5616 Vol. 9, 5616 –5619, November 15, 2003
Clinical Cancer Research
Use of 11p15 Mucins as Prognostic Factors in Small
Adenocarcinoma of the Lung
Noboru Nishiumi,1 Yoshiyuki Abe,3
Yoshimasa Inoue,2 Hiroyuki Hatanaka,2
Ken-ichi Inada,4 Hiroshi Kijima,2
Hitoshi Yamazaki,2 Masae Tatematsu,4
Yoshito Ueyama,2 Masayuki Iwasaki,1
Hiroshi Inoue,1 and Masato Nakamura2
Departments of 1General Thoracic Surgery and 2Pathology, Tokai
University School of Medicine, Isehara, Kanagawa; 3Department of
Respiratory Disease, National Kanagawa Hospital, Hadano,
Kanagawa; and 4Laboratory of Pathology, Aichi Cancer Center
Research Institute, Nagoya, Aichi, Japan
ABSTRACT
Purpose: Patients with small adenocarcinoma of the
lung (SACL) generally have a good prognosis. However,
some SACL cases show lymph node metastasis, with poor
prognosis. The expression pattern of 11p15 mucins (clustered on the p15 arm of the chromosome 11) is known to
change during carcinogenesis in lung cancer.
Experimental Design: We evaluated the expression of
the 11p15 mucins (MUC2, MUC5AC, and MUC6) in 79
surgical specimens of SACL cases by immunohistochemical
analysis. Lymph node metastasis was estimated by pathological staging.
Results: Six (7.6%) and 11 (13.9%) of the 79 SACL
cases showed MUC2 and MUC6 expression, respectively.
Three SACL cases showed both MUC2 and MUC6 expression, and a significant correlation was found between MUC2
and MUC6 expression (Fisher’s test, P ⴝ 0.033). Six (7.6%)
SACL cases showed MUC5AC expression. Five of the 6
cases with MUC2 expression and 6 of the 11 cases with
MUC6 expression were had lymph node metastasis. SACL
cases with MUC2 or MUC6 expression showed a significantly higher incidence of nodal metastasis than those without expression (P < 0.001 and P ⴝ 0.006, ␹2 test, respectively). There was no significant correlation between
MUC5AC expression and nodal involvement in SACL,
whereas three of the six cases with MUC5AC expression
showed lymph node metastasis. The SACL cases with MUC2
expression had a significantly poorer prognosis than those
without MUC2 expression (P ⴝ 0.011, log-rank test).
Received 3/10/03; revised 7/22/03; accepted 8/4/03.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Dr. Masato Nakamura, M.D., Department of
Pathology, Tokai University School of Medicine, Bohseidai, Isehara,
Kanagawa 259-1193, Japan. Phone: 81-463-93-1121, ext. 2570; Fax:
81-463-91-1370; E-mail: [email protected].
Conclusions: These results suggest that 11p15 mucins
MUC2 and MUC6 are related to lymph node metastasis in
SACL.
INTRODUCTION
Lung cancer is one of the most common malignant diseases
in the world, and its prognosis is generally poor. Among several
histological types of lung carcinoma, adenocarcinoma of the
lung makes up approximately half of the non-small cell lung
cancer and shows a variety of histopathological features.
SACL,5 measuring 2 cm or less at its greatest dimension, shows
generally good prognosis. However, a few SACL form metastases of the mediastinal lymph nodes or systemic organs and
showed very poor prognosis (1). It has been reported that the
histopathological features of SACL are various and that some
types of SALC showed poor prognosis (2). However, it is very
difficult to distinguish the above histological types by routine
histological examination, despite some immunohistochemical
analyses (3). If SACL cases with good prognosis could be
distinguished from those with poor prognosis, some cases of
SACL could be cured by limited surgical removal. It is therefore
very important to reveal the factors that distinguish between
SACL cases with good prognosis and those with poor prognosis.
Mucins have been postulated to be important molecules in
maintaining epithelial homeostasis in various diseases (4, 5). In
cancer, mucins are large O-glycoproteins expressed either at the
cell surface or as secreted molecules that form a protective gel
(6, 7). Core proteins for several human mucins (MUC1–MUC9
and MUC11–MUC13) have been identified (8 –12). The mucin
genes MUC2, MUC5AC, MUC5B, and MUC6 are clustered on
the p15 arm of chromosome 11 and are therefore called 11p15
mucin genes (13). The 11p15 mucin genes possess a cellspecific pattern of expression in normal lungs, and the expression pattern is altered during carcinogenesis (14).
In this study, we immunohistochemically examined the
11p15 mucins MUC2, MUC5A, and MUC6 in SACL and
explored the relationship between mucin expression and lymph
node metastasis and prognosis.
MATERALS AND METHODS
SACL Surgical Specimens. The 79 SACL specimens
were obtained by surgical resection from previously untreated
patients with their informed consent. Surgical specimens were
processed for routine histopathological analysis. Morphological
classification was based on histological typing of lung tumors.
The nodal metastases were evaluated histologically.
Immunohistochemistry. Immunohistochemical analysis
was performed with the Catalyzed Signal Amplification system
5
The abbreviation used is: SACL, small adenocarcinoma of the lung.
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Clinical Cancer Research 5617
monoclonal antibodies, 1:100 dilution): MUC2 glycoprotein/
clone (Ccp58), MUC5AC glycoprotein/clone (CLH2), and
MUC6 glycoprotein/clone (CLH5; Novocastra Laboratories
Ltd.). The immune complex on the sections was then amplified
with the biotinylated secondary antibodies (goat antimouse immunoglobulin; DAKO EnVision), streptavidin– biotin complex,
and streptavidin peroxidase. The amplified products were visualized by the 3,3⬘-diaminobenzidine tetrahydrochloride reaction.
Statistical Analysis. We examined the statistical significance of the relationship between the expression of the MUC
proteins by Fisher’s test. We examined the statistical significance of the relationship between MUC expression and lymph
node metastasis by ␹2. The survival rate of patients with SACL
was estimated from Kaplan–Meier life tables plotted to compare
survival and mucin production. The curves were analyzed for
statistically significant differences by the log-rank test.
RESULTS
Expression of MUC2, MUC5AC, and MUC6. We
evaluated the expression of MUC2, MUC5AC, and MUC6, in
79 surgical specimens by immunohistochemical analyses (Fig.
1). Six (7.6%) of 79 SACL cases showed MUC2 expression, and
11 (13.9%) of the 79 SACL cases showed MUC6 expression.
Three SACL cases showed both MUC2 and MUC6 expression,
and a significant correlation was noted between MUC2 and
MUC6 expression (Fisher’s test, P ⫽ 0.033). Six (7.6%) SACL
cases showed MUC5AC expression. No significant correlation
was observed between MUC expression and histopathological
features.
Five of the 6 SACL cases with MUC2 expression showed
lymph node metastasis, and 6 of the 11 cases with MUC6
expression showed lymph node metastasis (Table 1). SACL
cases with MUC2 or MUC6 expression showed a significantly
higher incidence of nodal metastasis than those without expression (P ⬍ 0.001 or P ⫽ 0.006, respectively, ␹2 test). Three of
the six cases with MUC5AC expression showed lymph node
metastasis. However, there was no significant correlation between MUC5AC expression and nodal involvement.
MUC Expression and Prognosis. Fig. 2 shows the survival curves of the 79 patients obtained with the Kaplan–Meier
method. The mean observation period was 5.1 years. The SACL
cases with MUC2 expression showed a significantly poorer
Table 1
Fig. 1 Immunohistochemical staining in SACL by specific antibodies
(⫻600). A, SACL with MUC2 expression; B, SACL with MUC5AC
expression; C, SACL with MUC6 expression.
(DAKO Co. Ltd., Kyoto, Japan) according to the manufacturer’s
recommendations. The tissue sample from the primary tumor
was embedded in paraffin. Endogenous peroxidase activity was
blocked with 0.3% hydrogen peroxide, and samples were
washed with 0.1 M PBS (pH 7.2). The sections were incubated
at 4°C for 10 h with the following primary antibodies (murine
Immunohistochemical analysis of MUC expression
in SACL
Lymph node metastasis, n (%)
MUC2
⫹
⫺
MUC5AC
⫹
⫺
MUC6
⫹
⫺
a
⫹ (n ⫽ 19)
⫺ (n ⫽ 60)
P
5 (26.3%)
14 (74.7%)
1 (1.7%)
59 (98.3%)
⬍0.001a
3 (15.8%)
16 (84.2%)
3 (5.0%)
57 (95.0%)
0.094
6 (31.6%)
13 (68.4%)
5 (8.3%)
55 (91.7%)
0.006a
Statistically significant by ␹2 test.
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5618 Mucin Expression in SACL
two-windows method (28). Few SACL have metastasis of the
mediastinal lymph nodes or systemic organs and show poor
prognosis (1). In this study, we showed that MUC2 and MUC6
production was related to lymph node metastasis and poor
prognosis. We conclude that these two mucins are markers for
predicting the prognosis of SACL. However, the sample size of
the mucin-positive specimens was rather small. The number of
SACL cases are increasing in our institute, and we are continuing studies with these SACL samples. We therefore hope to
confirm the results with a larger series in the future. At present
we are also examining the expression levels of the 11p15 mucin
genes by real-time PCR method.
ACKNOWLEDGMENTS
Fig. 2 Survival curves obtained with the Kaplan–Meier method for
SACL patients.
We thank Yuichi Tada, Johbu Itoh, and Koutatsu Nomura for
technical assistance.
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DISCUSSION
We examined the expression of 11p15 mucins in 79 patients with SACL. SACL cases with MUC2 (7.6%) and MUC6
(13.9%) expression showed a significantly higher incidence of
nodal metastasis than those without expression (P ⬍ 0.001 or
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Use of 11p15 Mucins as Prognostic Factors in Small
Adenocarcinoma of the Lung
Noboru Nishiumi, Yoshiyuki Abe, Yoshimasa Inoue, et al.
Clin Cancer Res 2003;9:5616-5619.
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