Download Glycoprotein nonmetastatic B as a prognostic indicator in small cell

APMIS
© 2013 APMIS Published by Blackwell Publishing Ltd.
DOI 10.1111/apm.12107
Glycoprotein nonmetastatic B as a prognostic indicator
in small cell lung cancer
YING-NA LI,1 LIN ZHANG,1 XIU-LI LI,1 DA-JIANG CUI,1 HUA-DONG ZHENG,1 SHUAN-YING
YANG2 and WEI-LIN YANG1
1
Department of Geriatrics, The Second Affiliated Hospital, Medical School of Xi’an Jiaotong University,
Xi’an; and 2Department of Respiratory Medicine, The Second Affiliated Hospital, Medical School of Xi’an
Jiaotong University, Xi’an, China
Li Y-N, Zhang L, Li X-L, Cui D-J, Zheng H-D, Yang S-Y, Yang W-L. Glycoprotein nonmetastatic
B as a prognostic indicator in small cell lung cancer. APMIS 2013.
Glycoprotein nonmetastatic melanoma B (GPNMB) is a type I transmembrane glycoprotein which is overexpressed in many tumors and seems to play a critical role in metastasis of malignant tumors. The purpose
of this study was to determine GPNMB expression in small cell lung cancer (SCLC) and analyze the prognostic value in patients with SCLC. A total of 132 cases of SCLCs were analyzed immunohistochemically
on tissue microarrays (TMAs). Patients were divided into weak-positive and strong-positive GPNMB
groups. In addition, serum GPNMB was evaluated by enzyme-linked immunosorbent assay (ELISA). The
average serum GPNMB concentration was 1054.15 363.71 pg/mL in the weak-positive group,
2611.52 457.57 pg/mL in the strong-positive group, and 427.61 273.9 pg/mL in the control. The
strong-positive group showed significantly higher serum GPNMB levels than the weak-positive group and
healthy control (p < 0.01). Overall survival in the weak-positive GPNMB group was significantly longer
than in the strong-positive group (27 months vs 15 months, p < 0.01). These results suggest that the
expression of GPNMB may be useful as a prognostic indicator in patients with SCLC.
Key words: GPNMB; prognostic indicator; small cell lung cancer; immunohistochemistry.
Wei-Lin Yang, Department of Geriatrics, the Second Affiliated Hospital, Medical School of Xi’an Jiaotong
University, Xi’an, China. e-mail: [email protected]; Shuan-Ying Yang, Department of Respiratory Medicine, the Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an, China.
e-mail: [email protected]
Lung cancer is one of the most common
malignancies and one of the leading causes of
cancer-related death in the world (1). Small
cell lung cancer (SCLC), which accounts for
15–20% of lung cancer cases, is the third
most common subtype of lung cancer (2).
SCLC is an aggressive cancer characterized by
its rapid doubling time and wide-spread metastatic behavior. The pathogenesis of SCLC is
strongly associated with cigarette smoking,
and both the smoking intensity and duration
increase the risk of SCLC development. WithReceived 10 January 2013. Accepted 17 March 2013
out treatment, the median survival of SCLC
patients is 2–4 months (3). Although SCLC is
responsive to chemotherapy, relapses are
common as patients rapidly develop chemotherapy-resistant disease (4). Due to the development of drug resistance, patients with
SCLC rarely achieve long-term survival. Therapies have developed involving systemic treatment over the last decade. However, there are
no effective early detection tools for SCLC,
and the prognosis for patients with SCLC is
poor (5). It has been suggested that a number
of factors play a prognostic role in SCLC, such
as C-reactive protein (6), Bcl-2, E-cadherin,
1
LI et al.
and integrin b1 (3). However, controversy
remains over which is the best prognostic
factor.
Glycoprotein nonmetastatic melanoma B
(GPNMB), also known as osteoactivin or dendritic cell heparin sulfate proteoglycan integrin-dependent ligand (DC-HIL), is a type I
transmembrane glycoprotein, that contains
three domains, a long extracellular domain
(ECD), a single transmembrane region, and a
short cytoplasmic domain (7, 8). The ECD
domain is made up of 12 potential N-glycosylation sites, a polycystic kidney disease (PKD)
domain, and a RGD motif that binds to integrin (9, 10). GPNMB has been identified as a
melanosome-specific structural protein. It
shares high homology with Pmel17, which is
reported to be involved in many biological
processes, including inflammation, tissue
regeneration, cell migration, and metastasis of
malignant tumors. It is known to be abnormally expressed in some aggressive cancers,
such as glioma (11), melanoma (12), and cancer of the stomach, lung, and breast (13).
Ectopic overexpression of GPNMB in hepatocellular carcinoma, glioma, and breast cancer
cells promotes the invasion and metastasis of
these cancers (14). This implies that GPNMB
is a potential molecular therapeutic target in
patients with many malignant tumors.
GPNMB-specific monoclonal antibody, generated against the GPNMB ECD domain was
designed to inhibit the growth of GPNMBpositive melanoma cells in vitro when linked to
the cytotoxic agent monomethylauristatin E
(MMAE) (15). Recent investigations suggest
that enhanced mRNA and protein levels of
GPNMB in glioblastoma multiforme patients
correlated with higher survival risk, indicating
that GPNMB may become a prognostic predictor of many malignant tumors that express
GPNMB (9). It has recently been demonstrated that GPNMB expression is a prognostic marker of poor outcome in patients with
breast cancer (16).
The role of GPNMB as a prognostic factor
in SCLC remains unknown. The purpose of
this study was to investigate the prognostic
significance of GPNMB expression in SCLC.
We used immunohistochemical staining on tissue microarray blocks containing tissue samples from 132 patients with SCLC. In this
2
study, we found that GPNMB was more
highly expressed in SCLC than in normal tissues. We also observed high levels of GPNMB
in the serum of SCLC patients. Our results
suggest that the expression of GPNMB indicates poor prognosis and may serve as a prognostic indicator for SCLC.
MATERIALS AND METHODS
Patient information
The study was approved by the Ethics Committee
of the second affiliated hospital of Xi’an Jiaotong
University. This study consisted of 132 patients
who were newly diagnosed with SCLC from June
2006 to October 2011 in the second affiliated hospital of Xi’an Jiaotong University. The diagnosis of
SCLC was confirmed by histology or cytology in all
cases. All patients with newly diagnosed SCLC had
a complete history. They all received platinumbased combination chemotherapy (platinum plus
etoposide or irinotecan). For control purposes,
serum from 26 normal volunteers was collected.
Before randomization, informed consent was
required from the patients and controls. Patients’
clinical information was obtained from medical
records. The data collected for each patient
included age, gender, disease stage, survival, smoking history, performance status, metastatic sites,
and follow-up data. All SCLC tissue samples were
immediately frozen and stored at 80 °C for tissue
microarray analysis.
Tissue microarray construction
Formalin-fixed, paraffin-embedded SCLC tissue
samples were obtained from the second affiliated
hospital of Xi’an Jiaotong University. SCLC tumor
microarrays were constructed using 132 SCLC
tumor samples as previously described (17, 18).
Corresponding normal samples or adjacent normal
tissues were used as the control. Tissue cores with a
2 mm diameter taken from each tissue block were
placed in a recipient block using a manual tissue
microarrayer (Beecher Instruments, Sun Prairie, WI,
USA). Sample analyses were performed in duplicate
for each specimen in the array. The TMA blocks
were then cut into 4 lm sections for immunohistochemical analysis.
Immunohistochemistry
Glycoprotein nonmetastatic melanoma B (GPNMB)
expression was evaluated by immunohistochemistry as previously described (19–21). Formalin-fixed
© 2013 APMIS Published by Blackwell Publishing Ltd
GPNMB AS A PROGNOSTIC INDICATOR IN SCLC
tissue blocks of SCLC were cut into 4 lm sections
and mounted on silane-coated slides, deparaffinized
in xylene, and dehydrated in a graded alcohol series.
The slides were treated with 3% hydrogen peroxide
to block endogenous peroxidase activity. Heat pretreatment at 95 °C for 20 min in citrate buffer at
pH 6 was applied for antigen retrieval. The slides
were then cooled at room temperature before immunohistochemistry staining. After washing with
deionized water, the slides were incubated with primary monoclonal antibodies to GPNMB (Sigma,
St. Louis, MO, USA) at 4 °C overnight. The slides
were then washed with phosphate-buffered saline
(PBS) three times, and incubated with HRP-labeled
anti-rabbit IgG. Slides were stained with diaminobenzidine and counterstained with hematoxylin.
Corresponding normal samples or adjacent normal
tissues were used as the control. To evaluate
GPNMB expression, the labeling score was
determined by the percentage of positive cells.
Cut-off points were chosen to categorize tumors as
positive or negative: <10% of cells with GPNMB
expression were regarded as GPNMB negative,
>10% and 50% were regarded as weakly positive,
>50% were considered as strongly positive. In this
study, scoring was performed by two investigators
independently.
ELISA
Serum from SCLC patients (n = 132) or healthy
volunteers (n = 26) was collected and stored at
80 °C until use. SCLC patients were divided into
two groups: GPNMB weak-positive group and
GPNMB strong-positive group. Serum GPNMB
levels were measured using a Human Soluble Osteoactivin/GPNMB ELISA kit (Aviscera Bioscience;
Santa Clara, CA, USA). GPNMB absorbance was
measured at 450 nm and corrected for plate artifact
at 570 nm.
Statistical analysis
Statistical analysis was performed using SAS software (StatView, version 5.0). The Student’s t-test
and Fisher’s exact probability test were used to
compare the data from the different groups. The
median ages of SCLC patients were analyzed using
the Mann–Whitney U-test. All data are presented as
median (range) or mean SD. Overall survival rate
was defined as the time from the date of diagnosis
until the date of death. Survival curves were
generated using the Kaplan–Meier method. The
statistical significance of differences between survival
curves was assessed using the log-rank test.
A p-value < 0.05 was considered as statistically
significant.
© 2013 APMIS Published by Blackwell Publishing Ltd
Table 1. Patient characteristics
Characteristics
Number
%
Total
132
100
Median age
68 (40–86)
Gender
Male
87
66
Female
45
34
Smoking history
Never
17
12.9
Former
21
15.9
Current
94
71.2
Performance status
ECOG 0-1
93
70.5
ECOG 2-4
39
29.5
Stage
LD
54
41
ED
78
59
Number of metastatic sites
0
79
59.8
1
36
27.3
2
12
9.1
3
5
3.8
Metastatic site
Bone
37
28.0
Bone marrow
2
1.5
Brain
18
13.6
Liver
26
19.7
Pleural effusion
17
12.9
Adrenal gland
10
7.6
Lung to lung
9
6.8
Pericardial effusion
5
3.8
Other
8
6.0
ECOG, Eastern Cooperative Oncology Group; LD,
limited disease; ED, extensive disease.
RESULTS
Patient characteristics
A total of 132 patients were included in this
study. Patient characteristics are summarized
in Table 1. The median age was 68 years
(range: 40–86). Seventeen patients (12.9%)
had never smoked, and most of the patients
had good performance status (70.4%). Fiftyfour patients had limited disease (LD, 41%),
and 78 had extensive disease (ED, 59%). The
most common metastatic site was bone
(28%). All patients received platinum-based
combination chemotherapy (platinum plus
etoposide or irinotecan). The median followup duration was 19 months (range, 1–60)
after the initial pathological diagnosis, and
median overall survival was 23 months.
Among the 132 patients, 118 (89.4%) died of
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LI et al.
their tumors and 7 (5.30%) were still alive at
the last follow-up.
A
Immunohistochemical analysis of GPNMB in SCLC
samples
Glycoprotein nonmetastatic melanoma B
(GPNMB) expression was observed in 132
SCLC tumor tissues. The percentage of tumor
staining for GPNMB was quantified, and
labeling scores were calculated by multiplying
the intensity by the percentage of tumor that
stained positive. GPNMB could be observed
in both normal tissues and tumor tissues.
Strong-positive expression of GPNMB was
observed in 73 cases (55.3%), and weakpositive expression of GPNMB in 59 cases
(44.7%) of SCLC. We found that GPNMB
was more highly expressed in SCLC than in
normal tissues as shown in Fig. 1.
B
Serum GPNMB expression
We next detected the serum GPNMB level of
the weak-positive group and the strong-positive
group. Serum GPNMB was evaluated using
enzyme-linked immunosorbent assay (ELISA),
and the serum of healthy volunteers was used
as the control. As shown in Fig. 2, healthy participants (n = 26) had a mean serum GPNMB
level of 427.61 273.9 pg/mL (range: 104–
1203 pg/mL, median: 348.5 pg/mL). The average serum GPNMB concentration was
1054.15 363.71 pg/mL (range: 150–1952 pg/
mL, median: 1078 pg/mL) in the weak-positive
group, and 2611.52 457.57 pg/mL (range:
1558–3773 pg/mL, median: 2611 pg/mL) in the
strong-positive group. The latter had significantly higher serum GPNMB levels than the
weak-positive group and healthy controls
(p < 0.01).
Elevated GPNMB expression is associated with
reduced overall survival
To analyze the effect of GPNMB expression
on SCLC prognosis, we studied all SCLC
cases by Kaplan–Meier analysis. All 132
patients were followed up for 1–60 months. As
shown in Fig. 3, patients with tumors that
showed weak-positive GPNMB expression
had a significantly longer overall survival time
4
C
Fig. 1. Representative immunohistochemical staining for Glycoprotein nonmetastatic melanoma B
(GPNMB) expression in small cell lung cancer tissues: (A) weak positive for GPNMB, (B) strong
positive for GPNMB. (C) Immunohistochemical
staining of GPNMB in healthy volunteers. (A)
and (B) were obtained at 200 9 magnification, and
(C) at 100 9 magnification.
than did those with strong-positive GPNMB
expression (27 months vs 15 months, p <
0.01). The 5-year survival rates were significantly
different between the GPNMB weak-positive
and GPNMB strong-positive group (p < 0.01,
log-rank test). The 5-year survival rate after
© 2013 APMIS Published by Blackwell Publishing Ltd
GPNMB AS A PROGNOSTIC INDICATOR IN SCLC
Fig. 2. Up-regulation of serum Glycoprotein nonmetastatic melanoma B (GPNMB) concentrations in
small cell lung cancer (SCLC) patients. The average
GPNMB level was 1054.15 363.71 pg/mL in the
GPNMB weak-positive group, 2611.52 457.57 pg/
mL in the GPNMB strong-positive group, and
427.61 273.9 pg/mL in healthy volunteers
(p < 0.01).
Fig. 3. Kaplan–Meier analysis of survival according
to Glycoprotein nonmetastatic melanoma B
(GPNMB) expression in SCLC. Patients with
tumors that showed weak-positive GPNMB expression (n = 59) showed significantly better overall survival than those with strong-positive GPNMB
expression (n = 73) (p < 0.01, log-rank test).
treatment in the weak-positive group and
strong-positive group was 7.7% and 4.6%,
respectively.
DISCUSSION
Small cell lung cancer (SCLC) accounts for
about 15–20% of all lung cancers and is one
of the most aggressive cancers with a very
poor prognosis (22, 23). Surgery cannot
© 2013 APMIS Published by Blackwell Publishing Ltd
achieve good clinical results because by the
time the cancer is discovered it has usually
spread. Chemotherapy and radiotherapy are
performed as the main treatments of limitedstage SCLC (LS-SCLC), with approximately
20% of patients achieving a cure (24,
25).Although chemotherapy has been quite
successful in improving the quality of life
in patients with extensive-stage SCLC
(ES-SCLC), little progress has been made in
the treatment of ES-SCLC, and subsequent
relapses are common because of drug resistance (26). The median survival of patients
who received current standard treatment was
only 9–10 months from diagnosis (27). To
improve the prognosis of SCLC, reliable prognostic indicators are needed.
Prognostic factors for SCLC include age,
performance status, and weight loss. Young
age and good performance status are associated with better prognosis in SCLC (6).
Patients with limited disease (LD) have a better prognosis than patients with extensive disease (ED). For patients with LD, a median
survival of 16–24 months and a 5-year survival
of 14% with current forms of treatment have
been reported. Other prognostic factors, such
as CYFRA21-1 (2), C-reactive protein (CRP)
(6), and E-cadherin (1) have also been identified as prognostic factors for SCLC in previous reports.
GPNMB is a type I transmembrane glycoprotein, that plays an important role in the
biosynthesis and transport of melanin. Previous studies have reported the association
between GPNMB expression and cancer.
GPNMB is overexpressed in various types of
tumor cells, such as melanoma, gliomas, hepatocellular carcinoma, and breast cancer.
Recent studies have demonstrated that
GPNMB expression in transformed human
astrocytes or rat hepatoma cells generated augmented invasiveness and metastasis capabilities.
It has been indicated that overexpression of
GPNMB promoted breast cancer metastasis to
bone in vivo (13). Therefore, GPNMB has been
identified as a potential therapeutic target for
malignant tumors.
Previous observations have demonstrated
that GPNMB is an independent prognostic
indicator and novel therapeutic target for
breast cancer (16). However, it is not known
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LI et al.
whether GPNMB contributes to the diagnosis
of SCLC. The results of this study show that
high expression of GPNMB appears to predict
a poor prognosis. To the best of our knowledge, this is the first report to evaluate the
clinical usefulness of GPNMB for predicting
survival of SCLC patients.
Using immunohistochemical analysis, we
found that GPNMB protein was higher in tissues of SCLC patients than in normal tissues
in this study. Then, we divided 132 SCLC
patients into two groups: weak-positive
GPNMB group and strong-positive GPNMB
group. We observed that serum GPNMB levels were significantly up-regulated in the
strong-positive group in comparison with the
weak-positive group and healthy control.
According to our statistical analyses, there
seems to be an association between GPNMB
expression and survival time. A total of 24.9%
of the patients in weak-positive group survived
for more than 3 years, whereas only 4.6% of
patients in the strong-positive group survived
for the same period of time. Analysis of the
GPNMB
weak-positive
group
showed
27 months median survival vs 15 months for
the strong-positive group. Elevated expression
of GPNMB was associated with poor survival.
Therefore, we speculated that GPNMB overexpression may be involved in the pathogenesis
of SCLC. GPNMB is highly expressed in
SCLC, but rarely in normal lung tissues, possibly demonstrating that high GPNMB is likely
to increase the invasiveness and metastatic
capabilities of SCLC.
In recent years, molecular target therapy has
brought about a breakthrough in the treatment
of cancer. Data presented in this study demonstrate that GPNMB may be a potential therapeutic target in SCLC. Tse reported that a
fully human monoclonal antibody CR011,
generated against the GPNMB ECD domain,
specifically inhibited the growth of GPNMBpositive melanoma cells in vitro (15). Another
investigation also reported that GPNMB is a
novel therapeutic target in breast cancer, and
found that a toxin-conjugated antibody CDX011 effectively inhibits the growth of GPNMBexpressing breast cancer cells (16). However,
the clinical usefulness of GPNMB in SCLC has
not been fully described and further studies are
needed to define the role of GPNMB in SCLC.
6
In conclusion, high expression of GPNMB
was correlated with poor prognosis, suggesting
that GPNMB could be used as a prognostic
indicator in SCLC patients. Our results might
provide a novel way to explore targeted therapy in SCLC patients.
DISCLOSURES
The authors have no financial conflicts of
interest.
This work was supported by Shaanxi Province science and technology research and development projects (No. 2008k09-01(5)).
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