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Natthakrit Anansitthikorn, M.D., Suchanan Hanamornroongruang, M.D.
Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ailand.
Two-Antibody Staining Method, A Cost-Saving
Strategy for Universal Lynch Syndrome Screening
in Endometrial Cancers
ABSTRACT
Objective: Lynch syndrome is an autosomal dominant disorder that increases the risk of cancers in many sites. In
women, endometrial cancer is oen a sentinel tumor and thus immunohistochemistry for mismatch repair (MMR)
proteins MLH1, MSH2, MSH6 and PMS2 is encouraged as a screening test. To reduce cost, staining for only 2
MMR proteins PMS2 and MSH6 has been proposed. is study aimed to determine whether a 2-antibody staining
test is enough to screen for Lynch syndrome in endometrial cancer patients.
Materials and Methods: Cases of endometrial carcinoma with immunohistochemistry for 4 MMR proteins were
reviewed. Results of immunohistochemistry screening were compared between all four antibodies and only two
(PMS2 and MSH6) antibodies.
Results: Loss of expression of any MMR proteins was detected in 51 out of 203 cases (25.12%). Twenty-three cases
(45%) showed loss of MLH1 and PMS2; 13 cases (25%) showed loss of MSH2 and MSH6; ve cases (10%) showed
loss of MSH6; seven cases (14%) showed loss of PMS2 and three cases (6%) showed loss of MSH2. e 2-antibody
method detected 48 cases (94%) with a MMR deciency but failed to detect three cases (6%) with an isolate loss
of MSH2. e screening results from the 2-antibody method are 98.5% (200/203) in accordance with the original
4-antibody method.
Conclusion: e 2-antibody method is a quite eective option to screen for Lynch syndrome in endometrial cancers.
However, MSH2 mutations may be missed in a few cases.
Keywords: Endometrial carcinoma; Lynch syndrome; MMR proteins; MSH2 loss (Siriraj Med J 2022; 74: 108-113)
Corresponding author: Suchanan Hanamornroongruang
E-mail: suchananice@hotmail.com
Received 12 October 2021 Revised 6 December 2021 Accepted 13 December 2021
ORCID ID: https://orcid.org/ 0000-0003-4392-0811
http://dx.doi.org/10.33192/Smj.2022.14
INTRODUCTION
Lynch syndrome (LS) is an autosomal dominant
disorder which is caused by a germline mutation in mismatch
repaired (MMR) genes (MLH1, MSH2, MSH6 and PMS2)
or EpCAM deletion.
1
is syndrome is associated with
cancer in many organs such as the lower gastrointestinal
tract, endometrium, ovary, stomach, pancreas and brain.
2
However, the two most well-known cancers associated
with LS are colorectal and endometrium. Women with
LS have a lifetime risk of developing colorectal cancer
and endometrial cancer at 50%-85% and 40%-60%,
respectively.
3,4
Although the prevalence of LS in the
general population remains elusive
1
, about 1.7%-5% of
endometrial cancers are associated with this syndrome.
1,5-10
For women with LS, endometrial cancer is oen a
sentinel tumor.
11
According to a study by Meyer et al,
61% of women with LS linked endometrial cancer had
a second primary cancer, mostly colorectal cancer.
3
Identication of LS patients is the rst step in achieving
proper cancer surveillance and management. Clinical
Anansitthikorn et al.
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Original Article
SMJ
screening criteria such as Amsterdam II and revised
Bethesda guidelines have failed to detect a signicant
number of LS patients.
2,10,12
us, tumor-based testing -
immunohistochemistry (IHC) for MMR proteins (MLH1,
MSH2, MSH6 and PMS2) and/or microsatellite instability
(MSI) - is recommended.
1-4,9,13,14
Both IHC and MSI have a high sensitivity and
specicity, however, IHC is more practical and cost
eective.
3,4
In addition, MSI is less sensitive to the MSH6
germline mutation.
1,2
Many studies claim that IHC for
only PMS2 and MSH6 is sucient for initial screening
15-18
due to the binding properties of MMR heterodimer
complexes; MSH2 binds with MSH6 and MLH1 binds
with PMS2. With a 2-antibody approach, universal LS
screening in endometrial cancers is easier to achieve,
especially in places with limited resources. According to
an international survey on LS screening in gynecologic
cancers by Ryan et al, most pathologists still prefer the
4-antibody method.
19
In our experience and personal
communication with pathologists and gynecologists, most
were not condent or did not acknowledge in this cost-
saving method. Moreover, most studies on a 2-antibody
approach were conducted in cases of colorectal cancer.
us, the purpose of this study was to determine the
utility of the 2-antibody method in cases of endometrial
cancer compared to the original 4- antibody method.
MATERIALS AND METHODS
e study was conducted at the Department of
Pathology, Faculty of Medicine Siriraj Hospital, Mahidol
University, Bangkok, ailand and was approved by the
Siriraj Institutional Review Board (COA no. Si058/2020).
All cases of endometrial carcinoma with an
immunohistochemistry conducted for the 4 MMR proteins
between January 1
st
, 2010 and December 31
st
, 2019 were
included in this study. Cases without available H&E and
immunostained slides were excluded. Immunohistochemical
staining was performed by the Ventana BenchMark
ULTRA autostainer. Monoclonal antibodies for MMR
proteins were as follows: anti-MLH1 (M1; Ventana),
anti-PMS2 (EPR3947; Cell marque; USA), anti-MSH2
(G219-1129; Cell marque; USA) and anti-MSH6 (44;
Ventana; USA). Intact expression was dened as positive
nuclear staining within tumor cells. Loss of expression
was dened as absence of nuclear staining within tumor
cells. Stromal cells and nonneoplastic epithelial cells
were used as internal control. Cases with absence of
staining in internal control cells were excluded from the
study. Focal and weak nuclear staining was considered
as “cannot be determined”.
All H&E and immunostained slides were reviewed.
Results of immunohistochemistry screening were recorded
and compared between all four antibodies against two
(PMS2 and MSH6) antibodies. Clinical information
including age at diagnosis, specimen type was retrieved
from database records.
RESULTS
A total 203 cases of endometrial carcinoma with
an age range of 23-62 were included in this study. Most
specimens (97.54%) were from total or subtotal hysterectomy.
Endometrioid carcinoma was the most common histologic
subtype (89.66%). Specimen characteristics are summarized
in Table 1. Loss of expression of any MMR protein was
detected in 51 out of 203 cases (25.12%). Of these 51
cases with MMR deciency, 23 cases (45%) showed
loss of MLH1 and PMS2; 13 cases (25%) showed loss of
MSH2 and MSH6; ve cases (10%) showed loss of MSH6;
seven cases (14%) showed loss of PMS2 and three cases
(6%) showed loss of MSH2 (Table 2). e 2-antibody
method detected 48 cases (94%) with MMR deciency
but failed to detect three cases (6%) with an isolate loss
of MSH2. Isolate loss of MLH1 was not observed. One
MSH2-absent/ MSH6-intact case was dedierentiated
carcinoma while the others were endometrioid type.
All three cases showed convincing MSH6 expression in
20-40% of tumor cells, although the staining intensity in
one case (case 2) was slightly less than internal control.
(Fig 1) Overall, 98.5% (200/203) of the results from the
2-antibody method were in accordance with the original
4-antibody method.
DISCUSSION
Immunohistochemistry for MMR proteins has
been acknowledged as the most practical screening test
for LS and is performed routinely in many developed
countries. Rates of MMR deciency in endometrial
cancer range from 19.8%- 35%.
4,7,10,12,17,18,20,21
Recently,
Puangsricharoen et al reported MMR deciency in 34.9%
of 166 endometrial cancer cases in ailand.
22
e rate
of MMR deciency in this study is 25.12% which is
lower than a previous ai study. However, population
selection for this retrospective study was based on the
presence or absence of immunohistochemistry for four
MMR proteins and not randomized.
is study supports that IHC testing for only PMS2
and MSH6 is acceptable for initial screening. We found
that PMS2 can detect all cases with loss of both MLH1
and PMS2 and PMS2 alone. While MSH6 can detect
all cases with loss of both MSH2 and MSH6 and MSH6
alone. In fact, the 2-antibody method failed to identify
three cases with an isolate loss of MSH2.
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TABLE 1. Specimen Characteristics (n = 203).
TABLE 2. Mismatch repair protein immunohistochemical staining pattern (n=203).
Characteristic Value
Ageatdiagnosis,average(range),years 45.06(23-62)
Specimen type
Total or subtotal hysterectomy 198 (97.54)
Endometrialsamplingorcurettage 5(2.46)
Tumor cell type
Endometrioidcarcinoma 182(89.66)
Endometrioidcarcinoma-grade1 96(47.29)
Endometrioidcarcinoma-grade2 63(31.03)
Endometrioidcarcinoma-grade3 21(10.34)
Endometrioidcarcinoma-notgraded 2(0.99)
Serous carcinoma 9 (4.43)
Mixedcarcinoma 6(2.96)
Clear cell carcinoma 3 (1.48)
Undifferentiated carcinoma 1 (0.49)
Dedifferentiated carcinoma 1 (0.49)
Carcinosarcoma 1 (0.49)
Immunohistochemicalpattern Number(%)
NolossofnuclearexpressionofMMRproteins 152(74.88)
LossofnuclearexpressionofanyMMRproteins 51(25.12)
LossofnuclearexpressionofMLH1andPMS2 23(11.33)
LossofnuclearexpressionofMSH2andMSH6 13(6.40)
LossofnuclearexpressionofMSH6only 5(2.46)
LossofnuclearexpressionofPMS2only 7(3.45)
LossofnuclearexpressionofMSH2only 3(1.48)
Selected studies on patterns of IHC for 4 MMR
proteins in endometrial cancers were reviewed (Table 3).
Modica et al reported one case of isolate MSH2 loss which
showed MSI-H in MSI testing.
20
Meanwhile, a study by
Crim el al reported one case of isolate MLH1 loss which
is impossible in the 2- antibody method, however, there
was no associated germline mutation.
18
Pearlman et al
also reviewed 1730 colorectal cancer cases with IHC
conducted to screen for LS and reported isolate MSH2
loss in 19 cases; eight had an ambiguous MSH6 expression
Anansitthikorn et al.
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Fig 1. ree cases with an isolate loss of MSH2.
TABLE 3. Literature reports on patterns of immunohistochemical staining for MLH1, MSH2, MSH6 and PMS2 in
endometrial carcinomas
IHCpatterns
Reference Total Lossofanynuclearexpression
MLH1and MSH2and MSH6 PMS2 MLH1 MSH2
Intact PMS2 MSH6 only only only only Others
Modica2007 85 37 23 6 9 6 0 1* 3
(43.53%) (27.06%) (7.06%) (10.59%) (7.06%) (0%) (1.18%) (3.53%)
Garg2009 71 39 19 9 4 0 0 0 0
(54.93%) (26.76%) (12.68%) (5.63%) (0%) (0%) (0%) (0%)
Backes2009 140 110 24 4 2 0 0 0 0
(78.57%) (17.14%) (2.86%) (1.43%) (0%) (0%) (0%) (0%)
Mojtahed2011 40 21 9 4 4 0 0 0 2
(52.50%) (22.50%) (10%) (10%) (0%) (0%) (0%) (5%)
Egoavil2013 173 115 42 5 7 1 0 0 3
(66.47%) (24.28%) (2.89%) (4.05%) (0.58%) (0%) (0%) (1.73%)
LongQ2014 173 132 10 21 7 3 0 0 0
(76.30%) (5.78%) (12.14%) (4.05%) (1.73%) (0%) (0%) (0%)
WatkinsJC2017 242 194 39 4 3 2 0 0 0
(80.17%) (16.12%) (1.65%) (1.24%) (0.83%) (0%) (0%) (0%)
Crim2017 116 92 15 1 3 2 1* 0 2
(79.31%) (12.93%) (0.86%) (2.59%) (1.72%) (0.86%) (0%) (1.72%)
Puangsricharoen 156 99 42 10 5 0 0 0 0
2020 (63.46%) (26.92%) (6.41%) (3.21%) (0%) (0%) (0%) (0%)
Ourstudy2021 203 152 23 13 5 7 0 3* 0
(74.88%) (11.33%) (6.40%) (2.46%) (3.45%) (0%) (1.48) (0%)
*cases in which the 2-antibody method could not detect defects compared to the 4-antibody method
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and 11 had convincing MSH6 expression. Germline
testing of these cases revealed MSH2 mutations in 7/8
cases with ambiguous MSH6 expression and 9/11 cases
with convincing MSH6 expression.
23
In clinical practice,
isolate MSH2 loss is unusual. Genetic consultation and
further investigations, such as MSI testing or germline
testing should be performed. Failure to identify these
rare cases by the 2-antibody method may lead to missed
opportunities for cancer surveillance and carrier testing
in relatives at risk.
IHC interpretation for MMR proteins can be dicult,
especially in cases with focal and weak staining. ere
is still no ocial guideline that provides the cut-o
proportion and staining intensity in tumor cells. us,
discordance results and incorrect interpretation are
possible pitfalls of IHC testing.
CONCLUSION
e results from the 2-antibody method are in
high accordance with the original 4-antibody method.
However, the 2-antibody method fails to detect a few
cases of isolate MSH2 loss which have a potential to
represent those with MSH2 germline mutation.
ACKNOWLEDGEMENTS
e authors thank Assistant professor Suwanit
Therasakvichya and Dr. Pornnapa Lomthong from
Department of Obstetrics and Gynecology, Faculty of
Medicine Siriraj Hospital, Mahidol University for sharing
data and experience.
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