Original Article

Yonago Acta medica 2015;58:151–156

Cytoplasmic Maspin Expression Correlates with Poor Prognosis of Patients with
Adenocarcinoma of the Uterine Cervix
Kanae Nosaka,* Yasushi Horie,† Tatsushi Shiomi,* Hiroaki Itamochi,‡ Tetsuro Oishi,§ Muneaki Shimada,§
Shinya Sato,§ Tomohiko Sakabe,* Tasuku Harada§ and Yoshihisa Umekita**Division of Organ Pathology, Department of Pathology, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503,
Japan, †Department of Pathology, Tottori University Hospital, Yonago 683-8504, Japan, ‡Department of Obstetrics and Gynecology,
School of Medicine, Iwate Medical University, Morioka 020-8505, Japan and §Division of Reproductive-Perinatal Medicine and Gynecologic Oncology, Department of Surgery, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan

ABSTRACT

present results suggest that cytoplasmic maspin expression could be an indicator of unfavorable prognosis inpatients with adenocarcinoma of the uterine cervix.

Background

Maspin is known to be a tumor suppressor protein and its prognostic significance in patientswith several types of cancer has been reported. To date,however, no study has focused on the association between maspin expression and the prognosis of patientswith adenocarcinoma of the uterine cervix. We exploredthe prognostic value of maspin expression with particular reference to its subcellular localization in patientswith adenocarcinoma of the uterine cervix.
Methods Paraffin-embedded tissue samples from 46
patients diagnosed as adenocarcinoma of the uterine
cervix were immunohistochemically analyzed using anantibody for maspin. The patients were followed up for 3
to 165 months (median: 64.2 months) and the prognostic
value was evaluated by the log-rank test and the Cox regression hazard model.
Results A sample was considered maspin-positive if
maspin was expressed in only the cytoplasm; 69.6% (32cases) of the specimens were maspin-positive, and there
was significant correlation between positivity and recurrence (P = 0.022). Maspin-positive patients had both
shorter disease free survival and shorter overall survivalby the log-rank test (P = 0.023, P = 0.043, respectively).
By Cox’s multivariate analysis, the International Federation of Obstetrics and Gynecology (FIGO) status was
the only independent prognostic factor for disease freesurvival and overall survival in patients with adenocarcinoma of the uterine cervix.
Conclusion This is the first report to reveal an association between cytoplasmic maspin expression and theprognosis of patients with adenocarcinoma of the uterine
cervix. Although further studies with a larger series ofpatients and a longer follow up period are necessary, theKey words

adenocarcinoma; immunohistochemistry;maspin; uterine cervix
Adenocarcinoma of the uterine cervix is a relatively
uncommon histological subtype of uterine cancer comprising 10–20% of all cervical cancer cases.1 Althoughsquamous cell carcinoma (SCC) of the uterine cervix
has declined markedly in developed countries, both therelative and absolute incidences of adenocarcinoma of
the uterine cervix have increased over the past four decades.1, 2 The majority of reports have shown that adenocarcinoma of the uterine cervix carries a worse prognosis than SCC in 5-year overall survival rates, and that its
survival rates are significantly lower than SCC’s in eachtumor stage.3–5 Differences have also been reported between SCC and adenocarcinoma of the uterine cervix inthe pattern of dissemination, recurrences and responseto chemotherapy.3 The important prognostic factors havebeen considered to be the International Federation of
Obstetrics and Gynecology (FIGO) stage, tumor size
and the occurrence of lymph nodes metastasis. However,the search for another predictive factor for adenocarcinoma of the uterine cervix may be important for deciding on a therapeutic strategy. Maspin, a member of theserine protease inhibitor (serpin) family, was originallydetected in normal breast epithelial cells.6 Maspin expression was previously shown to be down-regulated
as breast epithelial cells progressed to a higher grade ofbreast tumor and to be lost in metastasis and it has beenshown to have tumor suppressor activity attributable tothe inhibition of breast cancer cell motility, invasion andmetastasis.7 Although the precise biochemical pathways
leading to these biological endpoints remains to be elucidated, many reports describe the association between
maspin expression and clinicopathological factors inmany types of cancer.8 We have also reported on associations between maspin expression and clinicopatho-

Corresponding author: Kanae Nosaka
knosaka@med.tottori-u.ac.jp
Received 2015 September 2
Accepted 2015 October 2
Abbreviations: DAB, 3,3’-Diaminobenzidine; DFS, disease freesurvival; FIGO, The International Federation of Gynecology and
Obstetrics; OS, overall survival; SCC, squamous cell carcinoma

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K. Nosaka et al.

logical factors in several types of cancer such as breast
cancer,9–11 colorectal cancer,12 endometrioid endometrial
carcinoma,13 ovarian mucinous borderline tumor,14 lungadenocarcinoma and soft tissue sarcoma;15, 16 and wehave suggested that cytoplasmic maspin expression may
be an indicator of poor prognosis. To our knowledge,
this is the first report to investigate the association between maspin expression and the prognosis of patientswith adenocarcinoma of the uterine cervix. The aim of
this study was to clarify whether cytoplasmic expressionof maspin could be a predictive factor for patients withadenocarcinoma of the uterine cervix as well as othertypes of cancers.

(clone EAW24; diluted 1:50; Leica Biosystems, Newcastle, UK). The sections were incubated with biotinylated anti-mouse IgG antibody (BA-2000; diluted 1:150;
Vector Laboratories, Burlingame, VT) for 20 minutes,
followed by streptavidin biotinylated-HRP conjugate (diluted 1:150; Invitrogen, Camarillo, CA) for 20 minutes.
The sections were then incubated with 3,3’-Diaminobenzidine (DAB) solution (Liquid DAB+ Substrate, Imidazole-HCl buffer, pH 7.5, containing hydrogen peroxide
and an anti-microbial agent; Dako, Glostrup, Denmark)for 4 minutes, and finally were counterstained with hematoxylin.
Immunohistochemical evaluation

MATERIALS AND METHODS

To evaluate maspin expression status, the sections weredivided into two groups: maspin-positive and -negative.
Specimens were considered maspin-positive if staining
occurred only in the cytoplasm with a staining area of> 10% of the whole tumor for each section irrespectiveof staining intensity. The remaining specimens wereconsidered maspin-negative. These evaluations were
performed independently by two authors (K.N. and Y.U.),
who were blinded to the patients’ outcome data. Normal squamous epithelium served as an internal positive
control if it was present in the same specimen. Myoepithelial cells in normal breast tissue specimens were also
used as positive controls.

Tissue specimens

This study enrolled a total of 46 consecutive patientswith adenocarcinoma of the uterine cervix diagnosed at
the Department of Pathology Tottori University Hospital
between 1999 and 2010. The median follow up period
was 64.2 months (range: 3–165 months). Since a considerable number of patients had received neoadjuvant
chemotherapy, radiotherapy or both before tumor resection, we selected tissues mainly from biopsy specimens
to prevent therapy-related changes from disturbing histological or immunohistochemical assessment. The patients’ clinicopathological data were retrieved from thehospital’s computerized medical records system. Eachhistological diagnosis was re-evaluated and establishedaccording to the World Health Organization classification17 by experienced pathologists (K.N. and Y.U.). Most
patients underwent surgical treatments including total
hysterectomy with lymph node dissection (35 cases),radical trachelectomy (3 cases), and simple hysterectomy (1 case); the inoperable patients received either
chemoradiotherapy (5 cases) or chemotherapy only (2cases). Written informed consent was obtained from all
patients, and the study was performed with the approvalof the Ethics Committee of the Faculty of Medicine,
Tottori University (No. 1558).

Statistical analysis

All statistical analyses were performed using the Statistical Package for Social Sciences version 21 (IBM SPSS
Statistics; IBM, Armonk, NY). The association between
maspin status and clinicopathological parameters wasevaluated by non-parametric tests. The chi-square test
was used when there were two categorical variables ofinterest and the Kruskal-Wallis test when there were
three or more variables. Survival curves were calculatedusing the Kaplan-Meier method and compared by logrank test. The Cox proportional hazard regression model
was used to analyze the independence of each parameterof disease free survival (DFS) or overall survival (OS).

Immunohistochemical procedures

RESULTSAll specimens were fixed in 10% neutrally bufferedformalin and embedded in paraffin. A representative
block from each case containing an adequate amountof tumor tissue was selected. After the sections (4 μm
thick) were deparaffinized and endogenous peroxidase
activity was blocked, they were pretreated in citrate buffer (0.01 M, pH 6.0) using a microwave oven (RE-DD6S; Sharp, Osaka, Japan) for 20 minutes. After cooling toroom temperature, the sections were incubated at 4 ˚Covernight with monoclonal anti-human maspin antibody

Clinicopathological features

The clinicopathological profiles of the 46 patients are
summarized in Table 1. The age distribution was 22–74
years (median: 50.0 years). Thirty-three cases were at
FIGO Stage I/II and 13 cases were at Stage III/IV. In
23 cases (50%) the tumor was larger than 40mm, and in
10 cases (21.7%) lymph node metastasis was recognizedeither at operation or at first radiographic examination.
Nineteen cases (41.3%) had recurrent diseases: 6 distal
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Maspin expression in cervical adenocarcinoma

metastasis (lung: 4, brain: 2, liver: 1, bone: 1), 11 local
recurrences and 2 peritoneal disseminations.

(32 cases, 69.6%), pancellular staining (both nuclear andcytoplasm; 4 cases, 8.7%), nuclear staining only (4 cases,
8.7%) and no staining (6 cases, 13%). The latter threepatterns were judged as maspin-negative. Maspin-positive status was significantly correlated with recurrence (P= 0.022) and slightly correlated with the non-mucinous
subtype (P = 0.042) (Table 2).

Table 1. Clinicopathologic characteristics of patientswith adenocarcinoma of the uterine cervixn%Age (years old)
< 30
1
2.2
30–39
8
17.4
40–49
13
28.3
50–59
14
30.4
60–69
8
17.4
≥ 70
2
4.3
Recurrence
None
27
58.7
Local recurrence
11
17.4
Distant metastasis
6
19.6
Peritoneal dissemination
2
4.3
FIGO stage
IB
22
47.8
IIA
2
4.3
IIB
9
19.6
IIIA
1
2.2
IIIB
7
15.2
IV
5
10.9
Tumor size
< 40mm
21
45.7≥ 40mm23
50.0
Lymph node metastasis
Negative30
65.2
Positive10
21.7
Unknown6
13.0
Histological subtype
Mucinous adenocarcinoma
20
43.4
		 Endocervical type17 37.0
		 Intestinal type3
6.5
Endometrioid adenocarcinoma
16
34.8
Others5
10.8
Adenosquamous carcinoma
5
10.8
Treatment procedure
Resection only
13
28.3
Resection with neoadjuvant chemotherapy 13
28.3
Resection with adjuvant therapy only
13
28.3
Chemo-radiation therapy only
5
10.9
Chemotherapy only
2
4.3
FIGO, The International Federation of Gynecology and Obstetrics.

Table 2. Associations between maspin expression andclinicopathological factors in adenocarcinoma of theuterine cervix
Maspin expression
Positive
Negative

P-value

FIGO stage
I and II
20
13
0.072
III and IV
12
1
Tumor size
< 40mm
13
8
0.179≥ 40mm19
4
Lymph node metastasis
Present
1911
0.279
Absent11
2
Histological subtype
Mucinous
10100.042*
Endometrioid
13
3
Others9
1
Recurrence
Present
1720.022*
Absent15
12*P < 0.05. FIGO, The International Federation of Gynecology and
Obstetrics.

Survival analysis

During the follow up period, 19 patients experienced
recurrent disease and 18 patients died of the disease. In
the log-rank test, maspin-positive patients showed significantly shorter DFS (P = 0.023) and OS (P = 0.043)(Fig. 2). The 5 years DFS rates of the maspin-positiveand -negative groups were 45.5% (95% CI 0.27–0.64)
and 85.1% (95% CI 0.66–1.04), respectively. The 5 years
OS rates of the maspin-positive and -negative groups
were 54.0% (95% CI 0.36–0.72) and 82.1% (95% CI
0.59–1.07), respectively. There were significant differences in DFS and OS in the Kaplan-Meier curves
according to FIGO stage (P < 0.001, P < 0.001, respectively) and lymph node metastasis (P = 0.002, P < 0.001,
respectively). There was significant difference of OS in
Kaplan-Meier curves according to tumor size (P = 0.005).
According to Cox multivariate analysis, advanced FIGO
stage was the only independent prognostic factor forboth DFS (P = 0.001) and OS (P = 0.002) (Table 3).

Immunohistochemical findings and correlation
with clinicopathological factors

Representative staining patterns of maspin expression
are shown in Fig. 1. Subcellular localization was classified into three categories: cytoplasmic staining only
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K. Nosaka et al.

Fig. 1. Representative immunoreactivity for maspin in formalin-fixed paraffin-embedded adenocarcinoma tissues of the uterine cervix.
A: Normal squamous epithelium showed both cytoplasmic and nuclear staining, while normal columnar epithelium was not stained at thesquamo-columnar junction.
B: Mucinous adenocarcinoma showing only cytoplasmic staining for maspin.
C: Mucinous adenocarcinoma showing both cytoplasmic and nuclear staining for maspin.
D: Mucinous adenocarcinoma showing only nuclear staining for maspin.

P = 0.023

Cumulative servival

Cumulative servival

Maspin-negative (n = 14)

Maspin-positive (n = 32)

Disease-free servival (months)

Maspin-negative (n = 14)

Maspin-positive (n = 32)
P = 0.023

Overall servival (months)

Fig. 2. Disease-free survival (left) and overall survival (right) curves of 46 patients with uterine cervical adenocarcinoma according
to maspin positivity status.

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Maspin expression in cervical adenocarcinoma
Table 3. Multivariate analysis of clinicopathological factors for disease-free survival and overall survival
Disease-free survival
HR
95% ClP-value

Overall survival
HR
95% Cl

MaspinPositive vs. negative
2.733
0.609–12.262
0.189
3.505
0.684–17.953
FIGO stage
I,II vs. III, IV
0.070
0.015–0.324
0.001*
0.101
0.024–0.419
Tumor size
< 40mm vs. ≥ 40mm
0.958
0.345–2.665
0.935
2.350
0.693–7.968
Lymph node metastasis
Positive vs. negative
0.823
0.215–3.142
0.775
2.117
0.573–7.823* P < 0.05. CI, confidence interval; FIGO, The International Federation of Gynecology and Obstetrics; HR, hazard ratio.

DISCUSSION
It has been reported that adenocarcinoma of the uterine
cervix is clearly different from SCC based on its epidemiology, molecular pathogenesis and clinical behavior,including the response to chemotherapy and the patternsof recurrence.3 It has also been reported that adenocarcinoma is more likely to involve both lymph node anddistant metastasis compared to SCC resulting in a worse
prognosis.18, 19 The main prognostic factors are considered to be the presence of lymph nodes metastasis,tumor size and FIGO stage. However, few molecularalterations of adenocarcinoma of the uterine cervix
have been reported, in contrast to the case with SCC.
Therefore, it will be important to explore new molecularmarkers. The p53 gene is a candidate molecular marker
for adenocarcinoma of the uterine cervix. It has beenreported that nucleotide changes in the p53 gene weresignificantly more frequent in adenocarcinoma of theuterine cervix than in SCC, and the highest incidence of
p53 gene mutations was observed in adenocarcinoma of
the uterine cervix from Asia and Europe.20 On the other
hand, maspin is a member of the serpin family of protease inhibitors and was originally thought to be a tumor
suppressor due to its ability to inhibit invasion, motility, lymphangiogenesis and metastasis of mammary
tumors.6, 21 However, the loss of maspin expression inseveral cancers such as pancreatic, colorectal and ovarian cancers cannot typically be observed because of thelack of maspin expression in the corresponding normal
tissue. The most compelling data regarding the clinicalsignificance of maspin in cancer progression and metastasis emerged from survival studies of cancer patients.
Although the original studies revealed the associationof reduced maspin expression with cancer progression
and worse prognosis, it has been demonstrated that thiscorrelation was far more complex than originally suspected.8 Factors contributing to this complexity include
differences in cancer type (for example, adenocarcinoma

P-value
0.132
0.002*
0.170
0.261

vs. SCC), cut-off values of positive criteria, antibodies
used, methods of detection used, and subcellular maspin
distribution. Recent evidence indicates that nuclear
localization of maspin in cancer cells is necessary for
its tumor suppressor activity and that, when excluded
from the nucleus, maspin exhibited no tumor suppressor
activity.22, 23 These results suggest that maspin localized in the nucleus may result in better prognosis, while
cytoplasmic maspin accumulation may result in an unfavorable prognosis, which is consistent with our previous
reports suggesting that cytoplasmic maspin could bea predictor of worse prognosis in several types of cancers.9–16 In turn, it has been reported that p53 activatedthe maspin promoter by binding directly to the p53
consensus-binding site present in the maspin promoter,suggesting the close association between maspin andthe p53 gene.24 As nucleotide changes in the p53 gene
were significantly more frequent in adenocarcinoma of
the uterine cervix than in SCC,20 further investigation
concerning the gene or protein alteration of both maspin
and p53 may be intriguing. Although the present study
has the limitations of a small number of patients and a
retrospective nature, we for the first time suggested thatcytoplasmic maspin expression might be a candidate
molecular marker of progression in adenocarcinoma of
the uterine cervix.
In conclusion, to our knowledge, this is the first investigation of the association between maspin expression
and the prognosis of patients with adenocarcinoma of
the uterine cervix. Although further studies with a larger
series of patients and a longer follow-up period should
be undertaken, the present study suggests the potential
usefulness of cytoplasmic maspin expression as a prognostic factor for patients with adenocarcinoma of theuterine cervix. The immunohistochemical detection of
maspin could be a new marker of aggressive behavior inpatients with adenocarcinoma of the uterine cervix.
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K. Nosaka et al.
Acknowledgments: We are grateful to N. Itaki for his excellent
technical assistance.

13 Tsuji T, Umekita Y, Ohi Y, Kamino M, Douchi T, Yoshida H.
Maspin expression is up-regulated during the progression of endometrioid endometrial carcinoma. Histopathol.
2007;51:871-4. PMID: 18042077.
14 Tsuji T, Togami S, Douchi T, Umekita Y. Difference in subcellular localization of maspin expression in ovarian mucinous borderline tumour. Histopathol. 2009;55:130-2. PMID:
19614779.
15 Takagi Y, Matsuoka Y, Shiomi T, Nosaka K, Takeda C,
Haruki T, et al. Cytoplasmic maspin expression is a predictor of poor prognosis in patients with lung adenocarcinoma
measuring less than 3 cm. Histopathol. 2015; 66:732-9. PMID:
25322663.
16 Takeda C, Takagi Y, Shiomi T, Nosaka K, Yamashita H,
Osaki M, et al. Cytoplasmic maspin expression predicts poor
prognosis of patients with soft tissue sarcomas. Diagn Pathol
2014;30:205. PMID: 25358722.
17 Tavassoli FA, Devilee P: WHO Classification of Tumours ofBreast and Female Genital Organs, 3rd edition. Lyon: IARC
Press; 2003. p. 272-4.
18 Eifel PJ, Morris M, Oswald MJ, Wharton JT, Delclos L.
Adenocarcinoma of the uterine cervix. Prognosis and pattern of failure in 367 cases. Cancer. 1990;65:2507-14. PMID:
2337867.
19 Irie T, Kigawa J, Minagawa Y, Itamochi H, Sato S, Akeshima R, etal. Prognosis and clinicopathological characteristics of Ib-IIb
adenocarcinoma of the uterine cervix in patients who have hadradical hysterectomy. European J Surg Oncol. 2000;26:464-7.
PMID: 11016467.
20 Tornesello ML, Buonaguro L, Buonaguro FM. Mutations of
the TP53 gene in adenocarcinoma and squamous cell carcinoma of the cervix: A systematic review. Gynecol Oncol.
2013;128:442-8. PMID: 23168175.
21 Liu Z, Shi Y, Meng W, Liu Y, Yang K, Wu S, et al. Expression
and localization of maspin in cervical cancer and its role intumor progression and lymphangiogenesis. J Gynecol Oncol.
2014;289:373-82. PMID: 23959090.
22 Goulet B, Kennette W, Ablack A, Postenka CO, Hague MN,
Mymryk JS, et al. Nuclear localization of maspin is essential
for its inhibition of tumor growth and metastasis. Lab Invest.
2011;91:1181-7. PMID: 21502940.
23 Machowska M, Wachowicz K, Sopel M, Rzepecki R1. Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferationof normal epithelial cells. BMC Cancer. 2014;28:142. PMID:
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The authors declare no conflict of interest.

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