovarian cancers behave differently. Gynecol Oncol Rep 2017;22:108–9.
17 Barrow E, Evans DG, McMahon R, Hill J, Byers R. A comparative study of quantitative
immunohistochemistry and quantum dot immunohistochemistry for mutation carrier
identification in Lynch syndrome. J Clin Pathol 2011;64:208–14.
18 Parra- Herran C, Lerner- Ellis J, Xu B, Khalouei S, Bassiouny D, Cesari M, Ismiil N,
Nofech- Mozes S. Molecular- Based classification algorithm for endometrial carcinoma
categorizes ovarian endometrioid carcinoma into prognostically significant groups.
Mod Pathol 2017;30:1748–59.
19 Pal T, Permuth- Wey J, Kumar A, Sellers TA. Systematic review and meta- analysis of
ovarian cancers: estimation of microsatellite- high frequency and characterization of
mismatch repair deficient tumor histology. Clin Cancer Res 2008;14:6847–54.
20 Murphy MA, Wentzensen N. Frequency of mismatch repair deficiency in ovarian
cancer: a systematic review. Int J Cancer 2011;129:1914–22.
21 Leskela S, Romero I, Cristobal E, Pérez- Mies B, Rosa- Rosa JM, Gutierrez- Pecharroman
A, Caniego- Casas T, Santón A, Ojeda B, López- Reig R, Palacios- Berraquero ML, García
Ángel, Ibarra J, Hakim S, Guarch R, López- Guerrero JA, Poveda A, Palacios J. Mismatch
repair deficiency in ovarian carcinoma: frequency, causes, and consequences. Am J
Surg Pathol 2020;44:649–56.
22 Capoluongo E, Ellison G, López- Guerrero JA, Penault- Llorca F, Ligtenberg MJL,
Banerjee S, Singer C, Friedman E, Markiefka B, Schirmacher P, Büttner R, van Asperen
CJ, Ray- Coquard I, Endris V, Kamel- Reid S, Percival N, Bryce J, Röthlisberger B, Soong
R, de Castro DG. Guidance statement on BRCA1/2 tumor testing in ovarian cancer
patients. Semin Oncol 2017;44:187–97.
23 Vergote I, Banerjee S, Gerdes A- M, van Asperen C, Marth C, Vaz F, Ray- Coquard I,
Stoppa- Lyonnet D, Gonzalez- Martin A, Sehouli J, Colombo N. Current perspectives on
recommendations for BRCA genetic testing in ovarian cancer patients. Eur J Cancer
2016;69:127–34.
24 Crosbie EJ, Ryan NAJ, Arends MJ, Bosse T, Burn J, Cornes JM, Crawford R, Eccles
D, Frayling IM, Ghaem- Maghami S, Hampel H, Kauff ND, Kitchener HC, Kitson SJ,
Manchanda R, McMahon RFT, Monahan KJ, Menon U, Møller P, Möslein G, Rosenthal
A, Sasieni P, Seif MW, Singh N, Skarrott P, Snowsill TM, Steele R, Tischkowitz M,
Evans DG, Manchester International Consensus Group. The Manchester international
consensus group recommendations for the management of gynecological cancers in
Lynch syndrome. Genet Med 2019;21:2390–400.
on December 8, 2020 by guest. Protected by copyright.http://jmg.bmj.com/J Med Genet: first published as 10.1136/jmedgenet-2020-107270 on 11 September 2020. Downloaded from