10 1016j prp 2018 08 015

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Pathology - Research and Practice
journal homepage: www.elsevier.com/locate/prp
A novel case of endometrial dedifferentiated adenocarcinoma associated
with MLH1 promotor hypermethylation and microsatellite instability
Xinying Liua, Xiaodan Liua,1, Xiaoqiang Wangb, Rui Wub, Kuangen Zhanga, Beiying Liuc,
Qichen Liud, Yakun Shaoa, Ran Tanga, Jiangfeng Youa, Fei Peia,b,⁎
a Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, PR China
bDepartment of Pathology, Peking University Third Hospital, Beijing, 100191, PR China
c School of Mechanical Engineering, University of Science& Technology Beijing, Beijing, 100083, PR China
d College of Sciences, Georgia Institute of Technology, North Avenue, Atlanta, GA, 30332, United States
A R T I C L E I N F O
Keywords:
Endometrial dedifferentiated carcinoma
Microsatellite instability
MLH1
PD-1
PD-L1
A B S T R A C T
Endometrial dedifferentiated carcinoma is a rare, malignant tumor whose molecular alterations have not been
clarified yet. We report a novel case of a 61-year old woman who presented with irregular vaginal bleeding after
menopause and a 3 cm uterus mass. Histology revealed endometrial dedifferentiated adenocarcinoma, a rare
subtype comprised of undifferentiated adenocarcinoma. The patient still survived 1 year after surgery without
chemotherapy and radiotherapy. Immunohistochemistry revealed loss of MLH1/PMS2 expression and retained
MSH2/MSH6 expression. Consistently, microsatellite instability was detected indicative of high microsatellite
instability (MSI-H). No BRAF V600E, KRAS and POLE mutations were identified. Remarkably, the promoter
regions of mutL homolog 1(MLH1) were methylated. Furthermore, several tumor cells were PD-L1 positive in
this case with a concentration at the infiltrating tumor edge indicating MSI-H in endometrial dedifferentiated
adenocarcinoma is a potential predictive factor for response to immunotherapy targeting the PD-1 or its ligand
PD-L1.
1. Introduction
Endometrial cancers(EC) is a very common cancer of uterine corpus
which are pathogenetically divisible into type I and type II tumors.
Type I carcinomas is low-grade endometrioid adenocarcinoma and its
variants including well-differentiated (FIGO grade 1) and moderately
differentiated endometrioid carcinoma (FIGO grade 2). Over 10,000
women will die from endometrial cancer in the United States every
year, largely because of resistance to therapy in the advanced/recurrent
disease setting [1]. Combination chemotherapy in these circumstances
offers response rates (RR) ranging from 30 to 60% and median pro-
gression free survival of 5–14 months [2]. For this reason, it is critical to
identify molecular subgroups that are amenable to targeted therapies,
including immunotherapy.
Furthermore, undifferentiated carcinoma of the endometrium is a
malignant epithelial neoplasm with no differentiation. Dedifferentiated
carcinoma is composed of undifferentiated carcinoma and a second
component of either FIGO grade 1 or 2 endometrioid carcinoma.
Undifferentiated carcinomas are uncommon, and the behavior of these
tumors is highly aggressive, with recurrence or death from tumor in
55–95% of women. There is a possible association with Lynch syn-
drome and approximately on-half of tumors display high microsatellite-
instability with MLH1 promoter methylation and loss of expression of
MLH1 and PMS2.
Microsatellite instability (MSI), caused by loss-of-function defects in
DNA mismatch repair genes (MMRs), can lead to increased suscept-
ibility to a variety of neoplasms, including colorectal carcinoma (CRC)
and endometrial carcinoma [3]. Sporadic MSI is caused by hy-
permethylation of the promoter region of the repair gene MLH1, while
Lynch syndrome is caused by germline mutation of one of the MMRs
(MLH1, MSH2, MSH6, or PMS2) inherited in an autosomal dominant
pattern. Microsatellite instability-high(MSI-H) is a potential predictive
factor for response ton immunotherapy targeting the PD-1 or its ligand
PD-L1. However, the relationship between PD-L1 expression and MSI-H
https://doi.org/10.1016/j.prp.2018.08.015
Received 16 May 2018; Received in revised form 4 August 2018; Accepted 18 August 2018
⁎ Corresponding author at: 38 Xue Yuan Road, Haidian District, Beijing, 100191, PR China.
1 Xiaodan Liu contributed equally to the research as first author.
E-mail addresses: lxy920429@126.com (X. Liu), liuxiaodan@bjmu.edu.cn (X. Liu), xiaoerqd@126.com (X. Wang), raynwu98@163.com (R. Wu),
1158982958@qq.com (K. Zhang), byliu@me.ustb.edu.cn (B. Liu), iamliuqichen@gatech.edu (Q. Liu), 793118871@qq.com (Y. Shao),
tangran.hi@163.com (R. Tang), yjf107@126.com (J. You), peifei@bjmu.edu.cn (F. Pei).
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status remains poorly understood in endometrial cancers. In this study,
we investigated MSI status and PD-L1 expression in a patient suffered
from dedifferentiated EC, and analyzed MLH1 promoter hypermethy-
lation.
2. Case report
In May 2016, a 61-year-old woman presented at the department of
gynecology of the Peking University Third Hospital in Beijing, China,
complaining of paroxysmal abdominal pain for two weeks. After med-
ical examination, transvaginal ultrasonography (TVS) revealed en-
dometrial hyperplasia and echogenicity (abnormal echo). This finding
was corroborated by a female reproductive system pelvic magnetic
resonance imaging (MRI) enhanced by a contrast dye that showed
myometrium significantly enhanced, an irregularly mild enhanced area
and nodular shadow, with no evidences of metastasis to the other dis-
tant organs. Patient's medical history was largely asymptomatic besides
irregular vaginal bleeding after menopause for one more year. No signs
of diabetes, intestinal polyposis, or malignancies to other organs were
referred. None of the family members complained for intestinal poly-
posis or other malignancies. Surprisingly, all laboratory data on ad-
mission were in the reference range, including carcinoembryonic an-
tigen (CEA) and other tumor markers. The total hysterectomy with
bilateral saplingo-oophorectomy was performed along with removal of
all locoregional lymph nodes. During the surgery, the operative doctors
found that the uterine surface was smooth with normal size and good
mobility, but the endometrium texture was crisp during the curettage.
The double side accessories and the uterus-rectum-fossa did not have
significant changes. In addition, the doctors found that the appendix
widened cystic-solid, with the diameter up to 2.5 cm, and adhered
tightly to the ileocecal junction. Considering that the physical condition
of the patient, the attending doctor and the families made a decision
jointly. They reached agreement to give up the postoperative radio-
therapy and chemotherapy. For good news, until now the patient’s
status has been well except sometimes may felt a little pain in the
surgical incision without tumor recurrence and metastasis given by
gynaecological imaging.
3. Materials and methods
3.1. Immunohistochenmistry
Specimens from the surgically resected tumor were fixed in 10%
buffer formalin and routinely processed. Immunohistochemical staining
was performed using the avidin-biotin complex method for a panel of
antibodies raised againstCK-Pan-clone AE1/AE3; vimentin-clone VIM
3B4; p16-clone 6H12; Ki67-clone MIB-1; ER-clone SP1; PR-clone SP2;
P53-clone BP-53-12; CD3-clone PS1; CD4-clone SP35; CD8-clone 144B
(DAKO cytomation); CD99-clone Ho36; Chromogranin A-clone 5H7;
Synaptophysin-clone 27G12; neuronal-specific enolase(NSE)-clone
22C9(Novocastra Laboratories); CDX2-clone ab48009(Abcam com-
pany); MLH1-clone G168-728；MSH2-clone G219-1129；MSH6-clone
44; PD-L1-clone SP263 (Ventana company); PMS2-clone EPR3947(Cell
Marque company); PD-1-clone NAT(Abcam company).
3.2. Microsatellite instability
Multiplex PCR was used to amplify five mononucleotide repeat
markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) for MSI de-
termination (MSI Analysis System, Version 1.2, Promega). Two penta-
nucleotide repeat markers (Penta C and Penta D) were included to
confirm sample identity between tumor and normal. Fluorescently la-
beled PCR products were identified and sized by capillary electro-
phoresis on an ABI 3500XL and analyzed using Life Technologies Gene
Mapper software. Markers were scored for stability or instability by
comparison of marker allele sizes from tumor and normal samples. The
presence of a marker allele size in the tumor sample that was not pre-
sent in the normal sample was indicative of instability for that marker.
Samples with no allelic size variations in fewer than two of the mi-
crosatellites were classified as MSS. Tumors with allelic size variations
in two or more of the microsatellite markers were considered MSI-H.
3.3. BRAF, KRAS and POLE gene mutations
Genomic DNA was isolated from formalin-fixed, paraffin-embedded
(FFPE) tumor samples with a QIAamp DNA mini kit (Qiagen, Valencia,
CA, USA). BRAF mutation at codon 600 in exon 15, KRAS mutations at
codons 12 and 13 in exon 2 and POLE exonuclease domain (EDM)
mutations were detected by PCR/direct sequencing.
3.4. Analysis of MLH1 methylation
Promoter methylation of MLH1 was determined in tumors in an
effort to distinguish sporadic from familial dMMR patients. Tumor
DNA was extracted from FFPE tissue using the formalin-fixed paraffin
embedded (FFPE) tissue kit (Qiagen, Valencia, CA, USA), following
the manufacturer's instructions. Sodium bisulfite modification was
carried out using the EZ DNA Methylation Kit (Zymo Research Corp.,
Irvine, CA). PCR primers were designed to detect differences between
methylated and unmethylated DNA for the hMLH1 promoter. Primers
for the methylationspecific PCR assay included: methylated reaction
(5′-FAMAACGAATTAATAGGAAGAGCGGATAGCG-3′; 5′-CGTCCCTCC
CTAAAACGACTACTACCC-3′), unmethylated reaction (5′-NEDtaaaaa
tgaattaataggaagagtggatagtg-3′; 5′-AATCTCTTCATCCCTCCCTAAAAC
A-3′). The polymerase chain reaction (PCR) products were loaded onto
3% agarose gels, stained with ethidium bromide, and visualized under
an UV transilluminator.
4. Results
Gross observation of the tumor in the uterus, 3× 2.5×1 cm in size,
occupied the entire uterine cavity and completely presented invasion
reaching the muscular layer with no involvement of the lower uterine
segment. Additional, the appendix had a rupture full of numerous
mucus. At cut surface, it showed a moderate consistency.
Microscopically, the tissue sections showed mixed endometrial adeno-
carcinoma (Fig. 1A), 40% was well-differentiated endometrial adeno-
carcinoma, 40% was undifferentiated carcinoma, 10% was mucinous
adenocarcinoma, and another 10% was serous adenocarcinoma. Well-
differentiated Endometrioid carcinoma typically displays a grandular or
villoglandular architecture lined by stratified columnar epithelium with
crowded, complex, branching architecture. The lining cells are usually
columnar and share a common apical border with adjacent cells, re-
sulting in a smoothly contoured glandular lumen. The cytoplasm of the
neoplastic cells is eosinophilic and granular. Nuclear atypia is usually
mild to moderate, with inconspicuous nucleoli; whereas un-
differentiated carcinoma is composed of small to intermediated-sized,
dyshesive cells of relatively uniform size arranged in sheets without any
obvious nested or trabecular architecture resembling lymphoma or
small cell carcinoma. No gland formation is present. The nuclear
chromatin is usually condensed. Most cases have more than 25 mitotic
figures per 10 HPF. Although the stroma is generally unapparent, some
have a myxoid matrix. Tumor-infiltrating lymphocytes are often nu-
merous. Though the invasion of the cancer reached the muscular layer,
it was still less than half of it. Also, multiple intravascular cancer emboli
could be seen in different vessels. The surrounding tissue showed
atrophic endometrium with endometrial polyps. As for the appendix,
the pathology of the specimen revealed a low-grade mucinous tumor
and the invasion reaching the whole appendix wall. It also formed a
mucinous lake in the serosa, and there were still tumor cells in it.
Moreover, the formation of the mucinous lake could still be seen in the
broken end of the muscular layer of the appendix, but there wasn't any
X. Liu et al. Pathology - Research and Practice xxx (xxxx) xxx–xxx
2
neoplastic epithelium in it.
Immunohistochemically, in the area of the well-differentiated en-
dometrial adenocarcinoma, about 90% tumor cells were strongly po-
sitive (3+) for Pan-CK (Fig. 1B), moderately positive (2+) for ER
(Fig. 1C), and mildly positive (1+) for PR (Fig. 1D). Vimentin staining
was strongly/diffusely distributed throughout the area. Many tumor
cells' nuclei were positive for Ki-67, with a labeling index of 40%. Next,
in the area of the undifferentiated carcinoma, about 90% of the tumor
cells were mildly positive for Pan-CK (Fig. 1B), rare tumor cells were
mildly (1+) for ER (Fig. 1C) and PR (Fig. 1D) respectively. Almost all
the tumor cells' nuclei were positive for Ki-67, with a labeling index of
90%. Moreover, negative immunophenotypic findings for
CDX2(Fig. 1E), CgA (Fig. 1F), Syn (Fig. 1G) and P53 (Fig. 1H) help to
exclude other possible poorly differentiated neoplasms such as meta-
static colon tumor, neuroendocrine tumors or serous origin of the
tumor.
Interestingly, mutL homolog 1 (MLH1) immunostaining was nega-
tive in the whole cancer (Fig. 1I and Fig. 2B), which was the same as
PMS2 (Fig. 2E). Whereas mutS homolog 2 (MSH2) and mutS homolog 6
(MSH6) was diffuse positive in cancer cells (Fig. 2C and D). Besides,
tumor-associated immune infiltrates in this case were predominantly
located not only at invasive front but also in the tumor stroma and in
tumor cell nests (Fig. 2A). The cancer had significantly more CD3 and
CD8 T cells at tumor invasive front compared to CD4 and PD-1 T cells.
Moreover, the EC demonstrated tumoral PD-L1 expression. The ex-
pression pattern was predominantly focal, occurring in 10% of tumor
cells at the infiltrating tumor edge (Fig. 2F).
Furthermore, the tumor DNA demonstrated instability in five of five
mononucleotide marker loci after checking microsatellite instability
(MSI) with five satellite markers, BAT-25, BAT-26, NR-21, NR-24 and
MONO-27. This pattern is indicative of microsatellite instability-high
(MSI-H). To identify gene mutations possibly associated with the case,
we searched for KRAS, BRAF and POLE mutations. No KRAS mutations
were found at codons 12 and 13 in exon 2, no V600E BRAF mutation in
exon 15 and no POLE exonuclease domain (EDM) mutations suggesting
the EC case should be classified as microsatellite instability (MSI)
subgroup according to the Cancer Genome Atlas (TCGA). Further
testing revealed MLH1 promoter methylation and unmethylation both
Fig. 1. Pathologic findings of endometrial dedifferentiated adenocarcinoma. A, Microscopic findings of mixed endometrial adenocarcinoma, 40% was well-differ-
entiated endometrial adenocarcinoma, 40% was undifferentiated carcinoma (HE, X4). B, Immunohistochemically, in the area of the well-differentiated endometrial
adenocarcinoma, about 90% tumor cells were strongly positive (3+) for Pan-CK, whereas in the area of the undifferentiated carcinoma, about 90% of the tumorcells
were mildly positive for Pan-CK (IHC, X4). C, Immunohistochemical staining for ER (IHC, X4). D, Immunohistochemical staining for PR (IHC, X4). E, Negative
immunophenotypic findings for CDX2 (IHC, X4). F, Negative immunophenotypic staining for CgA (IHC, X4). G, Negative immunophenotypic staining for Syn (IHC,
X4). H, Negative immunophenotypic staining for P53 (IHC, X4). I, Negative immunophenotypic staining for mutL homolog 1 (MLH1) (IHC, X4).
X. Liu et al. Pathology - Research and Practice xxx (xxxx) xxx–xxx
3
in the case (Fig. 3). We speculated methylation of tumors and un-
methylation of non-cancer endometrial tissue.
5. Discussion
We report here a novel case of endometrial dedifferentiated carci-
nomas displaying an intense/diffuse MSH2/MSH6 expression accom-
panied by a negative immunostaining for MLH1/PMS2, and we also
have found that the MSI tests with five satellite markers showed MSI-H
status, which indicated the possibility of deficiency in MMR of this case.
Endometrial dedifferentiated cancers develop as a result of multiple
genetic and epigenetic alterations. Approximately one-half of en-
dometrial dedifferentiated cancers display high microsatellite-in-
stability with MLH1 promotor methylation and loss of expression of
MLH1 and PMS2 [4]. Also rare cases of undifferentiated carcinoma
arising individuals with Lynch syndrome have been described. Lynch
Fig. 2. Immunohistochemical stains from the patient’s endometrial dedifferentiated adenocarcinoma. A, Medium monocytes and lymphocytes were infiltrating in or
between the tumor cell nests in the undifferentiated carcinoma area (HEX10). B, The endometrial adenocarcinoma stained negative for MLH1 (IHCX10). C, The
endometrial adenocarcinoma stained diffuse positive for MSH2 (IHCX10). D, The endometrial adenocarcinoma stained diffuse positive for MSH6 (IHCX10). E, The
endometrial adenocarcinoma stained negative for PMS2 (IHCX10), the lymphocytes and stromal cells should stain positive and represent an internal positive control.
F, the EC demonstrated tumoral PD-L1 expression. The expression pattern was predominantly focal, occurring in 10% of tumor cells at the infiltrating tumor edge
(IHCX10).
Fig. 3. The MLH1 methylation assay showed MLH1 methylation and unmethylation both in the case. M: methylated primers; U: unmethylated primers; UDNA,
unmethylated positive control; MDNA, methylated positive control.
X. Liu et al. Pathology - Research and Practice xxx (xxxx) xxx–xxx
4
syndrome is an autosomal dominant syndrome characterized by mi-
crosatellite instability caused by defects in DNA MMR genes [5]. This
leads to increased susceptibility to a variety of cancers at a young age
(average<45 years). In addition, we believe a multi-disciplinary ap-
proach to the management of these individuals is crucial. So, it is im-
portant to identify MSI-H molecular mechanism of this case because of
different follow-up and treatment. In this case, we confirmed reduced
or abolished MLH1 and PMS2 expression due to MLH1 promoter hy-
permethylation. It is well known that a subset of endometrioid cancers
exhibits methylation at the promoter regions of a series of genes ac-
counting for the “CpG island methylator phenotype” (CIMP) [6], and
activating mutations in 2 members of the RAS/MAP kinase pathway,
KRAS and BRAF, have been detected in 20–26% of endometrioid car-
cinomas, especially BRAF mutations are frequently associated with the
CIMP+phenotype and MSI-H in cancers [7–9]. However, it is still
unclear in endometrial dedifferentiated adenocarcinoma. Thus, we
detected KRAS, BRAF and POLE mutations and found there were no
KRAS mutations at codons 12 and 13 in exon 2, no V600E BRAF mu-
tation in exon 15 and no POLE exonuclease domain (EDM) mutations.
As we known, mismatch repair (MMR)-deficient endometrial car-
cinomas (ECs) bearing Lynch syndrome (LS)-associated germline mu-
tations or sporadic MLH1 promoter hypermethylation (MLH1hm) are
highly immunogenic and may represent excellent candidates for
therapies targeting the programmed cell death(PD)/programmed cell
death ligand-1 (PD-L1) immune checkpoint pathway [7]. Thus we
evaluated PD-L1 expression in this case and found focal expression
occurring in 10% of tumor cells at the infiltrating tumor edge. Although
sporadic MLH1 hypermethylated EC were significantly less likely to
demonstrate tumor cell expression of PD-L1 relative to LS-associate
tumors, they often demonstrated high levels of PD-L1 positive immune
stromal infiltrates as in this case. It is well known that one of immune
checkpoint pathways is mediated by interactions between programmed
cell death-1 (PD-1) protein and programmed cell death receptor ligand
1 (PD-L1) protein. PD-1 is expressed on activated lymphocytes and its
ligand PD-L1 is upregulated on multiple antigen-presenting cell types in
response to cytokine mediators as part of a healthy immune response.
PD-1/PD-L1 interactions result in immune suppression through inhibi-
tion of T-cell proliferation, activation of regulatory T cells, and induc-
tion of T-cell anergy and apoptosis to curb ongoing inflammation
[10,11]. PD-L1 can also be constitutively or inducibly expressed on
tumor cells through oncogenic signaling or adaptive immune resistance.
Thus, while PD-1/PD-L1–mediated immune suppression provides a
safeguard against immunopathology following infections and other
inflammatory stimuli, it can also be coopted as a mechanism of tumoral
immune avoidance in the setting of malignancy. This checkpoint
pathway is of considerable clinical interest due to the recent success of
PD-1 and PD-L1 inhibitors in a variety of malignancies such as mela-
noma, non–small cell lung carcinoma, renal carcinoma, and bladder
carcinoma [12]. In the gynecologic tract, endometrial carcinoma(EC)
represents a potential target for PD-1/PD-L1 inhibition as early studies
have shown increased PD-L1 expression in endometrial tumors relative
to ovarian and cervical malignancies. ECs with MMR deficiencies re-
present particularly intriguing candidates for PD-1/PD-L1 inhibitor
therapies because of their immunogenicity, and clinical trials in-
vestigating these therapies in MMR deficient colorectal carcinomas are
encouraging [12–15].
In conclusion, we report a novel case of endometrial dediffer-
entiated carcinomas with high microsatellite-instability by MLH1 pro-
motor methylation, loss of expression of MLH1 and PMS2 and focal
expression of PD-L1 at the infiltrating tumor edge, which suggest that
MMR deficiency may be a better predictor of response to PD-1/PD-L1
inhibitor therapy.
Acknowledgements
The article was supportedby the National Sciences Foundation of
China (81572533). Thanks to Beiying Liu and Qichen Liu for data
analysis.
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http://refhub.elsevier.com/S0344-0338(18)30579-X/sbref0065
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http://refhub.elsevier.com/S0344-0338(18)30579-X/sbref0070
http://refhub.elsevier.com/S0344-0338(18)30579-X/sbref0070
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http://refhub.elsevier.com/S0344-0338(18)30579-X/sbref0075
	A novel case of endometrial dedifferentiated adenocarcinoma associated with MLH1 promotor hypermethylation and microsatellite instability
	Introduction
	Case report
	Materials and methods
	Immunohistochenmistry
	Microsatellite instability
	BRAF, KRAS and POLE gene mutations
	Analysis of MLH1 methylation
	Results
	Discussion
	Acknowledgements
	References