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Gynecologic Oncology 140 (2016) 226–233
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Gynecologic Oncology
journal homepage: www.elsevier.com/locate/ygyno
Clinicopathologic implications of DNA mismatch repair status in
endometrial carcinomas
Ayumi Shikama, Takeo Minaguchi ⁎, Koji Matsumoto, Azusa Akiyama-Abe, Yuko Nakamura, Hiroo Michikami,
Sari Nakao, Manabu Sakurai, Hiroyuki Ochi, Mamiko Onuki, Toyomi Satoh, Akinori Oki, Hiroyuki Yoshikawa
Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
H I G H L I G H T S
• 221 endometrial cancers were classified as sporadic or Lynch syndrome by MMR analyses.
• LS correlated with favorable prognostic factors and sensitivity to adjuvant therapies.
• Analyzing MMR and searching for LS may find patients with favorable survival and sensitivity to adjuvant therapies.
a r t i c l e
i n f o
Article history:
Received 23 August 2015
Received in revised form 24 November 2015
Accepted 27 November 2015
Available online 28 November 2015
Keywords:
Mismatch repair deficiency
Endometrial carcinoma
Lynch syndrome
Survival
MLH1 promoter methylation
a b s t r a c t
Objective. Endometrial carcinoma is the most common malignancy in women with Lynch syndrome caused
by mismatch repair (MMR) deficiency. We investigated the clinicopathologic significance of deficient MMR
and Lynch syndrome presumed by MMR analyses in unselected endometrial carcinomas.
Methods. We analyzed immunohistochemistry of MMR proteins (MLH1/MSH2/MSH6/PMS2) and MLH1 promoter methylation in primary endometrial carcinomas from 221 consecutive patients. Based on these results, tumors were categorized as sporadic or probable Lynch syndrome (PLS). Clinicopathologic variables and prognosis
were compared according to MMR status and sporadic/PLS classification.
Results. Deficient MMR showed only trends towards favorable overall survival (OS) compared with intact
MMR (p = 0.13), whereas PLS showed significantly better OS than sporadic (p = 0.038). Sporadic was significantly associated with older age, obesity, deep myometrial invasion, and advanced stage (p = 0.008, 0.01, 0.02
and 0.03), while PLS was significantly associated with early stage and Lynch syndrome-associated multiple cancer (p = 0.04 and 0.001). The trend towards favorable OS of PLS was stronger in advanced stage than in early
stage (hazard ratio, 0.044 [95% CI 0–25.6] vs. 0.49 [0.063–3.8]). In the subset receiving adjuvant therapies, PLS
showed trends towards favorable disease-free survival compared to sporadic by contrast with patients receiving
no adjuvant therapies showing no such trend (hazard ratio, 0.045 [95% CI 0–20.3] vs. 0.81 [0.095–7.0]).
Conclusions. The current findings suggest that analyzing MMR status and searching for Lynch syndrome may
identify a subset of patients with favorable survival and high sensitivity to adjuvant therapies, providing novel
and useful implications for formulating the precision medicine in endometrial carcinoma.
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
In the western world, endometrial carcinoma is the most common
gynecologic malignancy. Based on differences in clinicopathologic characteristics, there are two subtypes of endometrial carcinoma. Type I tumors, estrogen-related, are caused by the unopposed estrogen and
follow endometrial hyperplasias. This type of tumors is characterized
by endometrioid histology, occurrence in perimenopausal, obese
⁎ Corresponding author at: Department of Obstetrics and Gynecology, Faculty of
Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
E-mail address: [email protected] (T. Minaguchi).
http://dx.doi.org/10.1016/j.ygyno.2015.11.032
0090-8258/© 2015 Elsevier Inc. All rights reserved.
women, superficial myometrial invasion, and favorable prognosis. On
the contrary, type II tumors, estrogen-unrelated, develop from atrophic
endometrium. This type of tumors is generally characterized by nonendometrioid histology, occurrence in older, postmenopausal, thin
women, deep myometrial invasion, and poor prognosis. Defect in specific signal transduction pathways are known to drive endometrial carcinogenesis. These pathways include the phosphatidylinositol 3-kinase
(PI3K)-PTEN-Akt pathway, the p53 pathway, and the DNA mismatch repair (MMR) mechanism.
The MMR system is a strand-specific DNA repair mechanism. The
role of MMR gene is to maintain genomic integrity by correcting base
substitutions mismatches and small insertion-deletion mismatches
that are generated by errors in base pairing during DNA replication. Of
A. Shikama et al. / Gynecologic Oncology 140 (2016) 226–233
Table 1
Patient characteristics.
Characteristic
Number (n =
221)
Median age (range)
Median BMI (kg/m2 range)
FIGO stage
I
Ia
Ib
Ic
II
IIa
IIb
III
IIIa
IIIc
IV
IVa
IVb
Histotype
Endometrioid
G1
G2
G3
Serous
Adenosquamous
Clear cell
Poorly differentiated
Undifferentiated
Mixed epithelial
Myometrial invasion N 1/2
Lymophovascular space invasion
Carcinoma of the lower uterine segment
57.4 (26–84)
23.9 (17.0–43.9)
Primary treatment
Surgery
Lymphadenectomy
Lymph node sampling
Lymph node not removed
Adjuvant chemotherapy
TC
CAP
Adjuvant radiotherapy
Lynch syndrome-associated multiple cancer
%
227
results of our molecular analyses. The current findings will provide
novel and useful implications for the precision medicine in endometrial
carcinoma.
2. Materials and methods
2.1. Patients and specimens
128
22
76
30
26
10
16
43
20
23
24
2
22
196
115
56
25
12
4
4
1
1
3
81
84
13
221
171
21
29
60
55
4
58
15
58
10
34
14
12
5
7
19
9
10
11
1
10
89
52
25
11
5
2
2
0
0
1
37
38
6
100
77
10
13
27
25
2
26
7
Abbreviations: FIGO = International Federation of Gynecology and Obstetrics; TC =
paclitaxel and carboplatin combination; CAP = cyclophosphamide, doxorubicin, and cisplatin combination.
the MMR genes, heterodimeric complexes of MSH2 and MSH6 recognize mismatch nucleotides, and heterodimeric complexes of MLH1
and PMS2 are then recruited to excise the mismatched nucleotides.
MMR deficiency is detected as microsatellite instability (MSI) or loss
of MMR protein expression by immunohistochemistry (IHC). In Lynch
syndrome, MMR deficiency results from germline mutations of MLH1,
MSH2, MSH6, and PMS2. Deficient MMR is also detected in sporadic cancer due to hypermethylation of MLH1 promoter. MMR deficiency is reported to contribute to oncogenesis in some types of malignancy,
including colon, gastric and endometrial cancers. In patients with
colon cancer, multiple studies have reported that deficient MMR is associated with favorable prognosis [1,2]. Furthermore, colon cancers with
deficient MMR have been demonstrated to be more responsive to 5FU-based chemotherapy compared with tumors with intact MMR [1–
3]. In endometrial cancer, most studies reported on the association between MSI and clinical outcome and it is controversial whether MSI status improves patient survival [4–7]. A few studies reported on the
association between MMR protein expression and clinical outcome,
but prognostic significance of MMR protein expression is yet to be determined [8,9]. Moreover, germline mutation of BRCA1/2, another cancer predisposition gene, is reported to be associated with improved
survival and high response to platinum-based chemotherapy in ovarian
cancer [10,11]. Here, we have investigated the clinicopathologic significance of deficient MMR and presumed Lynch syndrome based on the
The Ethical Committee of the University of Tsukuba Hospital approved the study protocol. All patients diagnosed with endometrial carcinoma, who were treated in the Department of Obstetrics and
Gynecology at the University of Tsukuba Hospital between 1999 and
2009, were identified through our database. A total of consecutive 221
patients with endometrial carcinomas were included in the present
study, and their medical records were reviewed. A median follow-up
duration was 92 months (range, 3–181 months). All patients provided
written informed consent. Staging was performed based on the criteria
of International Federation of Gynecology and Obstetrics (FIGO, 1988).
Endometrioid adenocarcinomas were subclassified into three grades
(G1, G2, and G3) according to the FIGO criteria. Treatment of patients
was described elsewhere [12]. Table 1 summarizes the patient
characteristics.
We categorized patients as sporadic or probable Lynch syndrome
(PLS) based on the results of our MMR analyses as follows [13–15]. A patient with endometrial carcinoma with intact expression of all MLH1,
MSH2, MSH6, and PMS2 proteins was considered as having a sporadic
tumor. A patient with loss of MLH1 expression, but presence of MLH1
promoter methylation, was also considered sporadic. Patients with
loss of MSH2, MSH6, or PMS2 expression were considered as PLS. Patients with tumors with loss of MLH1 and absence of MLH1 promoter
methylation were also considered PLS.
2.2. Immunohistochemistry (IHC)
IHC procedures were conducted as described previously [16]. Monoclonal antibodies used are MLH1 (BD Pharmingen, Franklin Lakes, NJ),
MSH2 (EMD Millipore corporation, Billerica, MA, USA), MSH6 (Epitomics, Burlingame, CA, USA), and PMS2 (BD Pharmingen). The corresponding normal tissue provided an internal positive control, and
cases already known as mutated were used for negative controls for
MMR proteins. Loss of MMR protein expression was defined as the absence of nuclear staining in tumor cells in the presence of adjacent
non-neoplastic cells with nuclear staining. Blinded for clinical and pathologic parameters, immunoreaction was assigned independently by
two investigators (AS and TM), and any discrepancies were resolved
by conferring over a multiviewer microscope. Tumors were classified
as deficient MMR, if expression loss of one or more MMR proteins was
detected. Fig. 1 shows examples of IHC staining for MMR proteins in endometrial carcinomas. PTEN and p53 expressions were examined and
evaluated as previously described [12].
2.3. DNA extraction
Genomic DNA was extracted from tumors and corresponding normal areas of formalin-fixed, paraffin-embedded endometrial tissues
using blackPREP DNA kit (Analytik Jena, Jena, Germany) according to
the manufacturer's instructions. For isolating DNA from normal tissues,
we used FFPE tissue blocks (uterus, ovary, or omentum) containing only
normal tissues without any tumor tissues, which was confirmed by
checking corresponding slides by microscopy.
2.4. MSI analysis
MSI status was analyzed using five fluorescence-labeled microsatellite markers, BAT25, BAT26, D2S123, D5S346 and D17S250, recommended by the National Cancer Institute [17]. Tumors showing allelic
228
A. Shikama et al. / Gynecologic Oncology 140 (2016) 226–233
Fig. 1. IHC staining patterns of MLH1, MSH2, MSH6, PMS2 in endometrial carcinomas (×100).
shift at two or more markers were classified as MSI-high, at one marker
as MSI-low, and tumors with no allelic shift at any marker as microsatellite stable (MSS).
72 °C. The PCR products were resolved by electrophoresis on a 3% agarose gel and visualized by ethidium bromide staining and UV
illumination.
2.5. Methylation-specific PCR (MS-PCR)
2.6. Statistical analyses
Modification of genomic DNAs with sodium bisulfite was performed
using the EpiTect kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. MS-PCR was performed as described elsewhere
with slight modifications [18]. Bisulfite-modified DNAs served as templates using the primer sets specific for methylated and unmethylated
CpG islands in the MLH1 promoter. PCR mixture contained 1 × PCR
buffer (Perkin-Elmer, Foster City, CA), 2.5 mM MgCl2, 200 μM of each
dNTP, 0.5 μM of each PCR primer, 0.5 U of AmpliTaq Gold (PerkinElmer), and 50 ng of DNA in a final volume of 25 μl. Amplification
proceeded at 95 °C for 10 min, followed by 40 cycles at 94 °C for 45 s,
60 or 62 °C for 30 s, and 72 °C for 60 s and a final 5-min extension at
Differences in proportions were evaluated by the Fisher's exact test.
Kaplan-Meier survival curves were calculated and compared statistically using the log-rank test. Univariate and multivariate analyses for prognostic factors was conducted by the Cox proportional hazard model. For
all analysis p-value b 0.05 was considered statistically significant. All
statistical analyses were performed using SPSS version 21.
3. Results
In our IHC analyses and MLH1 MS-PCR in primary endometrial carcinomas from 221 unselected patients, deficient MMR was observed in 62
8 (89%)
144 (68%)
0.170
1 (11%)
36 (17%)
0.538
20 (83%)
132 (67%)
0.077
2 (8%)
35 (18%)
0.193
52 (84%)
100 (63%)
0.002
4 (6%)
33 (21%)
0.006
32 (80%)
120 (66%)
0.063
4 (10%)
33 (18%)
0.151
54 (78%)
98 (65%)
0.028
6 (9%)
31 (20%)
0.021
128 (66%)
24 (86%)
0.027
35 (18%)
2 (7%)
0.113
118 (65%)
12 (75%)
0.309
33 (18%)
2 (13%)
0.432
Abbreviations: IHC = immunohistochemistry; MSI = microsatellite instability; MS-PCR
= methylation-specific PCR; MMR = mismatch repair; PLS = probable Lynch syndrome.
3 (19%)
7 (44%)
1 (6%)
0 (0%)
3 (19%)
0 (0%)
0 (0%)
13 (81%)
2 (13%)
6 (38%)
8 (50%)
0 (0%)
10 (63%)
3 (19%)
4 (25%)
79 (44%)
51 (29%)
35 (19%)
29 (16%)
64 (35%)
18 (10%)
40 (22%)
161 (89%)
22 (12%)
74 (41%)
70 (39%)
11 (6%)
99 (55%)
60 (33%)
8 (5%)
0.008
0.58
0.11
0.01
0.12
0.27
0.05
0.19
0.14
0.02
0.52
0.51
0.04
0.03
0.001
5 (18%)
8 (29%)
2 (7%)
0 (0%)
6 (21%)
1 (4%)
2 (7%)
23 (82%)
1 (4%)
5 (18%)
11 (39%)
2 (7%)
21 (75%)
4 (14%)
7 (25%)
83 (43%)
55 (29%)
35 (18%)
30 (16%)
67 (35%)
18 (9%)
43 (22%)
173 (90%)
24 (12%)
76 (39%)
73 (38%)
11 (6%)
107 (55%)
63 (33%)
8 (4%)
0.018
0.11
0.21
0.044
0.53
0.38
0.11
0.019
0.11
0.17
0.47
0.17
0.45
0.42
0.53
68 (45%)
39 (26%)
28 (18%)
16 (11%)
50 (33%)
12 (8%)
27 (18%)
130 (86%)
14 (9%)
52 (34%)
57 (38%)
11 (7%)
89 (59%)
45 (30%)
10 (7%)
20 (29%)
24 (35%)
9 (13%)
14 (20%)
23 (33%)
7 (10%)
18 (26%)
66 (96%)
11 (16%)
29 (42%)
27 (39%)
2 (3%)
39 (57%)
22 (32%)
5 (7%)
0.055
0.34
0.06
0.16
0.08
0.54
0.40
0.51
0.005
0.018
0.029
0.07
0.41
0.30
0.11
77 (43%)
53 (30%)
34 (19%)
27 (15%)
64 (35%)
16 (9%)
38 (21%)
160 (88%)
15 (8%)
60 (33%)
63 (35%)
13 (7%)
106 (59%)
53 (29%)
10 (6%)
PLS
n = 16
Sporadic
n = 181
Sporadic
n = 193
Unmethylated
n = 152
229
Abbreviations: MMR = mismatch repair; MSI = microsatellite instability; PLS = probable Lynch syndrome; BMI = body mass index; MI = myometrial invasion; LVI = lymophovascular space invasion; LUS = carcinoma of the lower uterine segment; FIGO = International Federation of Gynecology and Obstetrics.
Lynch syndrome-associated multiple cancers include colorectal, endometrial, gastric, small bowel, ovarian, pancreatic, ureter and renal pelvic, biliary tract, and brain (glioblastoma in Torcot syndrome), sebaceous gland adenomas, and
keratoacanthomas in Muir-Torre syndrome.
0.012
11 (28%)
10 (25%)
3 (8%)
3 (8%)
9 (23%)
3 (8%)
7 (18%)
36 (90%)
10 (25%)
21 (53%)
21 (53%)
0 (0%)
22 (55%)
14 (35%)
5 (13%)
2 (5%)
35 (20%)
0.002
0.21
0.006
0.037
0.012
0.34
0.22
0.41
0.24
0.48
0.18
0.24
0.22
0.47
0.03
0.005
73 (46%)
48 (30%)
33 (21%)
26 (16%)
60 (38%)
15 (9%)
35 (22%)
140 (88%)
16 (10%)
59 (37%)
57 (36%)
11 (7%)
89 (56%)
49 (31%)
7 (4%)
36 (86%)
116 (64%)
15 (24%)
15 (24%)
4 (6%)
4 (6%)
13 (21%)
4 (6%)
10 (16%)
56 (90%)
9 (15%)
22 (35%)
27 (44%)
2 (3%)
39 (63%)
18 (29%)
8 (13%)
0.021
Age ≥ 60
Pre-menopause
Null Parity
BMI N 30
Hypertension
Hyperlipidemia
Diabetes
Endometrioid (vs. Non-endometrioid)
G3
MI N 1/2
LVI
LUS
FIGO stage I
FIGO stage III-IV
Lynch syndrome-associated multiple
cancer
2 (5%)
35 (19%)
MSI-H
n = 40
0.034
P-value
32 (82%)
120 (66%)
Intact
n = 159
P-value
Deficient
n = 62
Positive p53
MSI
P-value
MMR
Negative PTEN
Clinicopathologic variables
Expression
Table 3
Relationships between clinicopathologic variables and results of MMR analyses.
Table 2
Results of IHC, MSI and MLH1 MS-PCR.
MLH1
Negative (n = 39)
Positive (n = 182)
PMS2
Negative (n = 42)
Positive (n = 179)
MSH2
Negative (n = 9)
Positive (n = 212)
MSH6
Negative (n = 24)
Positive (n = 197)
MMR
Deficient (n = 62)
Intact (n = 159)
MSI
MSI-H (n = 40)
MSI-L + MSS (n = 181)
MLH1 promoter
Methylated (n = 69)
Unmethylated (n = 152)
Sporadic/PLS
Sporadic (n = 193)
PLS (n = 28)
Sporadic/PLS (+MSI)
Sporadic (n = 181)
PLS (n = 16)
Methylated
n = 69
MSI-L +
MSS
n = 181
P-value
MLH1 promoter
P-value
Sporadic/PLS
PLS
n = 28
P-value
Sporadic/PLS (+MSI)
P-value
cases (28%) and MLH1 promoter methylation in 69 cases (31%)
(Table 2). MLH1 promoter methylation was significantly associated
with loss of MLH1 expression by IHC (p = 2.2E-15; Table S1). We also
conducted MSI analysis in all cases. Deficient MMR was found to be significantly associated with MSI (p = 5.6E − 26; Table S1). Together,
these results support the validity of our IHC analyses. Comparison between expressions of MMR proteins and those of PTEN/p53 tumor suppressors showed that MMR deficiency was significantly associated with
negative PTEN and inversely with positive p53 (p = 0.002 and 0.006;
Table 2). Expressions of MLH1, PMS2, and MLH1 methylation also
showed the similar significant correlations with PTEN and p53
(Table 2). We next correlated our IHC results to clinicopathologic variables. MMR deficiency was found to be significantly associated with
age b 60 and Lynch syndrome-associated multiple cancer, and inversely
with null parity, BMI N30, and hypertension (p = 0.002, 0.03, 0.006,
0.037, and 0.012; Table 3). MLH1 methylation was found to be significantly associated with age b 60, BMI N 30, and endometrioid histology
(p = 0.018, 0.044, and 0.019; Table 3).
Subsequently we examined the prognostic significance of MMR and
MSI status in our survival analysis. Patients with deficient MMR showed
only a tendency towards better overall survival than those with intact
MMR without statistical significance (p = 0.13; Fig. 2A). MSI status
did not show any correlation with overall survival (data not shown).
Aiming to next investigate the possible clinicopathologic difference between sporadic and Lynch syndrome-associated endometrial carcinomas, we categorized tumors according to the results of MMR protein
expression and MLH1 promoter methylation. Consequently, 28 cases
(13%) were classified as PLS, and 193 cases (87%) were classified as sporadic. Comparison with clinicopathologic variables showed that sporadic group was significantly associated with older age (≥ 60), BMI N 30,
deep myometrial invasion (N 1/2), and advanced FIGO stage (III-IV)
(p = 0.008, 0.01, 0.02 and 0.03; Table 3), while PLS group was significantly associated with early FIGO stage (I) and Lynch syndromeassociated multiple cancer (p = 0.04 and 0.001; Table 3). Subsequent
survival analyses revealed that PLS group had significantly better overall
survival than sporadic group (p = 0.038; Fig. 2B). Interestingly, the
trend towards favorable overall survival of PLS group against sporadic
0.043
0.17
0.17
0.07
0.14
0.20
0.022
0.28
0.61
0.51
0.27
0.38
0.37
0.19
0.010
A. Shikama et al. / Gynecologic Oncology 140 (2016) 226–233
230
A. Shikama et al. / Gynecologic Oncology 140 (2016) 226–233
group was stronger in advanced-stage disease than in early-stage disease (Fig. 2C, D; hazard ratio, 0.044 [95% CI 0–25.6] vs. 0.49 [0.063–
3.8]; Table S2). In order to identify the mechanism underlying this prognostic difference between sporadic and PLS groups, we further conducted subset analyses on disease-free survivals (DFS) in patients who
received adjuvant therapies. In the patients receiving adjuvant therapies, PLS group showed trends towards favorable DFS compared to sporadic group, whereas no such trend was observed in patients receiving
no adjuvant therapies (Fig. 2E, F; hazard ratio, 0.045 [95% CI 0–20.3]
vs. 0.81 [0.095–7.0]; Table S2). We moreover compared the trends of
PLS towards favorable DFS between patients receiving adjuvant chemotherapies alone and those receiving adjuvant radiotherapies alone, but
both were found to be similar (Fig. 2G, H; hazard ratio, 0.043 [95% CI
0–66.2] vs. 0.043 [0–1303.2]; Table S2).
Our survival analyses showed that, in addition to the factor of PLS
against sporadic, factors of age b 60, endometrioid histology, G1, MI
≤ 1/2, absent LVI, FIGO stage I/II, and no adjuvant therapy received
were also significantly associated with favorable overall survival (p =
0.001, 2.2E−9, 1.8E− 5, 5.7E− 7, 2.9E−5, 9.5E− 11, and 5.0E− 9 by
the log-rank test). Among these significant prognostic factors, subsequent multivariate analysis exhibited that endometrioid histology,
early FIGO stage, and no adjuvant therapy received were found to be
significant and independent for favorable overall survival (p =
1.9E−4, 0.02, and 0.017; Table 4).
4. Discussion
Our IHC analyses showed that deficient MMR was significantly associated with negative PTEN and inversely correlated with positive p53
(Table 2), being consistent with previous publications [19−21]. However, deficient MMR was found to be significantly associated inversely
with null parity, obesity, and hypertension (Table 3). These associations
are generally uncommon in type I endometrial cancer where mutant
PTEN and wild-type p53 are frequent, suggesting that the characteristics
of MMR-deficient endometrial carcinoma are distinct from those of typical type I tumor. This may be because more diverse range of oncogenes
and tumor suppressor genes than just PTEN is targeted by MMR deficiency. By contrast, MLH1 methylation was significantly associated
with obesity and endometrioid histology (Table 3), probably reflecting
sporadic origin.
We next examined the prognostic significance of MMR and MSI status. MMR deficiency determined by IHC alone showed only trends towards better overall survival compared with intact MMR without
statistical significance (Fig. 2A). MSI status did not exhibit any correlation with overall survival (data not shown). Therefore, we further examined the clinicopathologic significance of PLS determined by MMR
IHC plus MLH1 promoter methylation, as germline mutation of BRCA1/
2, which is another cancer predisposition gene, is reported to be associated with improved survival and high response to platinum-based chemotherapy in ovarian cancer [10,11]. Sporadic group was significantly
associated with older age, obesity, deep myometrial invasion, and advanced FIGO stage, while PLS group was significantly associated with
early FIGO stage and Lynch syndrome-associated multiple cancer
(Table 3). Subsequent survival analyses revealed that overall survival
of PLS group was significantly better than that of sporadic group
(Fig. 2B). These findings suggest that favorable prognosis of PLS group
may be due to superficial myometrial invasion and early stage, possibly
reflecting the indolent biological tumor behavior. If the favorable prognostic impact by PLS group is mainly attributed only to early stage and
superficial myometrial invasion, the both prognostic effects by early
stage or superficial myometrial invasion should be diluted in
advanced-stage diseases than in early-stage diseases. However, our subset survival analyses showed that the tendency of PLS group towards favorable survival was observed in advanced-stage (III-IV) diseases, while
no such trend was observed in early-stage (I-II) diseases (Fig. 2C, D;
Table S2), suggesting that factors other than stage and depth of
myometrial invasion are also contributing to favorable prognosis. As
an attempt to identify other mechanisms of the prognostic difference
between PLS and sporadic groups, we next conducted subset analyses
on DFS in patients who received adjuvant therapies. Interestingly, the
trend of PLS group towards better DFS was observed in patients receiving adjuvant therapies, whereas no such trend was observed in patients
receiving no adjuvant therapies (Fig. 2E, F; Table S2). These observations implicate that the prognostic difference between PLS and sporadic
groups may be also attributed to higher sensitivity to adjuvant therapies
in PLS group. It can be theoretically thought that tumors with deficient
MMR are more vulnerable to genotoxic therapies than MMR-intact tumors. Sporadic group must include much less MMR-deficient tumors
than PLS group, possibly explaining the prognostic impact of PLS over
sporadic (Fig. 2B). Likewise, advanced-stage diseases include more patients receiving adjuvant therapies compared to early-stage diseases,
possibly explaining the prognostic difference of PLS between the advanced and early-stage diseases (Figs. 2C, D; Table S2). Regarding
MMR deficiency and chemosensitivity, Bertagnolli et al. reported on
colon cancer that defect MMR, determined by MSI testing or MMR
IHC, was significantly associated with improved DFS in stage III patients
treated with adjuvant irinotecan, fluorouracil, and leucovorin [22].
Zaanan et al. reported that defect MMR was significantly associated
with improved DFS in patients with stage III colon cancer receiving adjuvant 5-FU–oxaliplatin chemotherapy [23]. Sinicrope et al. reported on
colon cancer that MMR-deficient tumors with suspected germline mutations were associated with improved DFS after 5-FU-based treatment
compared with sporadic tumors where no benefit was observed [1]. As
for endometrial cancer, Kato et al. have recently reported that response
rate of first-line platinum-based chemotherapy in evaluable cases was
slightly higher in MMR-deficient cases (determined by MMR IHC; 67%
vs. 44%, p = 0.34), and MMR-deficient cases had significantly better
progression-free and overall survival compared with MMR-intact
cases [24]. In the current study, however, MMR deficiency in itself
showed only a tendency towards favorable overall survival without statistical significance (Fig. 2A). This discrepancy may be due to the difference of adjuvant treatment strategies between institutions. Regarding
MMR and sensitivity to adjuvant radiotherapy in endometrial cancer,
Resnick et al. reported a significant increase in overall (p = 0.003)
and progression-free (p = 0.004) survival in a subgroup of patients
with MMR-deficient, non-endometrioid tumors treated with adjuvant
radiotherapy compared to those with intact MMR [25]. Further studies
are warranted to clarify the prognostic significance of MMR status in endometrial carcinoma.
In addition to the factor of PLS against sporadic, our survival analyses
showed that younger age, endometrioid histology, G1, superficial
myometrial invasion, absent LVI, early FIGO stage, and no adjuvant therapy received were also significant prognostic factors for favorable overall survival. In the subsequent multivariate analysis, PLS against
sporadic was not left significant (Table 4), keeping in line with our
above findings that PLS showed significant correlations with other prognostic factors, i.e. younger age, superficial myometrial invasion, and
early stage (Table 3).
There are two publications which adopted the same criteria as ours
to predict Lynch syndrome among unselected endometrial cancers.
Bruegl et al. reported 10.5% [13] and Buchanan et al. reported 8.3%
[15] as predicted Lynch syndrome, both frequencies being equivalent
to ours (13%). The latter also examined germline MMR mutation, and
sensitivity, specificity, positive predictive value, and negative predictive
value were reported as 100%, 94.5%, 36.2%, and 100%, respectively. Accordingly, all of the germline MMR mutation carriers in the current
study are supposed to be categorized in the PLS group. Therefore, although positive predictive value may be not high, we consider that
the clinicopathologic associations of the current study are significant.
We also examined incorporating MSI status into the criteria to predict
PLS as follows [26,27]. Patients with MSI-L/MSS and intact all MMR expression, and patients with MSI-H, MLH1 loss, and MLH1 methylation
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231
232
A. Shikama et al. / Gynecologic Oncology 140 (2016) 226–233
References
Table 4
Multivariate analysis of favorable prognostic factors for overall survival.
Prognostic factor
HR
95% CI
P-value
PLS (vs. Sporadic)
Age b 60 (vs. ≥60)
Endometrioid (vs. Non-endometrioid)
G1 (vs. Others)
MI ≤1/2 (vs. N1/2)
LVI absent (vs. present)
FIGO stage I/II (vs. III/IV)
AdjTx not received (vs. received)
2.0E-06
0.94
0.23
0.61
1.01
0.93
0.35
0.19
0–∞
0.49–1.80
0.11–0.50
0.29–1.32
0.44–2.32
0.47–1.82
0.15–0.85
0.05–0.74
0.97
0.85
0.0002
0.21
0.97
0.82
0.02
0.017
Abbreviations: HR = hazard ratio; 95% CI = 95% confidence interval; PLS = probable
Lynch syndrome; MI = myometrial invasion; LVI = lymphovascular space invasion;
FIGO = International Federation of Gynecology and Obstetrics; AdjTx = adjuvant therapy.
were both considered sporadic. Patients with MSI-H and MSH2/MSH6/
PMS2 expression loss, and patients with MSI-H, MLH1 loss, and
unmethylated MLH1 were both considered PLS. Consequently, 181
cases were classified as sporadic, and 16 cases (8%) were classified as
PLS, designated as Sporadic/PLS (+MSI) (Table 2). Significant associations of PLS (+MSI) with Lynch syndrome-associated multiple cancer
remained (p = 0.01; Table 3), but those with early stage or superficial
myometrial invasion were lost (p = 0.37 and 0.51, respectively;
Table 3). PLS (+MSI) showed only trends towards favorable overall survival compared to sporadic (+ MSI) (p = 0.19, data not shown).
Whether or not MSI status should be incorporated into the criteria for
PLS is beyond the scope of the present study.
In conclusion, we have demonstrated here that categorizing patients
into PLS and sporadic based on MMR analyses exhibited significantly
better overall survival for PLS group compared to sporadic group. This
prognostic impact for favorable survival of PLS over sporadic may be attributed to higher sensitivity to adjuvant therapies as well as indolent
tumor characteristics such as early stage and superficial myometrial invasion. Accordingly, advanced-stage patients of PLS group may benefit
more from adjuvant therapies compared to sporadic group. Altogether,
our observations suggest that analyzing MMR status and searching for
Lynch syndrome may identify a subset of patients with higher sensitivity to adjuvant therapies and favorable survival in endometrial carcinomas. Besides, recent emerging evidence suggests that differentially
methylated microRNAs serve as markers for distinguishing MMRdeficient sporadic from Lynch-associated tumors and for early progression to tumorigenesis in endometrium and colon [28]. We believe that
further genetic and epigenetic MMR analyses will provide useful information for formulating precision medicine in endometrial carcinomas.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ygyno.2015.11.032.
Conflict of interest statement
The authors have no conflict of interest to disclose.
Acknowledgments
This study was partially supported by the Grant-in-Aid for Scientific
Research (No. 24592503) from the Ministry of Education, Culture,
Sports, Science, and Technology, Tokyo, Japan.
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