Osteopontin Expression Correlates With Prognostic Variables and Survival in
Clear Cell Renal Cell Carcinoma
Koviljka Matusan, MD1, Gordana Dordevic, MD1, Darko Stipic, MD1, Vladimir Mozetic,
PhD2, Ksenija Lucin, PhD1
1
Department of Pathology, Rijeka University School of Medicine, Rijeka, Croatia
2
Department of Urology, Clinical Hospital Center Rijeka, Rijeka, Croatia
Corresponding author: Ksenija Lucin, PhD: Department of Pathology, Rijeka University
School of Medicine, Brace Branchetta 20, 51 000 Rijeka, Croatia
tel. /fax. +385 51 325 805/+385 51 325 810
E-mail: ksenijal@medri.hr
Running head: OPN in Renal Cell Carcinoma
1
ABSTRACT
Background and Objectives: Osteopontin (OPN) is a phosphorylated glycoprotein with
diverse functions including tumorigenesis and tumor cell metastasis. Recently, it has been
detected in a growing number of human tumors, and assessed as a potential prognostic
marker. The aim of this study was to analyze the expression of osteopontin in normal renal
tissue and clear cell renal cell carcinomas (CRCC), and to assess its prognostic significance.
Methods: The expression of OPN protein was immunohistochemically analyzed in 171
CRCC and compared to usual clinicopathological parameters such as tumor size, nuclear
grade, pathological stage, Ki-67 proliferation index, and cancer specific survival.
Results: In normal renal parenchyma the expression of osteopontin was seen in distal tubular
epithelial cells, calcifications and some stromal cells. The upregulation of OPN was observed
in 61 CRCC (35.7%) in the form of cytoplasmic granular staining of various intensities.
Statistical analysis showed correlation of the OPN expression with tumor size (P<0.001),
Fuhrman nuclear grade (P<0.001), pathological stage (P=0.011), and Ki-67 proliferation
index (P<0.001). Moreover, patients with OPN positive tumors had significantly worse
prognosis in comparison to patients with tumors lacking OPN protein (P=0.004).
Conclusion: Our results suggest that overexpression of OPN is involved in the progression of
CRCC.
Key Words: adenocarcinoma; renal cell; osteopontin; immunohistochemistry; prognosis
2
INTRODUCTION
Osteopontin (OPN) is a highly phosphorylated and glycosylated protein secreted into
the extracellular matrix by a variety of cell types. OPN was originally identified in bone [1,2],
but is now known to be expressed in other tissues as well, including the epithelium of the
gastrointestinal tract, exocrine glands, and distal renal tubules [for review see ref. 3]. OPN is
upregulated in activated T cells, macrophages associated with inflammation and tissue repair,
and smooth vascular cells of atherosclerotic plaques [4]. Numerous functions have been
ascribed to osteopontin, including roles in bone remodelling, cell-mediated immunity, the
ability to act as a cytokine in cell signalling, resulting in proliferation and/or cell survival, and
as a cell-attachment protein [3,4]. Increased expression of OPN in many transformed cell lines
[5,6], and its induction during multistage carcinogenesis in mouse skin, implicate that this
protein participates in interactions of tumor cells with host matrix, especially in the invasion
and spreading of tumor cells [7,8].
OPN has also been detected in a number of human tumor tissues and assessed as a
potential marker of tumor progression [9-16]. However, until now, there are no detailed
reports on the OPN expression in renal cell carcinoma (RCC), which is a common cancer
worldwide with increasing incidence and variable clinical behaviour. At present, tumor stage
and nuclear grade are considered to be the main prognostic indicators [17]. However, in a
significant number of patients, these parameters are insufficient to predict biological
behaviour of the tumor, especially in clear cell renal cell carcinoma (CRCC), the most
common type of RCC. Therefore, additional prognostic factors are needed to identify the
patients at high risk of tumor progression.
In the present study, using immunohistochemical technique, we have analyzed the
expression of OPN in specimens of CRCC from a large group of patients with a long term
3
follow up, and its relationship with the usual prognostic factors, as well as the association
with cancer-specific survival.
MATERIALS AND METHODS
Tumor samples
A total of 171 renal cell carcinoma were obtained from patients surgically treated
between 1990 and 1998 at the Department of Urology, Clinical Hospital Center Rijeka.
Tumor samples were fixed in 4% buffered formalin, embedded in paraffin, and routinely
stained with haematoxylin and eosin. Since typing and grading of RCCs has markedly
changed in the past decade, all cases were reviewed by two pathologists. Tumors were
classified according to WHO criteria [18], and graded using the Fuhrman nuclear grading
system [19]. Tumor stage was defined according to the International Union Against Cancer
(IUCC) 2002 TNM classification [20]. Follow up information was obtained from patients’
medical records and from files of the Croatian Cancer Registry. Since 51 patients were lost
during the follow up, a total of 120 cases were included in survival analysis.
Immunohistochemistry
For each case of CRCC, a representative slide of the tumor with highest nuclear grade
and the corresponding paraffin block were selected. Five-micron sections were cut on glass
slides (DakoCytomation, Glostrup, Denmark) and air-dried during the night. Following
deparaffinization in xylene and rehydration in alcohols, heat-induced epitope retrieval was
achieved by immersing slides in 10mM citrate buffer (pH 6.0) and boiling for 10 minutes in a
pressure cooker. Slides were allowed to cool during 45 minutes, and then pre-incubated with
blocking solution containing normal donkey serum (Santa Cruz Biotechnology, Santa Cruz,
CA, USA) for 30 minutes. Indirect immunoperoxidase staining was performed using
4
DakoCytomation LSAB2 HRP system on the automatic immunostainer (DakoCytomation,
TechMateTM Horizon, Glostrup, Denmark), according to the manufacturer’s protocol. OPN
protein was detected by goat anti-human monoclonal antibody (clone K-20, Santa Cruz
Biotechnology, Santa Cruz, CA, USA, dilution 1:100), followed by donkey anti-goat IgG as
secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:250). For a
negative control, an irrelevant goat IgG was used (Santa Cruz Biotechnology, Santa Cruz,
CA, USA). Staining of the luminal portion of distal tubular cells and calcifications within
renal parenchyma served as a positive control. In some doubtful cases, staining with antiCD68 (clone KP-1, DakoCytomation, Glostrup, Denmark, 1:200) was performed to
distinguish between tumor cells and histiocytes, which, in activated state, are also OPN
positive.
MIB-1 (DakoCytomation, Glostrup, Denmark, dilution 1:50) was used to analyze
nuclear expression of the Ki-67 cell proliferation antigen.
Evaluation of immunohistochemistry
The immunohistochemical staining results were examined independently by two
pathologists, without knowledge of the nuclear grade or other clinicopathological parameters
of each individual case. Staining results were evaluated in a semi-quantitative manner, by
assessing the percentage of carcinoma cells with cytoplasmic staining, as described recently
[9]. Cases without any detectable staining were considered negative, as well as those with
only focal positivity found in less than 1% of tumor cells. Positive staining was defined as
cytoplasmic granular positivity found in more than 1% of tumor cells. The percentage of cells
expressing OPN was classified into six groups and scored as follows: negative, when no
staining was present or staining in less than 1% of tumor cells; borderline, 1-5% of cells
positive; intermediate, 5-25% of cells positive; moderate, 25-50% of cells positive; strong, 50-
5
75% of cells positive; and very strong, 75-100% of cells positive. For the purpose of two-way
statistical analysis, all positive cases were grouped together.
Ki-67 labelling index (percentage of positive cells) was determined by scoring 500 tumor
cells at high power field in tumor areas with the highest density of positive cells. The counting
was performed on image analyzer using ISSA 3.1 software (Vams, Zagreb, Croatia). Sample
was considered positive if any nuclear staining was seen.
Statistical analysis
Statistical analysis was performed using Statistica 6.1 software (StatSoft, Inc., Tulsa,
OK, USA). Pearson’s chi-square test was used to assess the significance of associations
between categorical data. The mean values of continuous data, such as Ki-67 proliferation
index and tumor size, were compared by Student’s t-test. Survival probabilities were
estimated by the univariate Kaplan-Meier method, and survival curves were compared by the
log-rank test. Multivariate survival analysis was performed by the Cox regression model.
Statistical differences with P value less than 0.05 were considered significant.
RESULTS
Clinicopathological data
There were 103 men and 68 women in the study. The mean size of tumors was 7 ± 3.5
cm. The Fuhrman nuclear grading distribution was as follows: 29 (17%) grade 1, 69 (40.4%)
grade 2, 45 (26.3%) grade 3 and 28 (16.3) grade 4 tumors. There were 117 (68.4%) tumors
limited to the kidney (pT1 and pT2) and 54 (31.6%) tumors that expanded outside the kidney
(pT3 and pT4). Follow up was available for 120 patients and ranged from 1 to 165 months
(median 85 months). Survival time was calculated from date of surgery to date of death or to
date of last follow up.
6
Assessment of immunohistochemical staining for OPN
In normal renal parenchyma the expression of osteopontin was seen in distal tubular
cells in a form of granular cytoplasmic positivity with preferential luminal localization.
Calcifications in tubules or interstitium were also stained (Figure 1A). Some inflammatory
stromal cells, like macrophages and plasma cells, were also positive.
Renal cell carcinomas showed heterogeneous staining pattern, ranging from focal
expression of low intensity to strong and diffuse staining of tumor cells. In tubule-forming
areas, OPN staining was present on the apical surface in malignant glands, similar to the
pattern of staining seen in normal renal tubules. The assessment of staining was made only on
tumor cells, however, positive staining was also present on stromal macrophages, mainly
around the necrotic areas, and plasma cells. These stromal cells, as well as calcifications
(Figure 1A and 1D), and staining of luminal parts of distal tubules served as a positive
control. Among 171 carcinomas examined, 105 (61.4%) were negative (Figure 1B), 5 (2.9%)
displayed borderline staining, 21 (12.3%) intermediate, 16 (9.3%) moderate (Figure 1C), 9
(5.3%) strong, and 15 (8.8%) very strong staining (Figure 1D and 1E). Frequency distribution
of immunohistochemical staining patterns for OPN is shown in Table I.
Association of OPN and clinicopathological parameters
The expression of OPN protein in carcinoma cells was compared to tumor variables
that represent prognostic factors in CRCC: tumor size, histological grade, pathological stage,
and Ki-67 proliferative index. We found statistically strong association between the level of
OPN expression and all prognostic variables, by grouping together all the positive samples as
shown in Table II. By using five grades of OPN staining based on the percentage of positive
cells in Pearson’s chi-square statistics, the association was less pronounced, although still
significant (data not shown). The mean size of OPN positive tumors was 8.2 ± 3.5 cm,
7
compared to 6.3 ± 3.3 cm for tumors lacking OPN protein (P<0.001). All of grade 1 CRCC
were negative for OPN protein, while the number of positive tumors increased with higher
nuclear grade (P<0.001) (Figure 2). Considering tumors confined within kidneys (i.e., pT1 or
pT2) and those invading beyond the kidneys (i.e., pT3 or pT4) as low-stage and high-stage
tumors, respectively, the incidence of predominant expression of OPN in high pathological
stage was significantly higher than that in low-stage tumors (P=0.011). We also found strong
association between OPN expression and tumor growth fraction expressed as Ki-67
proliferation index (P<0.001). Namely, the expression of OPN significantly increased with
increasing proliferative activity of tumor cells. The mean value of Ki-67 index in OPN
negative tumors was 4.7  4.5%, while it was significantly higher in the group of OPN
positive tumors and measured 9.8  9%.
Association of OPN and patient survival
The association of immunohistochemical positivity for OPN and the cumulative
proportion of patients surviving during the follow up are shown in Figure 3. The 5-year
survival rate was 71% for 75 patients who were classified as OPN-negative, in comparison
with 40% of the 45 patients classified as OPN positive. Over the time period of 165 months,
the survival of patients with OPN positive carcinomas was significantly worse than of those
with OPN negative tumors (P=0.004). Because tumor grading and staging are considered
major prognostic parameters in CRCC, we first analyzed their impact on postoperative
survival. We found a significant inverse correlation between survival and tumor grading
(P<0.001) or staging (P<0.001). Although univariate survival analysis showed pathological
stage, nuclear grade, and OPN expression to be the significant predictive factors, only
pathological stage (P=0.013) remained significant in multivariate analysis, while nuclear
grade and OPN expression did not show independent prognostic value (Table III).
8
DISCUSSION
The role of OPN in tumor progression was first analyzed in experimental animal
models, where it was identified as phosphoprotein secreted by transformed cells, and
associated with increased metastatic potential in rodents [5,7]. Following this observation,
OPN mRNA and protein were also analyzed in several types of human cancer. Firstly, OPN
RNA was found to be produced primarily by tumor-associated macrophages rather than tumor
cells themselves, while both tumor cells and macrophages immunohistochemically stained for
OPN protein [10]. Authors suggested that OPN secreted by macrophages might bind to tumor
cells, possibly through the RGD-binding domain in OPN. Later on, tumor cells were also
identified as a source of OPN [12,13,21]. Until now, OPN RNA and protein have been found
to be overexpressed in a number of human tumor types, relative to normal tissue, and the
results of this investigations support the hypothesis that OPN detected within tumor cells has
a potential utility as a prognostic marker [9,13,22-25]. However, there are limited data
regarding the OPN expression in human renal cell carcinoma [10,14]. In order to better define
the role of OPN in the progression of CRCC, we performed an immunohistochemical staining
for OPN protein in large cohort of CRCC specimens, and assessed its potential prognostic
significance.
We have found that 39% of CRCC stained positively by the goat mAb to human OPN.
This percentage is largely in agreement with the overall staining levels of OPN reported
previously [10]. The staining was also observed in some reactive stromal cells, mainly
macrophages and plasma cells, and was particularly pronounced near areas of tumor necrosis,
as described by Brown et al. [10]. In tubule-forming areas, the OPN staining was present on
the apical surface in malignant glands, similar to the pattern of staining seen in the normal
distal tubules.
9
Brown et al. analyzed distribution of OPN mRNA and protein in 14 renal cell
carcinomas, and found strong expression of OPN mRNA in 13 cases, and strong and diffuse
cytoplasmic staining for OPN protein in 7 cases [10]. In their study, all tumors were
moderately differentiated clear cell renal cell carcinoma, except for one well-differentiated
papillary carcinoma, which was also positive. In our study, the level of OPN expression
strongly correlated with tumor variables reported previously to be associated with patient
outcome: histological grade, pathological stage, tumor size, and Ki-67 proliferation index.
While all of grade 1 tumors were negative for OPN protein, the positivity increased with
transformation to higher nuclear grade. In the recent work of Coppola et al., osteopontin was
also found to be significantly associated with tumor stage in 36 RCC, including tumors of
bladder, colon, kidney, larynx, mouth, and salivary gland [14]. They used tissue arrays to
assess OPN protein levels in 350 tumors from 23 body sites, compared with 113 normal
tissues. In that study, OPN was found to be elevated in tumors, relative to normal tissues, and
correlated significantly overall with tumor stage, when considering all tumor sites, as well as
with tumor stage for several sites individually. This association between OPN and tumor stage
most likely reflects the effect of OPN on cell migration. OPN contains an Arg-Gly-Asp
(RGD) sequence that binds to v1, 3, and 5 integrins, and is capable of promoting cell
attachment, chemotaxis, and signal transduction in several different cell types [26]. Due to the
presence of this sequence, it is probable that high expression of OPN by tumor cells may play
a role in tumor cell invasion and metastasis, a process in which adhesive interactions between
tumor cells and extracellular matrix are critical.
In our study, the level of OPN expression was higher in large tumors, as well as in
tumors with high growth fraction, expressed as Ki-67 proliferation index. Interestingly, in this
regard, recent experiments suggest that OPN acts in concert with several growth factors,
including hepatocyte growth factor [27], and epidermal growth factor (EGF) [28], to induce
10
malignant properties. Moch et al. described the relationship between EGF-receptor (EGFR)
expression and Ki-67 index in RCC, and their association with poor prognosis [29]. In the
light of these findings, it would be interesting to analyze the relationship between Ki-67
index, EGFR, and OPN protein in RCC. Also, several studies have demonstrated that OPN
delivers a prosurvival, antiapoptotic signal to the cell [30,31]. Since tumor size is largely
defined by the number of cells in proliferation and cells that undergo apoptosis, the
mechanisms described previously could explain the association of OPN and tumor size
observed in our study.
We have also found significant association of OPN expression and poor survival of
CRCC patients. So far, there were no reports on the impact of the OPN expression to the
clinical outcome in RCC. However, OPN overexpression has been significantly associated
with patient survival in lung carcinoma [15,16,22], and breast carcinoma [9,13].
The mechanisms by which osteopontin could promote tumor progression are still
unknown. Through its adhesive properties OPN can induce changes in tumor cell gene
expression, including induction of proteolytic enzymes, particularly urokinase plasminogen
activator (uPA) [32], and activation of growth factor kinases, which in turn may lead to
increased cell motility and invasion. Also, the role of OPN protein in angiogenesis could be
the one of proposed mechanisms through which OPN can mediate tumor progression and
metastasis. OPN augments endothelial cell migration induced by vascular endothelial growth
factor (VEGF) in an v3 integrin-dependent manner [33], and enhances survival of
endothelial cells [34]. In this regard, Shijuba et al. have shown the role of VEGF and OPN
coexpression in clinical outcome of patients with stage I lung carcinoma [16].
In conclusion, in this study we have shown for the first time the upregulation of OPN
protein in a large group of CRCC, and its association with the parameters of poor prognosis
11
and with shorter survival. Significance of increased OPN expression in predicting the
biological behavior of CRCC is unknown at present, and has to be more evaluated in future.
ACKNOWLEDGEMENT
This work was supported by the Ministry of science, education and sport of the
Republic of Croatia (grant 0062066). We thank Mr. Ozren Stanfel for excellent technical
support and Prof. Nives Jonjic for critical reading of the manuscript.
12
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TABLE I. Frequency distribution of immunohistochemical
staining of conventional renal cell carcinoma for osteopontin
Percentage of carcinoma
cells staining
Number of
carcinomas
Percentage of
carcinomas
<5
5-25
25-50
50-75
75-100
Total
110
21
16
9
15
171
64.3
12.3
9.4
5.3
8.7
100
TABLE II. Association of immunohistochemical staining for osteopontin (OPN) with other tumor
variables
Fuhrman nuclear
Pathological stage,
grade, No. (%)
No. (%)
Tumor size (cm)
Ki-67 index (%)
(mean ± SD)
(mean ± SD)
Grade 1,2 Grade 3,4
pT* 1,2
pT* 3,4
OPN79 (71.8) 31 (28.2)
negative
OPN19 (31.1) 42 (68.9)
positive
P value
<0.001
*Pathological stage
6.3 ± 3.3
83 (75.5)
27 (24.5)
4.7 ± 4.5
8.2 ± 3.5
34 (55.7)
27 (44.3)
9.8 ± 9
<0.001
0.011
<0.001
TABLE III. Multivariate analysis of prognostic factors
in conventional renal cell carcinoma patients
Relative
risk
95% confidence
interval
P value
Osteopontin
1.6
1.1 - 2.4
0.081
pT*
1.8
1.2 - 2.7
0.013
Nuclear grade
1.7
1.2 - 2.5
0.108
Variable
*Pathological stage
16
FIGURE LEGENDS
Figure 1
Immunohistochemical staining for osteopontin (OPN) in clear cell renal cell carcinoma
(CRCC). (A) Normal renal tissue showing staining of epithelial cells in distal tubules and
luminal calcifications. (B) Tumor cells are negative for OPN protein, while stromal cell,
probably macrophage, is positive. (C) Tumor sample showing moderate staining. (D) Tumor
cells showing diffuse staining of low intensity, and stromal calcifications strongly positive for
OPN protein. (E) Tumor cells diffusely and strongly positive. (F) Tumor cells with low
nuclear grade are negative, while those inside lymphocapillary space, and showing
transformation to higher nuclear grade, are strongly positive for OPN protein. Magnification
x100 (A), x200 (B-E)
Figure 2
Frequency distribution of immunohistochemical staining for osteopontin in relation to
Fuhrman nuclear grade. White bars represent negative, while black bars represent positive
cases. All carcinomas showing nuclear grade I morphology are negative, while the proportion
of positive cases increases in the course of transformation to higher nuclear grade. P<0.001.
Figure 3
Kaplan-Meier cumulative survival analysis according to staining for osteopontin (OPN). The
log-rank test showed significantly shorter overall survival in patients with OPN positive
tumors. P=0.004.
17
18
60
Number of cases
50
40
30
20
10
0
1
2
3
4
Fuhrman nuclear grading
19
1.0
0.9
0.8
0.7
OPN negative
0.6
0.5
OPN positive
0.4
0.3
Cumulative proportion surviving
0.2
0.1
0.0
0
20
40
60
80
100
120
140
160
180
Survival time (months)
20