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

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DOI: 10.1186/1756-0500-5-110 · Source: PubMed
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.
RESEARCH ARTICLE Open Access
Immunohistochemical expression of epithelial
and stromal immunomodulatory signalling
molecules is a prognostic indicator in breast
cancer
Elin Richardsen
1,2*
, Rebecca Dale Uglehus
1
, Stein Harald Johnsen
3,4
and Lill-Tove Busund
2,1
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 lo w 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 facilita te tumor progression in breast cancer.
Background
Epithelial-stromal interactions are important for tumor
developme nt 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 select ion of cells with altered survival char-
acteristics. In normal mamm ary tissue stroma plays a
major role in control a nd regulation of physiological
processes in t he breast [4]. The comp lexity 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
1
Department 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
Richardsen et al. BMC Research Notes 2012, 5:110
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© 2012 Rich ardsen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creativ e
Commons Attribution License (http://creativecommons.org/license s/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
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 progre ssion
[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 pro liferation of fibro-
blast-like cells and extracellular matrix (ECM);
inflammation and immune responses represented by lym-
phocytes, macrophages, den dritic cells; and tumor ang io-
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 o f inter leukin 10 (IL- 10) within the
tumor microenvironment is debated because it is depen-
dent of the malignant cells, tumor-infiltrating macro-
phages and lymphocyte s [19,20]. IL-10 derived from
regulatory T-cel ls in the tumor or fro m cells i n the
tumor microenvironment may stimu late tumor prog res-
sion [21,22]. In tumor tissue, IL-10 has both immuno-
suppressive properties (potentially cancer promoting due
to inhibitory effects on antigen presen ting capacity) and
anti-angiogenic properties (potentially cancer inhibiti ng)
[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 highli ghted 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.
Methods
Patients
Primary tum or 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 Northe rn Nor-
way (UNN) from 2000 to 2003. The i nitial diagnoses
were based on fine-needles biopsies, lumpectomy speci-
mens and resection speci mens. 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 Resear ch Ethics appr oved
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 te n 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 observatio ns up to the diagnosis o f 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 recept or 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 collect ed 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 f or 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 m in. 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 (fi nal 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 Ig G 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. F or each a ntibody, 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. Appro priate positive and negative
controls were included in each antibody run according
to the manufacturers 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 cel ls in ce ntral pa rts 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 Mass on Tr ichrome (collag en fib res),
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, M aiden-
head, UK), accor ding to a previo usly 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 e stimated 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 w as 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. L eica 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 t umor
stromal areas was quantified by measuring the colour
value of red, green and blue colours (RGB), expressed in
composite units. Densit y thresholds of RGB were set to
quantify pos itive immunoreactivity of the red, green and
blue colour components and these thresholds were fixed
during the study. The number of pixels falling w ithin
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 allo wed 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, I L-10 and Ki67. Positive
staining of COX-2 was assessed by the presence of
marked diffuse brown cytoplasm in cancer cells (Figure
1). T he staining intensity of COX-2 was scored as 0
(negative staining), 1 (weak), 2 (moderate), or 3 (strong
staining) and the proportion o f 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 w ere 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 (a bove median) and low (below medi an)
staining inte nsity groups was co mpared 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. Pearsons produc t moment correlation coefficient
and Spearman s rank correlation were used in the
reproducibility analysis. A two-sided p value < 0.05
was considered statistically signi ficant. 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 c haracte ristics are listed in T able 1. Mean age
was 61.3 y ears (range, 39-89). Fifteen patient s 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 p ro-
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 pri mary specimen s 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 s tromal 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 wa s expressed in cells with
morphological features of macrophages and lympho-
cytes. The stromal expression was highest in the pri-
mary tumo rs (p < 0.001), wher eas 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
g
roup.
Primary tumor Metastatic tumor Primary tumor Metastatic tumor
Tumor ce
ll
areas Tumor stroma
l
areas
Negative
Weak
Moderat
e
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 e xpressions of the different markers
Were categorized into a high v ersus low staining group.
High expression of COX-2 was seen in tumor stromal
areas of both primary tumors and the metastases (Figure
3andTable3,p =0.020,p = 0.003, l og 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 r eceptor pos itivity the risk
estimate was weakened, but remained borderline signifi-
cant. The corre sponding 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).HighIL-10expressionintumorcell
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 bord erline
associated (p = 0.057). Ki67 e xpression 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 tumo r stroma is believed to contribute in actively
gen erat ing 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 cance rs [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 e mbryogenesis, 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 brea st cancer m ortality in thi s
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=20 n=12
Hormone receptor status
ER+ 5 19 5
ER- 1 1 8
PGR+ 3 11 0
PGR- 3 9 6
Her2-Neu+ 1 0 6
Her2-Neu- 2 4 2
Tumor size (mm)
0 - 10 - 1 1
11 - 20 2 9 3
21 - 30 1 6 4
31 - 40 - 3 -
>40 3 1 4
Mean 2 4 3
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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 act ivity 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-ȕ
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 a re interrelat ed. Some experimen tal studies hav e
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,andIL-10
expression in ma mmary tumor cell area s 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 IL-10 Ki67
TP Median (IQR) 760 (580-862) 395 (204-715) 676 (545-764)
SP Median (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 count ed 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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tumor stromal areas than in tumor cell areas. Our find-
ings put emphasize on the surrounding stroma, support-
ing the hypothesis that the microenvironment
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 s ingle 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 regarde d as be ing a
more objective method with a higher sensitivity and
reproducibility than light mi croscope 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
assessment of protein expression. We excluded fields
with tissue folding or overlapping from analysis. A
shortcoming o f 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
1
Department of Clinical Pathology, University Hospital of Northern Norway,
N-9038 Tromsø, Norway.
2
Department of Medical Biology, University of
Tromsø, N-9037 Tromsø, Norway.
3
Department of Neurology and
Neurophysiology, University Hospital of Northern Norway, N-9038 Tromsø,
Norway.
4
Department 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 test Unadjusted 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 = 0.020 3.9 (1.1-14.2), P = 0.036 3.5 (0.8-14.7), P = 0.059
TM P = 0.074 2.3 (0.8-6.9), P = 0.121 1.5 (0.4-6.0), P = 0.592
SM P = 0.003 6.8 (1.5-30.4), P = 0.013 8.3 (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 = 0.839 1.1 (0.4-3.3), P = 0.842 5.2 (1.1-24.0), P = 0.035
TM P = 0.052 0.4 (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 = 0.046 2.3 (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 = 0.400 1.1 (1.0-1.8), P = 0.003
TM P = 0.009 5.7 (1.3-25.6), P = 0.023 4.7 (0.8-25.5), P = 0.076
SM P = 0.035 4.4 (1.0-19.7), P = 0.053 4.0 (0.8-20.7), P = 0.057
Ki67 TP P = 0.257 1.8 (0.6-5.3), P = 0.275 1.2 (0.3-4.7), P = 0.771
SP P = 0.369 1.7 (0.5-6.1), P = 0.387 1.1 (0.2-6.5), P =
0.959
TM P = 0.001 7.7 (1.7-35.2), P = 0.008 7.4 (1.4-33.3), P = 0.020
SM P < 0.000 3.6 (1.0-12.9), P = 0.031 1.1 (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: tum or stromal areas of primary tum ors; 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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Page 8 of 10
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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    • "reement with the work of Desruisseau et al. who reported that a high TGFβ1 protein level measured by enzyme-immunoassay in breast cancer tissue was an independent poor prognostic marker for disease free survival [21]. Richardsen et al. also reported that high stromal expression of TGFβ in breast cancer areas was associated with increased mortality. [22]. However, other studies showed opposing results with better DFS and OS in patients with high TGFβ1 and TGFβ receptor type II expression [18] and lower recurrence rates with patients expressing TGFβ1 and pSMAD2/3 [20]. Our data may help to provide explanations for some of the discrepancies in the results of previous studies testing TGFβ1"
    [Show abstract] [Hide abstract] ABSTRACT: The Transforming growth factor β (TGFβ) signaling has a paradoxical role in cancer development and outcome. Besides, the prognostic significance of the TGFβ1, SMAD4 in breast cancer patients is an area of many contradictions. The transcriptional intermediary factor 1γ (TIF1γ) is thought to interact with the TGFβ/SMAD signaling through different mechanisms. Our study aims to define the prognostic significance of TGFβ1, SMAD4 and TIF1γ expression in breast cancer patients and to detect possible interactions among those markers that might affect the outcome. Immunohistochemistry was performed on tissue microarray (TMA) blocks prepared from samples of 248 operable breast cancer patients who presented at Centre Léon Bérard (CLB) between 1998 and 2001. The intensity and the percentage of stained tumor cells were integrated into a single score (0-6) and a cutoff was defined for high or low expression for each marker. Correlation was done between TGFβ1, SMAD4 and TIF1γ expression with the clinico-pathologic parameters using Pearson's chi-square test. Kaplan-Meier method was used to estimate distant metastasis free survival (DMFS), disease free survival (DFS) and overall survival (OS) and the difference between the groups was evaluated with log-rank test. 223 cases were assessable for TIF1γ, 204 for TGFβ1 and 173 for SMAD4. Median age at diagnosis was 55.8 years (range: 27 to 89 years). Tumors were larger than 20 mm in 49.2 % and 45.2 % had axillary lymph node (LN) metastasis (N1a to N3). 19.4 % of the patients had SBR grade I tumors, 46.8 % grade II tumors and 33.9 % grade III tumors. ER was positive in 85.4 %, PR in 75.5 % and Her2-neu was over-expressed in 10 % of the cases. Nuclear TIF1γ, cytoplasmic TGFβ1, nuclear and cytoplasmic SMAD4 stainings were high in 35.9 %, 30.4 %, 27.7 % and 52.6 % respectively. TIF1γ expression was associated with younger age (p = 0.006), higher SBR grade (p < 0.001), more ER negativity (p = 0.035), and tumors larger than 2 cm (p = 0.081), while TGFβ1 was not associated with any of the traditional prognostic factors. TGFβ1 expression in tumor cells was a marker of poor prognosis regarding DMFS (HR = 2.28; 95 % CI: 1.4 to 3.8; p = 0.002), DFS (HR = 2.00; 95 % CI: 1.25 to 3.5; p = 0.005) and OS (HR = 1.89; 95 % CI: 1.04 to 3.43; p = 0.037). TIF1γ expression carried a tendency towards poorer DMFS (p = 0.091), DFS (p = 0.143) and OS (p = 0.091). In the multivariate analysis TGFβ1 remained an independent predictor of shorter DMFS, DFS and OS. Moreover, the prognostic significance of TGFβ1 was more obvious in the TIF1γ high patient subgroup than in the patients with TIF1γ low expression. The subgroup expressing both markers had the worst DMFS (HR = 3.2; 95 % CI: 1.7 to 5.9; p < 0.0001), DFS (HR = 3.02; 95 % CI: 1.6 to 5.6; p < 0.0001) and OS (HR = 2.7; 95 % CI: 1.4 to 5.4; p = 0.005). There is a crosstalk between the TIF1γ and the TGFβ1/SMAD4 signaling that deteriorates the outcome of operable breast cancer patients and when combined together they can serve as an effective prognostic tool for those patients.
    Full-text · Article · Jun 2015
    • "To the best of our knowledge only two groups carried out studies of the prognostic value of the COX-2 expression in the stromal cells of human breast cancer [16,19]. Nakopoulou et al. [19] did not find significant correlation between the COX-2 expression in the stromal cells and the patient survival while Richardsen et al. [16] found that high stromal staining intensity in the primary tumours was associated with significantly higher risk of death compared to the low staining group. Our study demonstrated that stromal expression of COX-2 evaluated according to the algorithms ALG1 (this work) and ALG3 (used according to [16]) could be considered an independent prognostic factor for the breast cancer. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Prognostic value of enhanced COX-2 expression in breast cancer has been controversial for a long time. The opinions vary widely between studies. Moreover, significant majority of studies considered only COX-2 expression in cancer epithelial cells. Methods We examined the prognostic value of COX-2 expression in both epithelial and stromal cells using three different antibodies and three algorithms of immunohistochemical scoring and categorizing the tumours into COX-2 overexpressing groups. Results Our results demonstrate that COX-2 expression in stromal cells is independent prognostic factor indicating worse overall survival of patients. Such a result was obtained using each of the three antibodies and two of the algorithms used for evaluations of COX-2 expression levels. We also show that immunohistochemical assessment of the prognostic value of COX-2 expression in cancer epithelial cells depends to a large extent on a combination of primary antibodies and algorithms used for determination of the COX-2 over-expressing tumours. Conclusions Our results indicate that stromal expression of COX-2 is independent prognostic parameter relatively insensitive to variations in sensitivity of antibodies used for its determination. Wide scatter of the published results concerning prognostic value of COX-2 expression in breast cancer tissues seems to be due to a large extent to multitude of antibodies and scoring algorithms used by different groups.
    Full-text · Article · Sep 2014
    • "In addition, a highly significant association between TβRII expression and reduced survival has been detected in patients bearing estrogen receptor negative breast cancer [68]. Richardsen et al. recently published immunohistochemical data from 38 cancer patients: high TGF-β levels can be detected in both primary and metastatic tumors and high stromal TGF-β expression is associated with increased mortality [69]. "
    [Show abstract] [Hide abstract] ABSTRACT: Breast cancer is the most prevalent cancer among females worldwide leading to approximately 350,000 deaths each year. It has long been known that cancers preferentially metastasize to particular organs, and bone metastases occur in ~70% of patients with advanced breast cancer. Breast cancer bone metastases are predominantly osteolytic and accompanied by increased fracture risk, pain, nerve compression and hypercalcemia, causing severe morbidity. In the bone matrix, transforming growth factor-β (TGF-β) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteoclastic bone resorption. TGF-β, in turn, stimulates bone metastatic tumor cells to secrete factors that further drive osteolytic bone destruction adjacent to the tumor. Thus, TGF-β is a crucial factor responsible for driving the feed-forward vicious cycle of cancer growth in bone. Moreover, TGF-β activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-β signaling pathway to interrupt this vicious cycle between breast cancer and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. This review will describe the role of TGF-β in breast cancer and bone metastasis, and pre-clinical and clinical data will be evaluated for the potential use of TGF-β inhibitors in clinical practice to treat breast cancer bone metastases.
    Full-text · Article · Oct 2013
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