Immunohistochemical expression of epithelial and stromal immunomodulatory signalling molecules is a prognostic indicator in breast cancer.

Abstract

The immune system has paradoxical roles during cancer development and the prognostic significance of immune modulating factors is controversial. The aim of this study was to determine the expression of cyclooxygenase 2 (COX-2), transforming growth factor-beta (TGF- beta), interleukin-10 (IL-10) and their prognostic significance in breast cancers. Ki67 was included as a measure of growth fraction of tumor cells.
On immunohistochemical stained slides from 38 breast cancer patients, we performed digital video analysis of tumor cell areas and adjacent tumor stromal areas from the primary tumors and their corresponding lymph node metastases. COX-2 was recorded as graded staining intensity.
The expression of TGF-beta, IL-10 and Ki67 were recorded in tumor cell areas and adjacent tumor stromal areas. In both primary tumors and metastases, the expression of COX-2 was higher in the tumor stromal areas than in the tumor cell areas (both P < 0.001). High stromal staining intensity in the primary tumors was associated with a 3.9 (95% CI 1.1-14.2) times higher risk of death compared to the low staining group (P = 0.036). The expression of TGF-beta was highest in the tumor cell areas of both primary tumors and metastases (both P < 0.001). High stromal expression of TGF-beta was associated with increased mortality. For IL-10, the stromal expression was highest in the primary tumors (P < 0.001), whereas in the metastases the expression was highest in tumor cell areas (P < 0.001). High IL-10 expression in tumor- and stromal cell areas of primary tumors predicted mortality. Ki67 was higher expressed in tumor stromal areas of the metastases, and in tumor cell areas of the primary tumors (P < 0.001). Ki67 expression in tumor cell areas and stromal areas of the metastases was independently associated with breast cancer mortality.
Stromal expression of COX-2, TGF-beta and Ki67 may facilitate tumor progression in breast cancer.

Full text

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RESEARCH ARTICLE Open Access
Immunohistochemical expression of epithelial
and stromal immunomodulatory signalling
molecules is a prognostic indicator in breast
cancer
Elin Richardsen1,2*†, Rebecca Dale Uglehus1†, Stein Harald Johnsen3,4and Lill-Tove Busund2,1†
Abstract
Background: The immune system has paradoxical roles during cancer development and the prognostic
significance of immune modulating factors is controversial. The aim of this study was to determine the expression
of cyclooxygenase 2 (COX-2), transforming growth factor-beta (TGF- beta), interleukin-10 (IL-10) and their
prognostic significance in breast cancers. Ki67 was included as a measure of growth fraction of tumor cells.
Methods: On immunohistochemical stained slides from 38 breast cancer patients, we performed digital video
analysis of tumor cell areas and adjacent tumor stromal areas from the primary tumors and their corresponding
lymph node metastases. COX-2 was recorded as graded staining intensity.
Results: The expression of TGF-beta, IL-10 and Ki67 were recorded in tumor cell areas and adjacent tumor stromal
areas. In both primary tumors and metastases, the expression of COX-2 was higher in the tumor stromal areas than
in the tumor cell areas (both P < 0.001). High stromal staining intensity in the primary tumors was associated with
a 3.9 (95% CI 1.1-14.2) times higher risk of death compared to the low staining group (P = 0.036). The expression
of TGF-beta was highest in the tumor cell areas of both primary tumors and metastases (both P < 0.001). High
stromal expression of TGF-beta was associated with increased mortality. For IL-10, the stromal expression was
highest in the primary tumors (P < 0.001), whereas in the metastases the expression was highest in tumor cell
areas (P < 0.001). High IL-10 expression in tumor- and stromal cell areas of primary tumors predicted mortality. Ki67
was higher expressed in tumor stromal areas of the metastases, and in tumor cell areas of the primary tumors (P <
0.001). Ki67 expression in tumor cell areas and stromal areas of the metastases was independently associated with
breast cancer mortality.
Conclusions: Stromal expression of COX-2, TGF-beta and Ki67 may facilitate tumor progression in breast cancer.
Background
Epithelial-stromal interactions are important for tumor
development and progression [1]. The stroma surround-
ing solid tumors contains activated and recruited cells
like fibroblasts, innate and adaptive immune cells, and
endothelial cells which can be supportive and responsive
agents in tumorigenesis [1,2]. An abnormal stroma may
cause dysfunction of epithelial-mesenchymal interactions
which promotes progression of preneoplastic lesions to
malignancy [3]. Changes in the stroma environment
may lead to selection of cells with altered survival char-
acteristics. In normal mammary tissue stroma plays a
major role in control and regulation of physiological
processes in the breast [4]. The complexity of stromal
reaction and the signalling mechanisms between tumor
and stromal cells in breast cancer is incompletely under-
stood, not least because stroma is continuously remo-
delled during tumor progression [5,6].
The cytokine cyclooxygenase-2 (COX-2) is frequently
expressed by cancerous cells. It is Not constitutively
* Correspondence: elin.richardsen@unn.no
† Contributed equally
1Department of Clinical Pathology, University Hospital of Northern Norway,
N-9038 Tromsø, Norway
Full list of author information is available at the end of the article
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© 2012 Richardsen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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expressed, but can be rapidly induced by oncogenes, other
cytokines, chemokines, growth factors, hypoxia, ultraviolet
light, and epidermal growth factors. The transcription fac-
tors, nuclear factor-?B (NF-?B), hypoxia-inducible factor
1a (HIF1a), and activator of transcription 3 (STAT3) coor-
dinate the production of COX-2 and prostaglandins [7-11].
In early tumor outgrowth elevated transforming growth
factor-b (TGF-b) is tumor suppressive, whereas at later
stages it may act as a promoter of tumor progression
[12,13]. TGF-b induces a-smooth muscle actin and col-
lagen production in culture fibroblasts [14] and is a poten-
tial mediator of desmoplastic responses in tumors.
Desmoplasia in invasive tumors and metastases is morpho-
logically characterized by extensive proliferation of fibro-
blast-likecells andextracellular
inflammation and immune responses represented by lym-
phocytes, macrophages, dendritic cells; and tumor angio-
genesis [15]. Loss of TGF-b sensitivity in carcinoma cells is
frequently accompanied by increased expression of TGF-b
in the same cells [16]. TGF-b is elevated in cancer cells
compared to normal epithelial cells, and appears to be
even more elevated in poorly differentiated tumors [17,18].
The significance of interleukin 10 (IL-10) within the
tumor microenvironment is debated because it is depen-
dent of the malignant cells, tumor-infiltrating macro-
phages and lymphocytes [19,20]. IL-10 derived from
regulatory T-cells in the tumor or from cells in the
tumor microenvironment may stimulate tumor progres-
sion [21,22]. In tumor tissue, IL-10 has both immuno-
suppressive properties (potentially cancer promoting due
to inhibitory effects on antigen presenting capacity) and
anti-angiogenic properties (potentially cancer inhibiting)
[23-25].
Ki67 is a nuclear protein, which is expressed during all
the active phases of cell cycle (G1, S, G2 and mitoses),
but is absent from the resting phase (G0). Ki67 is strictly
associated with cell proliferation during the cell cycle
interphase. The Ki67 antigen can be exclusively detected
within the cell nucleus, whereas in mitosis most of the
protein is relocated to the surface of chromosomes. Stu-
dies have shown that Ki67 is associated with tumor
aggressiveness in breast cancer [26].
Previous studies have highlighted the epithelial-stro-
mal interactions and emphasized the stromal-epithelial
interface as critical mediators of tumor progression
[27-31]. In this study, we evaluated the prognostic sig-
nificance of the immunomodulatory signalling molecules
COX-2, TGF-b, IL-10 and Ki67 in tumor epithelium
and stromal areas of human breast cancer.
matrix (ECM);
Methods
Patients
Primary tumor tissues and tissues from the correspond-
ing lymph node metastases were investigated in 38
untreated breast cancer patients. The specimens were
diagnosed at the University Hospital of Northern Nor-
way (UNN) from 2000 to 2003. The initial diagnoses
were based on fine-needles biopsies, lumpectomy speci-
mens and resection specimens. To be included, patients
must have a confirmed diagnosis of breast cancer. In
case of death, the Causes of Death Registry/Population
Registry of Norway was consulted regarding the
assumed cause of death. Follow-up time was assigned
from the date of initial diagnosis to the beginning of
February 2010 with date of death as censoring points.
The Regional Committee for Research Ethics approved
the study. The Regional Committee approved that writ-
ten consent from the patients for their information to
be stored in the hospital database and used for research
was not needed because most of the material was more
than ten years old and many of the patients are now
dead.
Tissue samples
Representative formalin-fixed paraffin-embedded tissue
blocks were obtained from the archives of the Depart-
ment of Pathology at UNN. Clinical, biochemical and
radiological observations up to the diagnosis of metas-
tases were from the patients journals. Histological classi-
fication and grading of breast cancer was made in
accordance to the World Health Organizations criteria
and TMN-classification of malignant tumors [32,33].
Histological diagnoses were ductal carcinoma (34 cases)
and lobular carcinoma (4 cases). Hormone receptor sta-
tus was recorded at the time of the initial diagnose. His-
tological verification of metastases to axillary lymph
nodes was performed at the Department of Pathology,
UNN.. The material was collected from our approved
biobank for paraffin embedded material and slides. All
material was anonymously collected. The data were ana-
lyzed anonymously.
Immunohistochemistry
Antigen retrieval of COX-2 and IL-10 was performed
with Protease I, in a final dilution of 1:100 for 4 and 16
minutes, respectively. Antigen retrieval of TGF-b was
performed in a microwave oven with Tris/EDTA buffer,
pH 9.0, for intervals of 2 × 10 min. Antigen retrieval of
Ki67 was performed with steamer with Citrate buffer,
pH 7, for 32 min. The slides were then transferred to a
Ventana Benchmark®, XT automated slide stainer (Ven-
tana Medical System, France). Tissue sections were
incubated with primary polyclonal goat antibody against
COX-2 (final dilution 1:100), monoclonal rabbit antibo-
dies against TGF-b (final dilution 1:50) and monoclonal
mouse antibodies against IL-10 (final dilution 1:20). All
antibodies were from Santa Cruz Biotechnology Inc,
CA, USA. The mouse monoclonal antibody against Ki67
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was optimized for use in a Ventana automated slide stai-
ner in combination with Ventana detection kit. As sec-
ondary antibodies, biotinylated goat-anti-mouse IgG and
IgM, and goat-anti-rabbit IgG, both 200 μg/ml, were
used. For endogenous peroxidase blocking, the I-
VIEW™ DAB Detection Kit (Ventana) was used. Finally,
all slides were counterstained with haematoxylin to
visualize the nuclei. For each antibody, including nega-
tive staining controls, all staining was performed in a
single experiment. As negative staining controls, the pri-
mary antibodies were replaced with the primary anti-
body diluents. Appropriate positive and negative
controls were included in each antibody run according
to the manufacturer’s recommendations. Single stromal
cells within groups of epithelial cancer cells were
defined as belonging to tumor cell areas. The tumor
stromal areas were defined as stromal tissue surround-
ing groups of epithelial cancer cells in central parts of
the tumor and were negative by IHC staining with anti-
bodies directed against Cytokeratin (CK, Ventana). Stro-
mal tissues in the periphery of the tumors were not
investigated. For stromal cell characterisation, the slides
were stained by Masson Trichrome (collagen fibres),
Giemsa (granulocytes), Vimentin (fibroblasts), CD34
(vessels), CD20 (B-lymphocytes), CD3, CD4 and CD8
(T-lymphocytes), CD68 (macrophages), CD56 (NK-cells)
and CD1a (dendritic cells) (all antibodies were fromVen-
tana). Oestrogen receptors (ER) and progesterone recep-
tors (PRs) were visualized using antibody 1D15 (Dako)
and antibody NCL-PGR (Abbott Laboratories, Maiden-
head, UK), according to a previously published protocol
(34). The demonstration of oestrogen receptors (ER)
were visualized using antibody 1D15 (Dako), and pro-
gesterone receptors (PRs), with antibody NCL-PGR
(Abbott Laboratories, Maidenhead, UK), according to a
previously published protocol [34]. The staining of ER
and PRs was estimated using the “quick score” techni-
que [34] as follows: slides were assessed for both the
proportion of cells stained and staining intensity. Pro-
portions were scored as 0 (no cells staining); 1) 1-25%;
2) 26-50%; 3) 50-75%; or 4) > 75% stained cells. The
intensity was scored as 0, (no staining); 1, (weak); 2,
(moderate); or 3, (strong staining). The two scores were
added to give a final score of 0-7. A final score < 3 was
regarded as negative.
Digital video analysis
Microscopic images for quantitative analysis were
recorded with a Leitz Aristoplane microscope equipped
with a Leica DFC 320 digital camera. The Leica QWin
Image Analysing system (Leica Microsystems Digital
Imaging Solutions Ltd, Cambridge, UK) was used for
morphometric analysis. Leica DFC320 is based on a 3.3
megapixel sensor. The staining intensity of COX-2,
TGF-b, IL-10 and Ki67 in tumor cell areas and tumor
stromal areas was quantified by measuring the colour
value of red, green and blue colours (RGB), expressed in
composite units. Density thresholds of RGB were set to
quantify positive immunoreactivity of the red, green and
blue colour components and these thresholds were fixed
during the study. The number of pixels falling within
each threshold (1 pixel = 0.172 μm) indicated the
immunoreactivity reaction of each field and was
recorded. The intensity of immunoreaction for each
slide was expressed as the mean of RGB. Each slide was
initially examined at 10× and 20× magnifications for an
overall view. This practice allowed an area to be chosen
as the most representative, with no tissue folding or
overlapping, and minimal background staining. In each
slide, ten different areas along a projected Z-line at
400× magnification comprising both tumor cell and
stromal areas were systematically evaluated for the
expression of COX-2, TGF-b, IL-10 and Ki67. Positive
staining of COX-2 was assessed by the presence of
marked diffuse brown cytoplasm in cancer cells (Figure
1). The staining intensity of COX-2 was scored as 0
(negative staining), 1 (weak), 2 (moderate), or 3 (strong
staining) and the proportion of positive stained cells
within each group were assessed. A combined staining
index, SI, was calculated by multiplying the staining
intensity (0, 1, 2, or 3) by the percentage of positively
stained cells within each group (Figure 1) [34]. The
expression of TGF-b, IL-10 and Ki67 was recorded in
single non-epithelial cells within groups of epithelial
cancer cells (tumor cell areas) and in the surrounding
tumor stromalareas in central parts of the tumor (tumor
stromal areas).
Statistical analysis
Differences in staining intensity between primary
tumors and metastases were analyzed By Wilcoxon
signed rank test. Disease-specific survival was deter-
mined from the date of initial diagnosis to the time of
breast cancer death. The risk of death from breast can-
cer in high (above median) and low (below median)
staining intensity groups was compared by Kaplan-
Meier survival analysis and log-rank test. COX-propor-
tional hazards regression models were used to model
the outcome death as a function of staining intensity.
Age, histological grade (1-3), tumor size, oestrogen-
and progesterone receptor positivity (yes/no), were
included in the models in separate analyses to adjust
for possible confounding. Her2-neu was done in few
patients and was therefore excluded from these ana-
lyses. Pearson’s product moment correlation coefficient
and Spearman’s rank correlation were used in the
reproducibility analysis. A two-sided p value < 0.05
was considered statistically significant. The SPSS 16.0
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software package was used in all analyses (SPSS Inc.,
Chicago, IL, USA). This study was approved by the
Regional Committee for Research Ethics (REK,
ref.200303108- 3/IAY/400), and this study was sup-
ported by grants from the Northern Norway regional
Health Authority (Helse Nord RHF).
Results
Clinical features
Patients’ characteristics are listed in Table 1. Mean age
was 61.3 years (range, 39-89). Fifteen patients died dur-
ing the follow-up, and mean survival time was 45.6
months (range, 36-120 months). Histological grade 2
was the most common (53%). Except for two, all
patients had metastases to axillary lymph nodes at initial
diagnose. Tumor size was > 10 mm in 36 (94.7%)
patients. Mean tumor size at histological grade 1, 2 and
3 was 2, 4, and 3 mm, respectively. Oestrogen and pro-
gesterone hormone-receptor status were done in all
tumors, 29 was oestrogen positive and 15 were proges-
terone positive.
Expression of COX-2, TGF-b, IL-10 and Ki67
Figures 1 and 2, and Table 2 illustrate the COX-2, TGF-
b, IL-10 and Ki67 staining pattern in tumor cell areas
and tumor stromal areas of the primary specimens and
their corresponding lymph node metastases. The expres-
sion of COX-2, TGF-b, and IL-10 were predominantly
cytoplasmic. The overall expression of COX-2 was
higher in the metastases compared to the primary
tumors (p < 0.001) (Figure 1). In primary tumors as well
as in the metastases, the expression of COX-2 was high-
est in the tumor stromal areas (both p < 0.001). The
staining indexes (SI) for COX-2 in tumor stromal areas
were significantly higher in the metastases (121.3) com-
pared to the primary tumors (91.2).
The expression of TGF-b was highest in the tumor
cell areas of both primary tumors and metastases (both
p < 0.001) (Table 2). IL-10 was expressed in cells with
morphological features of macrophages and lympho-
cytes. The stromal expression was highest in the pri-
mary tumors (p < 0.001), whereas the tumor cell
expression was highest in the metastases (p < 0.001)
0
20
40
60
80
100
1
Distribution of staining intensity (%)
Abbreviation: SI= staining index: multiplications of staining intensity by percentage of positively stained cells within each
group.
Primary tumor Metastatic tumor Primary tumor Metastatic tumor
Tumor cell areas Tumor stromal areas
Negative
Weak
Moderate
Strong
SI:82.5 SI:105.2 SI:91.2 SI:121.3 (P= 0.001, compared to primary
tumors)
Figure 1 Expression of COX-2 in primary and metastatic tumors.
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(Table 2). Ki67 expression (Table 2) was higher in stro-
mal areas of the metastases (p < 0.001), whereas in pri-
mary tumors, the expression was highest in tumor cell
areas (p < 0.001). Using the median as cut-off, the
tumor and stromal expressions of the different markers
Were categorized into a high versus low staining group.
High expression of COX-2 was seen in tumor stromal
areas of both primary tumors and the metastases (Figure
3 and Table 3, p = 0.020, p = 0.003, log rank test). High
stromal staining intensity in the primary tumors was
associated with a 3.9 (95% CI 1.1-14.2) times higher risk
of death compared to the low staining group (p = 0.036)
(Table 3). After adjustment for age, histological grade,
oestrogen and progesterone receptor positivity the risk
estimate was weakened, but remained borderline signifi-
cant. The corresponding unadjusted and fully adjusted
risk estimates for the stromal areas in the metastases
were 6.8 (1.5-30.4) and 8.3 (4.2-27.7) (Table 3). High
stromal expression of TGF-b in primary tumors wasas-
sociated with increased mortality (HR 5.2, 95% CI 1.1-
24.0, P = 0.035). High IL-10 expression in tumor cell
areas (p = 0.018) and stromal areas (p = 0.003) in the
primary tumors predicted mortality, whereas high stain-
ing intensity in stroma of the metastases was borderline
associated (p = 0.057). Ki67 expression in tumor cell
areas and stromal areas of the metastases was indepen-
dently associated with breast cancer mortality.
Discussion
In both primary tumors and metastases, we observed
higher expression of COX-2 in the Tumor stromal areas
than in the tumor cell areas. For IL-10, a higher stromal
expression was seen in the primary tumors only,
whereas for Ki67 the stromal expression was highest in
the metastases. High stromal expression of COX-2 and
Ki67 in metastases, as well as high stromal expression of
TGF-b and IL-10 in primary tumors were independently
associated to breast cancer mortality.
Mammary stromal tissue has a major role in the con-
trol and regulation of physiological processes in the
breast. Likewise, during breast carcinoma development
of the tumor stroma is believed to contribute in actively
generating transformed lesions and tumors [35,36]. Evi-
dence from genetic and clinical studies indicates that
COX-2 up-regulation is one of the key steps in several
preneoplastic lesions and cancers [37-41]. The role of
COX-2 in the pre-invasive stages of breast tumorigen-
esis has been highlighted after recent publications,
which linked the use of NSAIDs to decreased risk of
breast cancer [42,43]. Our finding strengthens COX-2 as
an important marker of breast cancer aggressiveness.
TGF-b signalling pathways are involved in many bio-
logical processes during embryogenesis, tissue homeos-
tasis and mammary epithelial growth [44,45]. During
transformation of a normal cell into a cancer cell, var-
ious components of the TGF-b signalling pathway may
mutate, making the cell resistant to the effects of TGF-b
[46,47]. TGF-b may suppress tumor growth in early
stages, whereas at later stages TGF-b may enhance
tumor growth [48]. In spite of a higher TGF-b expres-
sion in tumor cell areas, it was the stromal expression
that was associated with breast cancer mortality in this
Table 1 Patients demographics and clinical characteristics (N = 38)
Histological differention grade
123
Mean age (range), years 52.1 (39-60)62.3 (45-88)63.8 (45-89)
n = 6 n = 20n = 12
Hormone receptor status
ER+5195
ER-118
PGR+3 110
PGR-396
Her2-Neu+106
Her2-Neu-242
Tumor size (mm)
0 - 10-11
11 - 20293
21 - 30164
31 - 40-3-
> 40314
Mean243
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study. This may indicate that stromal expression of
TGF-b is particularly important in the early stage of
tumor progression. Proliferative activity defined by Ki67
staining is associated with cancer progression and poor
prognosis in a number of malignant tumors, including
breast cancer [49]. Our study is in line with these
reports, showing the highest expression of Ki67 in
stroma of the metastases.






COX-2
TGF-?
IL-10
Ki67
Primary tumors Metastases
Figure 2 Expression of COX-2, TGF-b, IL-10 and Ki67 in tumor cells and stromal cells areas of primary and metastases.
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The production and action of COX-2, TGF-b and IL-
10 are interrelated. Some experimental studies have
shown that Th2 lymphocytes release high levels of IL-10
and thereby induce COX-2 expression [22]. IL-10 and
TGF-b also cooperate to down-regulate immune
responses. Studies on intestinal epithelial cells transgenic
for IL-10 have shown that high TGF-b production also
controls the ability to respond to TGF-b [21]. There are
few studies comparing COX-2, TGF-b, and IL-10
expression in mammary tumor cell areas and tumor
stromal areas. Except for TGF-b, the expression of
COX-2, IL-10 and Ki67 in this study was higher in
Table 2 Distribution of TGF-b, IL-10 and Ki67 in tumor
cell areas and tumor stromal areas and their
corresponding metastases.
TGF-b
TP Median (IQR) 760 (580-862)
IL-10Ki67
395 (204-715)676 (545-764)
SPMedian (IQR)250 (119-377)610 (291-791)326 (238-454)
TM Median (IQR)781 (693-904) 656 (433-887)419 (165-723)
SM Median (IQR)220 (92-273) 426 (255-273)751 (539-855)
The values are pr 1000 counted cells.
Abbreviation; IQR: interquartile range; TP: tumor cell areas of primary tumors;
SP: tumor stromal areas of primary tumors; TM: tumor cell areas of metastatic
tumors; SM: stromal cell areas of metastatic tumors.
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Figure 3 Disease-specific survivals in tumor cell areas and tumor stromal areas of metastatic tumors.
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tumor stromal areas than in tumor cell areas. Our find-
ings put emphasize on the surrounding stroma, support-
ingthehypothesisthat
surrounding tumor epithelium plays an important role
in breast cancer progression. In future studies, both the
tumor cell areas and the stromal areas should be investi-
gated in primary breast cancer with or without
metastases.
Most studies on human cancer tissues have evaluated
immunohistochemical treated hotspot areas, where the
staining intensity is highest. This is based on the
assumption that one single section is representative for
and reflects the histopathological pattern of the entire
specimen. Moreover, the hotspot areas are evaluated
without distinguishing between the tumor areas and the
stromal areas. Instead of evaluating the protein expres-
sion in hotspot areas, we evaluated ten consecutively
chosen fields along a projected Z-line in each tumor
specimen. This approach was chosen to achieving a
more representative picture of the tumor specimens and
to put emphasizes on the role of stromal tissue in
tumorigenesis.
In general, digital video analysis is regarded as being a
more objective method with a higher sensitivity and
reproducibility than light microscope and with better
responsiveness to changes in cell counts [50,51]. How-
ever, there are technical pitfalls in this method, includ-
ing background of the haematoxylin-eosin stained slides,
the thickness of the slide, and tissue folding or overlap-
ping, all which may cause bias in cell counting or
themicroenvironment
assessment of protein expression. We excluded fields
with tissue folding or overlapping from analysis. A
shortcoming of this study is the small sample size. Our
findings are on archived material, but should warrant a
larger prospective analysis.
Conclusions
In this study, immunohistochemical stromal expression
of COX-2, TGF-b, IL-10 and Ki67 was associated to
breast cancer mortality. Our findings heighten stroma as
an active participant in the carcinogenesis of breast
cancer.
Acknowledgements
We want to thank the participating laboratory technicians at Department of
Pathology, UNN, for their support and technical skills. Especially we want to
thank Andreas Lindal, Mona Pedersen, Hege Marthe Hoe, Lena Myreng and
Evy K. Johnsen for their very careful work. Financial support was given by
The Northern Norway Regional Health Authority (Helse Nord RHF) and The
Norwegian Cancer Society.
Author details
1Department of Clinical Pathology, University Hospital of Northern Norway,
N-9038 Tromsø, Norway.2Department of Medical Biology, University of
Tromsø, N-9037 Tromsø, Norway.3Department of Neurology and
Neurophysiology, University Hospital of Northern Norway, N-9038 Tromsø,
Norway.4Department of Clinical Medicine, University of Tromsø, N-9037
Tromsø, Norway.
Authors’ contributions
ER did the collection of tumor samples, and evaluated the
immunohistochemical staining, the digital video analysis performed the
statistical analysis and drafted the manuscript. RDU and ER performed the
microscopic images for quantitative analysis. RDU performed the digital
images. ER, LTB re-examined and histological graded the specimens. LTB
Table 3 Risk of death from breast cancer in high staining intensity groups compared to low staining intensity groups
Log Rank testUnadjusted HR (95% CI)Multivariate adjusted* HR (95% CI)
COX-2
TP
P = 0.322 0.6 (0.2-1.7), P = 0.339 0.6 (0.2-2.2), P = 0.485
SP
P = 0.0203.9 (1.1-14.2), P = 0.0363.5 (0.8-14.7), P = 0.059
TM
P = 0.0742.3 (0.8-6.9), P = 0.121 1.5 (0.4-6.0), P = 0.592
SM
P = 0.0036.8 (1.5-30.4), P = 0.0138.3 (4.2-27.7), P 0.024
TGF-b
TP
P = 0.984 1.0 (0.3-2.8), P = 0.989 2.2 (0.6-8.8), P = 0.249
SP
P = 0.8391.1 (0.4-3.3), P = 0.8425.2 (1.1-24.0), P = 0.035
TM
P = 0.0520.4 (0.1-4.1), P = 0.069 1.4 (0.3-6.6), P = 0.701
SM
P = 0.032 3.0 (1.0-9.2), P = 0.0462.3 (0.7-8.0), P = 0.174
IL-10
TP
P = 0.130 2.2 (0.8-6.4), P = 0.149 5.2 (1.3-20.6), P = 0.018
SP
P = 0.384 1.7 (0.5-5.3), P = 0.4001.1 (1.0-1.8), P = 0.003
TM
P = 0.0095.7 (1.3-25.6), P = 0.023 4.7 (0.8-25.5), P = 0.076
SM
P = 0.0354.4 (1.0-19.7), P = 0.0534.0 (0.8-20.7), P = 0.057
Ki67
TP
P = 0.2571.8 (0.6-5.3), P = 0.2751.2 (0.3-4.7), P = 0.771
SP
P = 0.3691.7 (0.5-6.1), P = 0.3871.1 (0.2-6.5), P = 0.959
TM
P = 0.0017.7 (1.7-35.2), P = 0.0087.4 (1.4-33.3), P = 0.020
SM
P < 0.0003.6 (1.0-12.9), P = 0.0311.1 (1.0-1.8), P = 0.002
*Adjusted for age, histology (grade 1, 2 and 3, tumor size, oestrogen- and progesterone receptor positivity (yes/no).
Abbreviations: TP: tumor cell areas of primary tumors; SP: tumor stromal areas of primary tumors; TM: tumor cell areas of metastases; SM: tumor stromal areas of
metastases
Richardsen et al. BMC Research Notes 2012, 5:110
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participated in study design and extensively reviewed the manuscript. SHJ
participated in data interpretation and reviewed the manuscript. All authors
have read and approved the manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 23 August 2011 Accepted: 21 February 2012
Published: 21 February 2012
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doi:10.1186/1756-0500-5-110
Cite this article as: Richardsen et al.: Immunohistochemical expression
of epithelial and stromal immunomodulatory signalling molecules is a
prognostic indicator in breast cancer. BMC Research Notes 2012 5:110.
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