Clinical & Experimental Pathology
El Aggan et al., J Clin Exp Pathol 2012, 2:7
http://dx.doi.org/10.4172/2161-0681.1000133
Research Article
Open Access
Macrophage Inflammatory Protein-1α (MIP-1 α) in Hepatitis C VirusRelated Hepatocellular Carcinoma: Relation to Clinical Staging and Tumour
Angiogenesis
Hoda A. El Aggan1, Myriam A. Helmy2, Nevine M.F. El Deeb3*, Ahmed E. Zeid1 and Mohamed F. A. Yehia1
Department of Medicine (Hepatobiliary Unit 1), Faculty of Medicine, University of Alexandria, Egypt
Department of Clinical Pathology, Faculty of Medicine, University of Alexandria, Egypt
3
Department of Pathology, Faculty of Medicine, University of Alexandria, Egypt
1
2
Abstract
Study background: Hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular
carcinoma (HCC), however, the mechanism of hepatocarcinogenesis in HCV infection is still undefined. Chemokines,
which induce leukocyte migration and activate inflammatory/immune responses, have recently been implicated in the
regulation of tumour growth. This work was designed to study the role of macrophage inflammatory protein-1α (MIP1α), a potent macrophage chemo-attractant, in the development and progression of HCV-related HCC.
Methods: Thirty patients with HCV-related cirrhosis (15 patients with HCC who underwent surgical hepatic
resection and 15 patients without HCC) and 15 healthy subjects were enrolled in the study. Immunohistochemical
staining of HCC and adjacent non-neoplastic liver tissue was performed using antibodies against MIP-1α, CD68 [for
assessment of tumour-associated macrophage (TAM) count] and CD105 [for determination of microvessel density
(MVD)]. Pre-operative serum MIP-1α levels were measured using enzyme linked immunosorbant assay kit.
Results: HCV-related HCCs showed significantly higher MIP-1α expression, CD68+ TAM count and CD105-MVD
compared with adjacent non-neoplastic liver tissue. Serum MIP-1α levels were significantly higher in patients with and
without HCC than in healthy subjects and in HCC patients than in patients without HCC. The sensitivity and specificity
of serum MIP-1α in discriminating cirrhotic patients with and without HCC were 100% and 93.3% respectively at a
cut-off value of 17.5 pg/ml. The MIP-1α expression in HCCs showed positive correlations with serum MIP-1α levels;
tumour size, stage and histopathological grade; and intratumoural CD68+ TAM count and CD105-MVD. Moreover,
CD68+ TAM count had direct correlation with CD105-MVD in HCC.
Conclusion: MIP-lα plays an important role in the development and progression of HCC in chronic HCV infection,
possibly through recruitment of macrophages into tumour microenvironment and fostering tumour angiogenesis. MIPlα may also serve as a potential serum biological marker and a useful therapeutic target in HCV-related HCC.
Keywords: Hepatocellular carcinoma; Macrophage inflammatory
protein-1 alpha; Tumour-associated macrophages
Abbreviations: HCV: Hepatitis C Virus; HCC: Hepatocellular
Carcinoma; MIP-1α: Macrophage Inflammatory Protein-1 alpha;
TAM: Tumour-Associated Macrophages; MVD: Microvessel Density;
MELD: Model for End Stage Liver Disease; CLIP: Cancer of the Liver
Italian Program; HAI: Histological Activity Index; AST: Aspartate
Aminotransferase; ALT: Alanine Aminotransferase; GGT: Gamma
Glutamyl Transpeptidase; AFP: Alpha-Fetoprotein; INR: International
Normalized Ratio; ELISA: Enzyme Linked Immunosorbant Assay;
PBS: Phosphate Buffered Saline; DAB: 3,3’-diaminobenzidine
Introduction
Chronic hepatitis C virus (HCV), infection is a major risk factor for
development of hepatocellular carcinoma (HCC) [1]. The incidence of
HCV-associated HCC is predicted to increase over the next several years
as a result of the epidemic of chronic HCV [2]. However, the precise
molecular mechanism underlying the progression of chronic HCV
infection to HCC is not clearly established. Recently, a critical role for
chronic inflammation in tumourigenesis has generally been accepted
and it has become evident that an inflammatory microenvironment
is an essential component of all tumours [3]. In patients with chronic
HCV infection, HCC usually arises in the setting of chronic hepatic
inflammation and cirrhosis or bridging fibrosis, which favours the
initiation and progression of HCC. Moreover, a persistent, nonspecific, and ineffective activation of the immune system within
the chronically-inflamed liver in chronic HCV infection is thought
J Clin Exp Pathol
ISSN: 2161-0681 JCEP, an open access journal
to promote hepatocarcinogenesis [4]. Furthermore, inflammation
increases vascular permeability and promotes formation of new blood
vessels (angiogenesis), which is closely related to the development of
HCC [5]. There is increasing evidence that the inflammatory milieu, by
virtue of both its soluble signals like chemokines and cellular infiltrates
may alter the behaviour of HCC [6].
The importance of chemokines in cancer-associated inflammation
has been highlighted. Chemokines are responsible for the migration of
various types of chemokine receptor-positive leukocytes to the tumour
site [7]. Both tumour cells and stromal cells such as fibroblasts and
macrophages elaborate chemokines in the tumour microenvironment
that can promote tumour growth via cell proliferation, angiogenesis,
invasion and metastasis [8]. Chemokines can also suppress immune
responses resulting in tumour escape from immune surveillance [9].
*Corresponding author: Nevine M.F. El Deeb, Department of Pathology,
Faculty of Medicine, University of Alexandria, Alexandria, Egypt, E-mail:
[email protected]
Received November 17, 2012; Accepted December 29, 2012; Published
December 31, 2012
Citation: El Aggan HA, Helmy MA, El Deeb NMF, Zeid AE, Yehia MFA (2012)
Macrophage Inflammatory Protein-1α (MIP-1 α) in Hepatitis C Virus-Related
Hepatocellular Carcinoma: Relation to Clinical Staging and Tumour Angiogenesis.
J Clin Exp Pathol 3:133. doi:10.4172/2161-0681.1000133
Copyright: © 2012 El Aggan HA, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Volume 2 • Issue 7 • 1000133
Citation: El Aggan HA, Helmy MA, El Deeb NMF, Zeid AE, Yehia MFA (2012) Macrophage Inflammatory Protein-1α (MIP-1 α) in Hepatitis C VirusRelated Hepatocellular Carcinoma: Relation to Clinical Staging and Tumour Angiogenesis. J Clin Exp Pathol 3:133. doi:10.4172/21610681.1000133
Page 2 of 8
Macrophage inflammatory protein-1α (MIP-1α), also known as
CC chemokine ligand 3 (CCL3), is a member of the pro-inflammatory
CC chemokine subfamily [10]. It binds to and activates cells via two
chemokine receptors, namely CCR1 and CCR5, which are expressed
by a variety of cells [11]. Several lines of evidence indicate that MIP-1α
and its receptors may be involved in tumour growth and associated
inflammation and may determine the destination of metastasis of
different types of tumours [12-14]. Moreover, MIP-1α secreted
by endothelial cells may promote vessel remodeling and tumour
neovascularization through its potent angiogenic activities [15].
Locally-produced MIP-1α is a potent chemoattractant of different
immune cells, particularly macrophages from the circulation to
inflammatory sites and tumour microenvironment [16]. Tumourassociated macrophages (TAMs) recruited by MIP-1α form a major
component of the inflammatory infiltrate seen in the stroma of many
tumours and can affect different aspects of the neoplastic process [17].
Therefore, the present work was designed to study the role of MIP1α in the pathogenesis of HCV-related HCC in relation to tumour
progression and angiogenesis.
Subjects and Methods
Thirty treatment-naïve patients with HCV-related cirrhosis
(15 patients with HCC who underwent surgical hepatic resection
and 15 patients without HCC) referred to the Hepatobiliary Unit,
Department of Medicine, Main Alexandria University Hospital; and
15 age- and sex-matched healthy subjects were enrolled in this study.
The diagnosis of HCC was based on serum levels of alpha-fetoprotein
(AFP), ultrasonography/triphasic computed tomography (CT) and/or
dynamic MRI and was confirmed by histopathological examination of
surgically-resected tumours. The presence of cirrhosis was determined
by clinical, biochemical and ultrasonographical evidences and by
histopathological examination of adjacent non-neoplastic liver tissue in
patients with HCC. Exclusion criteria included patients with hepatitis
B virus infection; history of alcohol consumption; concomitant
schistosomiasis; evidence of hepatocellular decompensation;
inflammatory disorders; other malignancies; cardiac, respiratory or
renal diseases and known chronic diseases such as diabetes mellitus
or connective tissue disorders. None of the patients has previously
received anti-viral therapy or treatment for HCC such as systemic
anti-cancer therapy, locoregional therapy or transcatheter arterial
embolization before the study. The study protocol was approved by
the Research Review Committee of the Alexandria Faculty of Medicine
and was conformed to the 1975 Declaration of Helsinki. An informed
consent was obtained from each subject included in the study.
Upon entry into the study, the following clinical and biochemical
parameters were assessed: age, gender, the presence of palpable focal
hepatic lesions, symptoms and signs of chronic liver disease, liver
and spleen size, serum albumin, serum bilirubin, serum aspartate and
alanine aminotransferases (AST and ALT respectively), serum gamma
glutamyl transpeptidase (GGT), prothrombin time, international
normalized ratio and serum creatinine. Serum AFP levels were
measured using standardized enzyme linked immunosorbant assay
kit (ELISA). The severity of liver disease in patients with cirrhosis
was graded according to the Child-Pugh classification [18] and the
Model for End Staging Liver Disease (MELD) score [19]. Abdominal
ultrasonographic and triphasic CT examination were used for initial
diagnosis of HCC and for assessment of tumour characteristics, and
the presence of cirrhosis, ascites and splenomegaly. The HCC stage
J Clin Exp Pathol
ISSN: 2161-0681 JCEP, an open access journal
was determined by the scoring system proposed by the Cancer of the
Liver Italian Program (CLIP) investigators [20]. This program includes
Child-Pugh class of hepatic function, where classes A , B and C are
scored as 0, 1 and 2 respectively; tumour morphology, where uninodular
tumours involving ≤50% of liver are scored as 0, multinodular tumours
involving ≤ 50% of liver are scored as 1 and diffuse tumours involving
>50% of liver are scored as 2; alpha fetoprotein level, where <400 ng/
ml and ≥400 ng/ml are scored as 0 and 1 respectively; and portal vein
thrombosis, which is scored as 0 if absent and as 1 if present. Tumours
are divided into: CLIP stages 0-6 as follows: CLIP 0, 0 points; CLIP 1, 1
point; CLIP 2, 2 points; etc
Measurement of serum MIP-1α levels
Pre-operative serum levels of MIP-1α were measured using
commercially-available solid phase sandwich ELISA kit (Invitrogen
Corporation, USA) according to the manufacturer’s instructions
[21]. Fifty μl of serum samples (obtained from all patients and healthy
subjects), standards and controls were added to the appropriate
microtiter wells coated with human MIP-1α and 50 µl of the standard
diluent buffer were added to the zero standard wells. Wells reserved
for chromogen blank were left empty. Fifty μl of biotinylated antiMIP-1α (Biotin conjugate) solution were then pipetted into each
well except the chromogen blank(s) and the plate was incubated for 2
hours at room temperature. Then, 100 μl of streptavidin-horseradish
peroxidase working solution were added to each well except the
chromogen blank(s) and the plate was incubated for 30 minutes at
room temperature. Solution from wells was thoroughly aspirated and
discarded and the wells were washed 4 times after each step. Then, 100
μl of stabilized chromogen were added and the plate was incubated for
30 minutes at room temperature in the dark. The liquid in the wells
began to turn blue. Finally, 100 μl of stop solution were added. The
solution in the wells changed from blue to yellow. The absorbance of
each well at 450 nm was read having blanked the plate reader against a
chromogen blank composed of 100 μl each of stabilized chromogen and
stop solution. The plate was read within 2 hours after adding the stop
solution. A curve-fitting software was used to generate the standard
curve, from which the concentrations of MIP-1α for serum samples
and controls were read. Serum MIP-1α was measured in pg/ml.
Pathological examination of HCC and adjacent nonneoplastic liver tissue
Surgically-resected specimens from patients with HCV-related
HCC were examined grossly for determination of tumour size and
multiplicity. Representative samples of tumours and adjacent nonneoplastic liver tissue were fixed in 10% formalin, embedded in
paraffin, sectioned (5μm) and stained with hematoxylin and eosin for
histopathological diagnosis and determination of tumour grade and
presence of capsule. Tumours were graded according to Edmondson
and Steiner’s criteria [22], as follows: grade 1 HCC (well differentiated):
the nuclear cytoplasmic ratio is nearly normal and the tumour is
recognized by the overall growth pattern; grade 2 HCC (moderately
differentiated) has larger more hyperchromatic nuclei, esinophilic
cytoplasm and acini, trabecular or papillary patterns; grade 3 HCC
(poorly differentiated) has more variable larger hyperchromatic nuclei
with multiple nucleoli, with a loss of trabecular pattern, more numerous
giant cells and less evident bile plugs; grade 4 HCC has less mature cells
with large nuclei and little cytoplasm, and is hard to recognize as being
of HCC origin. The adjacent non-neoplastic liver tissue was assessed
as regards presence of cirrhosis and steatosis as well as the grade and
stage of hepatitis using modified histological activity index (HAI)
Volume 2 • Issue 7 • 1000133
Citation: El Aggan HA, Helmy MA, El Deeb NMF, Zeid AE, Yehia MFA (2012) Macrophage Inflammatory Protein-1α (MIP-1 α) in Hepatitis C VirusRelated Hepatocellular Carcinoma: Relation to Clinical Staging and Tumour Angiogenesis. J Clin Exp Pathol 3:133. doi:10.4172/21610681.1000133
Page 3 of 8
[23]. Steatosis was evaluated by estimating the approximate amount
of parenchyma involved as follows: ‘-’=absent; ‘+’=mild, less than one
third; ‘++’=moderate, one third to two thirds and ‘+++’=marked, more
than two thirds [24].
stained cells in non-overlapping microscopic fields, and scored as
follows: ‘-’ = <10% of cells were stained; ‘+’ = 10-20% of cells showed
positive reaction; ‘++’ = 20-50% of cells exhibited positive reaction; and
‘+++’ = >50% of cells were positive [25].
Immunohistochemistry for MIP-1α, CD68 and CD105
Determination of macrophage count: The three areas of densest
macrophage infiltration (hot spots) were first identified by scanning
the entire CD68-stained section at low power (x100), then all CD68positive cells were counted in three high power microscopic fields
(x400), and the mean count was calculated [26].
Immunohistochemical staining for MIP-1α, CD68 and CD105
was performed as previously described [25-27] on 5 μm-thick paraffin
sections cut from HCCs and adjacent non-neoplastic hepatic tissue. The
tissue sections were deparaffinized in xylene, rehydrated in descending
grades of alcohol, then immersed in 0.3% hydrogen peroxide in
methanol for 20 minutes to inhibit endogenous peroxidase activity.
The following primary mouse monoclonal antibodies were used: antihuman MIP-1α/CCL3 antibody, clone 93321 (R&D Systems, USA)
diluted 1:20; anti-CD68 Ab-3, clone KP1 (Neomarkers, Labvision,
USA) (ready to use) for determination of macrophage count; and
anti-CD105/endoglin Ab-3 clone SN6h (Neomarkers, Labvision, USA)
(ready to use) for assessment of microvessel density (MVD).
Before immunostaining, antigen retrieval was performed as
follows: for anti-human MIP-1α antibody, slides were placed in
citrate buffer (0.01 M, pH 6.0) in a 700W microwave oven, and then
allowed to cool to room temperature; for anti-CD68 and anti-CD105
antibodies, enzymatic digestion was performed using protease XXV
at 1mg/ml phosphate-buffered saline (PBS) for 5 minutes at 37°C.
Then, Ultra V block was applied for 3-5 minutes to block nonspecific
background staining. Thereafter, tissue sections were incubated with
the primary antibodies at 4°C overnight in a humid chamber. Slides
were then incubated with biotinylated goat anti-polyvalent (linking
reagent), followed by peroxidase-conjugated streptavidin, each for 20
minutes at room temperature. Tissue sections were washed with PBS
for 5 minutes after each step. The reaction product was developed using
3,3’-diaminobenzidine (DAB) mixture for 10 minutes. The slides were
finally dehydrated, counterstained with hematoxylin and mounted.
Negative control sections (where the primary antibody has been
omitted), were included in each run.
Assessment of MIP-1α expression: MIP-1α immunostaining was
evaluated semiquantitatively according to the percentage of positively-
Determination of microvessel density: CD105-stained sections
were first screened at low magnification (x100) to identify the three
areas with the highest vascularity (hot spots). Then, the absolute
number of microvessels in three x200-power fields, one in each hot
spot, was counted. The mean of these 3 counts was considered as the
MVD, expressed as the absolute number of microvessels per 0.74mm2
(x200 field) [27]. Any brown-stained endothelial cell or endothelial
cell cluster that was clearly separate from adjacent microvessels was
counted as one microvessel irrespective of the presence of a vessel
lumen [26,27].
Statistical analysis
All data were statistically analyzed using the SPSS program (version
13.0) (SPSS Inc., Chicago, IL, USA) for Windows. The Student’s t test
was used for comparison between two arithmetic means and the MannWhitney U-test was used for comparison between two means of nonnormally distributed variables. The one-way ANOVA test was used for
comparing the three groups with post hoc comparisons. Comparison
between proportions was determined by the Chi square (χ2) test or
Fisher’s Exact test (FET). Correlations between variables were analyzed
by using Pearson’s correlation coefficient or Spearman’s rank test
where appropriate. Analysis was statistically significant at P<0.05. The
sensitivity and specificity of serum MIP-1α in discriminating cirrhotic
patients with and without HCC were assessed by plotting a receiveroperating characteristic (ROC) curve and determining its cut-off value.
Results
The clinical and biochemical characteristics of cirrhotic patients
Parameters
HCV patients with HCC (n=15)
HCV patients without HCC (n=15)
Healthy Subjects (n=15)
P value
Age (years)
51.27 ± 5.56
52.73 ± 7.23
50.93 ± 6.77
0.844
Gender:
- Male (%)
- Female (%)
15 (100.0)
0 (0.0)
15 (100.0)
0 (0.0)
15 (100.0)
0 (0.0)
AST (U/L; mean ± SD)
68.40 ± 30.38a b
50.87 ± 25.11a
20.13 ± 3.78
<0.0001
ALT (U/L; mean ± SD)
71.47 ± 29.44a b
40.53 ± 19.18a
20.40 ± 4.55
<0.0001
GGT (U/L; mean ± SD)
102.27 ± 80.4 a b
43.47 ± 21.62
20.93 ± 3.20
0.0001
3.53 ± 0.20a
3.49 ± 0.18a
4.60 ± 0.46
<0.0001
Serum bilirubin (mg/dl; mean ± SD)
1.08 ± 0.13 a
1.05 ± 0.23 a
0.64 ± 0.16
<0.0001
INR (mean ± SD)
1.19 ± 0.08 a
1.25 ± 0.13 a
1.07 ± 0.04
<0.0001
1302.36 ± 1890.19a b
6.00 ± 2.35
4.60 ±1.60
0.002
11 (73.3)
4 (26.7)
7 (46.7)
8 (53.3)
NA
0.132
Serum albumin (g/dl; mean ± SD)
AFP (ng/ml; mean ± SD)
Child-Pugh class:
- Class A (%)
- Class B (%)
MELD score
Serum MIP-1α (pg/ml)
7.13 ± 1.19
8.40 ± 1.96
NA
0.119
27.33 ± 4.58a b
8.67 ± 6.11a
1.67 ± 2.44
<0.0001
AST: Aspartate Amino Transferase; ALT: Alanine Amino Transferase; GGT: Gamma Glutamyl Transpeptidase; INR: International Normalized Ratio; AFP: AlphaFetoprotein; MELD: Model for End-Stage Liver Disease; MIP-1α: Macrophage Inflammatory Protein-1α
NA=not applicable
a=Statistically-significant difference from healthy subject;
b=Statistically-significant difference from cirrhotic patients without HCC.
Table 1: Clinical and biochemical characteristics of HCV-related cirrhotic patients with and without Hepatocellular Carcinoma (HCC) and healthy subjects.
J Clin Exp Pathol
ISSN: 2161-0681 JCEP, an open access journal
Volume 2 • Issue 7 • 1000133
Citation: El Aggan HA, Helmy MA, El Deeb NMF, Zeid AE, Yehia MFA (2012) Macrophage Inflammatory Protein-1α (MIP-1 α) in Hepatitis C VirusRelated Hepatocellular Carcinoma: Relation to Clinical Staging and Tumour Angiogenesis. J Clin Exp Pathol 3:133. doi:10.4172/21610681.1000133
Page 4 of 8
with and without HCC and healthy subjects are summarized in Table
1, and the radiological and histopathological features of patients with
HCC are shown in Table 2.
ROC Curve
1.00
Serum levels of MIP-1α
.75
The serum levels of MIP-1α ranged between 20-35 pg/ml and
0-20 pg/ml in cirrhotic patients with and without HCC respectively
and between 0-5 pg/ml in healthy subjects. Serum MIP-1α levels were
significantly higher in patients with and without HCC than in healthy
subjects (27.33 ± 4.58 pg/ml and 8.67 ± 6.11 pg/ml vs 1.67 ± 2.44 pg/
ml) and in patients with HCC than in those without HCC (P<0.0001)
(Table 1).
Sensitivity
.50
By plotting a ROC curve, the sensitivity and specificity of serum
MIP-1α in discriminating cirrhotic patients with and without HCC
were found to be 100% and 93.3% respectively at a cut-off value of 17.5
pg/ml (Figures 1 and 2).
In HCCs, positive cytoplasmic immunostaining for MIP-1α
was demonstrated in the tumour cells in 14 (93.3%) patients. The
distribution of MIP-1α expression in HCCs was as follows: ‘-’ in 1
(6.7%) patient; ‘+’ in 3 (20.0%) patients; ‘++’ in 4 (26.7%) patients; and
‘+++’ in 7 (46.7%) patients (Table 3, Figures 3A and 3B). In the adjacent
non-neoplastic liver tissue, the distribution of MIP-1α expression in
hepatocytes was as follows: ‘-’ in 9 (60.0%) patients and ‘+’ in 6 (40.0%)
patients (Table 3 and Figure 3C). In addition, strong MIP-1α staining
Parameters
Tumour size (cm):
- Range
- Mean ± SD
HCV patients with HCC (n=15)
4.0–8.0
5.60 ± 1.21
0.00
0.00
.25
.50
.75
1.00
Specificity
Figure 1: Receiver Operating Characteristic (ROC) curve of serum
Macrophage Inflammatory Protein-1α (MIP-1α) levels in discriminating
cirrhotic patients with and without Hepatocellular Carcinoma (HCC).
40
Serum MIP - 1 alpha (pg/ml)
Expression of MIP-1α in HCC and adjacent non-neoplastic
liver tissue
.25
30
20
Cut off value =17.5 pg/ml
10
Number of nodules:
- Uninodular (%)
- Multinodular (%)
10 (66.7)
5 (33.3)
Echopattern:
- Hypoechoic (%)
- Heterogenous (%)
10 (66.7)
5 (33.3)
Tumour Location:
- Left lobe (%)
- Right lobe (%)
15 (100.0)
0 (0.0)
Tumour Extension:
- < 50% (%)
- > 50% (%)
15 (100.0)
0 (0.0)
Tumour encapsulation (%)
11 (73.3)
CLIP stage:
- CLIP 0 (%)
- CLIP 1 (%)
- CLIP 2 (%)
3 (20.0)
7 (46.7)
5 (33.3)
HCC Grade:
- Grade 1 (%)
- Grade 2 (%)
- Grade 3 (%)
Macrophage count in HCC and adjacent non-neoplastic liver
tissue
2 (13.3)
7 (46.7)
6 (40.0)
In HCCs, CD68+ TAMs were numerous, and were primarily
distributed at the periphery of the tumour (Figure 4A). The CD68+
TAM count (at x 400 magnification) ranged between 28.7-108.7 and
15.7-55.0 in HCC and adjacent non-neoplastic liver tissue respectively.
The CD68+ TAM count was significantly higher in HCCs than in the
adjacent non-neoplastic liver tissue (73.47 ± 23.01 vs 31.79 ± 11.55,
P<0.001) (Table 3).
Adjacent non-neoplastic liver tissue:
- HAI grade (Range)
- Steatosis (%):
- (%)
+ (%)
++ (%)
+++ (%)
6-10
4 (26.7)
5 (33.3)
6 (40.0)
0 (0.0)
CLIP: Cancer of the Liver Italian Program; HAI: Histological Activity Index
‘-’=Absent; ‘+’=Mild; ‘++’=Moderate; ‘+++’=Marked.
Table 2: Radiological and histopathological characteristics of patients with
Hepatocellular Carcinoma (HCC).
J Clin Exp Pathol
ISSN: 2161-0681 JCEP, an open access journal
0
Cirrhotics without HCC
Cirrhotics with HCC
Figure 2: Distribution of serum Macrophage Inflammatory Protein-1α (MIP1α) levels around the cut-off value (17.5 pg/ml) in cirrhotic patients with and
without Hepatocellular Carcinoma (HCC).
was observed in vascular endothelial cells and bile duct epithelial cells
(Figure 3D). The MIP-1α expression was significantly higher in HCCs
than in the adjacent non-neoplastic liver tissue, (χ2=18.40, P=0.0004)
(Table 3).
Microvessel density in HCC and adjacent non-neoplastic
liver tissue
CD105 immunostaining highlighted the cell membrane of
endothelial cells. All of the HCC specimens were immunoreactive
Volume 2 • Issue 7 • 1000133
Citation: El Aggan HA, Helmy MA, El Deeb NMF, Zeid AE, Yehia MFA (2012) Macrophage Inflammatory Protein-1α (MIP-1 α) in Hepatitis C VirusRelated Hepatocellular Carcinoma: Relation to Clinical Staging and Tumour Angiogenesis. J Clin Exp Pathol 3:133. doi:10.4172/21610681.1000133
Page 5 of 8
Parameters
MIP-1α expression:
- (%)
+ (%)
++ (%)
+++ (%)
HCC
(n=15)
1 (6.7)
3 (20.0)
4 (26.7)
7 (46.7)
Adjacent
non-neoplastic liver P value
tissue
9 (60.0)
6 (40.0)
0 (0.0)
0 (0.0)
0.0004
CD68+ TAM count (/x400 field):
- Range
28.7–108.7
- Mean ± SD
73.47 ± 23.01
15.7–55.0
31.79 ± 11.55
<0.001
CD105-MVD (/x200 field):
- Range
- Mean ± SD
5.7-21.0
13.05 ± 4.62
<0.001
13.3-58.7
39.55 ± 12.55
‘-‘ =<10% of cells are stained; ‘+’ = 10-20 % of cells are positive; ‘++’ = 20-50% of
cells are positive; ‘+++’ = >50% of cells are positive.
Table 3: Macrophage Inflammatory Protein-1α (MIP-1α) expression, CD68-positive
Tumour-Associated Macrophage (TAM) count and CD105-Microvessel Density
(MVD) in Hepatocellular Carcinoma (HCC) and the adjacent non-neoplastic liver
tissue.
A
B
Statistical correlations between MIP-1α expression, CD68+
TAM count and CD105-MVD in HCC and other studied
parameters: (Table 4)
The MIP-1α expression in HCCs showed positive correlations
with serum MIP-1α levels (r=0.671, P=0.006), intratumoural CD68+
TAM count (r=0.844, P<0.001) and CD105-MVD (r=0.615, P=0.015).
Also, CD68+ TAM count and CD105-MVD in HCCs were directly
correlated (r=0.917, P<0.001). Moreover, positive correlations were
found between MIP-1α expression, CD68+ TAM count and CD105MVD in HCCs on one hand and HCC maximum diameter (r=0.730,
P=0.002; r=0.642, P=0.010 and r=0.641, P=0.010 respectively), CLIP
stage (r=0.637, P=0.011; r=0.602, P=0.018 and r=0.625, P=0.013
respectively) and histopathological grade (r=0.728, P=0.002; r=0.838,
P<0.001 and r=0.762, P=0.001 respectively) on the other hand. In the
mean time, there were no statistically significant correlations between
intratumoural MIP-1α expression, CD68+ TAM count and CD105MVD with serum levels of AST, ALT, GGT and AFP, Child-Pugh
score, MELD score and HAI grade and steatosis in the adjacent nonneoplastic liver tissue (P>0.05).
Discussion
C
D
Figure 3: Immunohistochemical expression of Macrophage Inflammatory
Protein-1α (MIP-1α): (A) Hepatocellular Carcinoma (HCC) demonstrating
positive staining for MIP-1α in less than 50% of tumour cells (++); (B) HCC
demonstrating positive staining for MIP-1α in more than 50% of tumour cells
(+++); (C) Non-neoplastic liver tissue adjacent to HCC showing negative
staining for MIP-1α; (D) Positive MIP-1α immunostaining of bile duct
epithelium and vascular endothelium. Original magnifications: (A,B) x200; (C)
x 100;(D) x200.
with anti-CD105 antibody. The density of microvessels was higher
in the peripheral part of the tumour than in the central areas. Three
patterns of expression were encountered: sinusoid-like, branching
and small without apparent lumina (endothelial sprouts) (Figure 4B).
Occasionally, CD105 highlighted vessels containing tumour emboli
(Figure 4C). In the adjacent non-neoplastic liver tissue, CD105 stained
scattered hepatic sinusoidal endothelial cells predominantly around the
tumour, whereas portal veins, hepatic arteries and bile ducts showed no
CD105-positivity (Figure 4D).
The CD105-MVD (at x 200 magnification) ranged between 13.358.7 and 5.7-21.0 in HCC specimens and adjacent non-neoplastic liver
tissue respectively. The CD105-MVD was significantly higher in HCC
tissues than in the adjacent non-neoplastic liver tissue (39.55 ± 12.55 vs
13.05 ± 4.62, P<0.001) (Table 3).
J Clin Exp Pathol
ISSN: 2161-0681 JCEP, an open access journal
Using immunohistochemical analysis, the present study
demonstrated that MIP-1α expression in HCC cells was significantly
higher than in the adjacent non-neoplastic liver tissues in patients
with HCV-related HCC unrelated to the severity of liver disease.
Vascular endothelial cells and bile duct epithelial cells were stained
as well. Previous investigators also found that MIP-1α was aberrantly
expressed in hepatoma cells as well as in vascular endothelium, and
small bile duct epithelium in surgically-obtained HCC tissues from
HCV-infected patients whereas their immunoreactivities were weakly
detected in normal liver tissues [25]. In murine hepatocarcinogenesis
A
B
C
D
Figure 4: Macrophage count and microvessel density in hepatocellular
carcinoma (HCC) and adjacent non-neoplastic liver tissue: (A) A dense
population of CD68 positive macrophages infiltrating among HCC cells,
with greater density at the periphery of the tumour (inset); (B) Highlighted
microvessels by CD105 in HCC showing sinusoid-like, and branching
patterns of expression. Inset: a small endothelial sprout without apparent
lumen demonstrating positive CD105 expression (arrow); (C) Positive
staining for CD105 in vascular endothelium of a vessel showing a tumour
embolus from HCC; (D) Branch of bile duct (thick arrow) and hepatic artery
(double arrows) not stained by CD105, in contrast to microvessels of the
adjacent HCC seen at the lower right (arrows). Original magnifications: (A)
x100; (B) x200, inset x400; (C) x400; (D) x 200.
Volume 2 • Issue 7 • 1000133
Citation: El Aggan HA, Helmy MA, El Deeb NMF, Zeid AE, Yehia MFA (2012) Macrophage Inflammatory Protein-1α (MIP-1 α) in Hepatitis C VirusRelated Hepatocellular Carcinoma: Relation to Clinical Staging and Tumour Angiogenesis. J Clin Exp Pathol 3:133. doi:10.4172/21610681.1000133
Page 6 of 8
Parameters
MIP-1α expression*
CD68+TAM count (/x400 field)
CD105- MVD (/x200 field)
"r" value
P value
"r" value
P value
"r" value
P value
Serum AST (U/L)
0.221
0.428
0.301
0.275
0.289
0.296
Serum ALT (U/L)
0.333
0.225
0.348
0.204
0.303
0.273
Serum GGT (U/L)
0.126
0.655
0.149
0.596
0.121
0.666
Serum AFP (ng/ml)
0.489
0.065
0.279
0.314
0.276
0.320
Child-Pugh score*
0.326
0.236
0.133
0.637
0.175
0.533
MELD score*
0.260
0.349
0.196
0.484
0.317
0.249
Serum MIP-1α (pg/ml)
0.671
0.006
0.410
0.129
0.401
0.138
HCC diameter (cm)
0.730
0.002
0.642
0.010
0.641
0.010
CLIP stage*
0.637
0.011
0.602
0.018
0.625
0.013
HCC grade*
0.728
0.002
0.838
<0.001
0.762
0.001
HAI grade
-0.314
0.255
-0.206
0.461
-0.165
0.556
Steatosis*
-0.203
0.468
-0.224
0.422
-0.175
0.533
CD68+ TAM count
0.844
<0.001
-
-
-
-
CD105-MVD
0.615
0.015
0.917
<0.001
-
-
AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; GGT: Gamma glutamyl transpeptidase; AFP :Alpha-fetoprotein; MELD: Model for End-stage Liver
Disease; MIP-1α: Macrophage Inflammatory Protein-1α; CLIP: Cancer of the Liver Italian Program; HAI: Histological Activity Index.
*Spearman’s rank test
Table 4: Statistical correlations (“r” value) between Macrophage Inflammatory Protein-1α (MIP-1α) expression, CD68-positive Tumour-Associated Macrophage (TAM)
count and CD105-Microvessel Density (MVD) in Hepatocellular Carcinoma (HCC) and other studied parameters.
models, aberrant expression of MIP-1α and its receptor CCR1 has been
demonstrated in HCC cells arising in N-nitrosodiethylamine (DEN)treated mice and HBsAg transgenic mice [28]. In addition, Takai et
al. [29] revealed that MIP-1α gene was significantly up-regulated in
tumour cells in mouse HCC using microarray and quantitative realtime polymerase chain reaction analyses. The finding that MIP-1α has
been detected within HCC cells may indicate that the tumour itself is
responsible for secretion of this chemokine.
In the mean time, the present study also showed a significant
increase in serum levels of MIP-1α in cirrhotic patients with HCC
compared with those without HCC and uninfected subjects. These
findings suggest that MIP-1α may play a role in the development of
HCC. Rajkumar et al. [30] detected a higher level of MIP-1α in the
plasma from gastric cancer patients than in patients with normal/
non-malignant gastric conditions, which showed a significant drop
after surgical resection. Also, increased serum levels of MIP-1α have
been reported in patients with chronic lymphocytic leukemia [31].
Moreover, the present study demonstrated that serum MIP-1α levels
were directly correlated with the intratumoural chemokine expression
in patients with HCC and showed high sensitivity and specificity in
discriminating cirrhotic patients with and without HCC at a cut-off
value of 17.5 pg/ml, suggesting that quantitative estimation of MIP-1α
in serum may reflect the chemokine production in HCC and could be a
potential diagnostic biomarker for HCC.
The possible role of HCV infection in enhancing MIP-1α production
in the liver may be linked directly or indirectly to HCV. Even before the
development of HCC, MIP-1α production has been found to increase
either in the liver or in serum of patients with different stages of HCVassociated liver disease [32]. In the present study, serum levels of MIP1α were significantly higher in HCV-positive cirrhotic patients without
HCC than in uninfected subjects, with a further increase observed in
patients with HCC suggesting that MIP-1α production is increasing
with the progression of liver disease in chronic HCV infection.
Furthermore, the current work showed that MIP-1α expression
in HCC cells was directly correlated with tumour size, stage and
histopathological grade suggesting that MIP-1α may play a role
in HCC progression. Several lines of evidence suggest a potential
contribution of MIP-1α to tumour growth [13,28,33]. Yang et al. [28]
J Clin Exp Pathol
ISSN: 2161-0681 JCEP, an open access journal
also demonstrated that tumour foci number and sizes were dramatically
reduced in CCL3- and CCR1-deficient mice compared with those in
wild-type mice after treatment with DEN, a known inducer of HCC.
Given that MIP-1α was expressed by HCC cells, it is likely that this
chemokine may interact with CCR1 expressed on hepatoma cells in
an autocrine and/or paracrine manner resulting in tumour progression
[28].
An additional role for MIP-1α during tumourigenesis might be
the regulation of tumour angiogenesis [34]. The current work showed
that CD105-MVD was significantly higher in HCC than in the adjacent
non-neoplastic liver tissue and was positively correlated with tumour
size, stage and histopathological grade suggesting that MIP-1α plays a
role in tumour neovascularization, an indispensable process for HCC
growth [5]. A previous study showed that tumour angiogenesis was
markedly reduced in CCL3- and CCR1-knockout mice compared with
wild-type mice in murine hepatocarcinogenesis [28]. Also, a deficiency
of the MIP-1α gene reduced neovascularization in a lung metastasis
model [34]. MIP-1α may affect neovessel formation by directly
activating endothelial cells expressing CCR1 and CCR5 in a paracrine
manner [25,28].
Being a potent chemoattractant factor, MIP-1α may indirectly
promote tumourigenesis by recruitment of various types of
leukocytes particularly macrophages into tumour tissues [16,17].
The current study demonstrated the presence of a large number
of infiltrating CD68+ macrophages in HCCs compared with the
adjacent non-neoplastic liver tissue, which was positively correlated
with intratumoural MIP-1α expression. Enrichment of macrophages
in tumour tissues compared with pericancerous tissues has been
previously reported in HCC [35-37]. Hussein et al. [36] found that the
transitions from normal liver to the subsequent lesional steps (chronic
hepatitis, cirrhotic nodules, dysplastic nodules and HCCs) in chronic
HCV infection were associated with significantly increased density
of tumour infiltrating CD68+ macrophages. It has been reported that
HCV can replicate in monocytes/macrophages causing their prolonged
activation and accumulation of inflammatory cytokines that contribute
to DNA damage and carcinogenesis [38]. The role of MIP-1α as a key
macrophage chemoattractant in the tumour microenvironment, has
been evidenced in previous studies. Yang et al. [28] found that the
Volume 2 • Issue 7 • 1000133
Citation: El Aggan HA, Helmy MA, El Deeb NMF, Zeid AE, Yehia MFA (2012) Macrophage Inflammatory Protein-1α (MIP-1 α) in Hepatitis C VirusRelated Hepatocellular Carcinoma: Relation to Clinical Staging and Tumour Angiogenesis. J Clin Exp Pathol 3:133. doi:10.4172/21610681.1000133
Page 7 of 8
numbers of macrophages in tumour foci in chemically-induced HCC
were remarkably depressed in both CCL3- and CCR1-deficient mice
compared with that of wild-type mice. Also, MIP-1α was detected in
tumour cells during murine lung metastasis process together with an
infiltration of macrophages expressing CCR5 [34].
In addition, the present study demonstrated that the number of
infiltrating CD68+ macrophages in HCC tissues was correlated with
tumour size, stage and histopathological grade suggesting that TAMs
are linked to HCC progression. There is substantial evidence that
macrophages play an important role in promoting tumour growth.
Ding et al. [35] showed that high intratumoural macrophage infiltration
predicts poor prognosis in patients with HCC. In experimental murine
hepatocarcinoma, stimulation of mouse tissue macrophages one day
before intravenous injection of tumour cells, increased the number and
weight of implants (experimental metastases) in the liver [39]. Also,
Zhang et al. [40] reported that treating HCC by ecteinascidin-743, an
anti-neoplastic alkaloid agent, can suppress tumour growth through
attacking tumour cells and TAMs. In the mean time, soluble mediators
like VEGF and TNF-α secreted from primary tumours may stimulate
macrophages to produce MIP-1α, which in turn stimulates primary
tumour cells in a paracrine manner to enhance tumour progression
[41]. Thus, both MIP-1α and TAMs may have a mutual relationship,
which favours their fundamental role in hepatocarcinogenesis [42].
Moreover, TAMs can promote tumour growth by stimulating
angiogenesis [8]. The present study showed that the number of CD68+
macrophages and CD105-MVD were positively correlated in HCC
tissues as previously demonstrated [35-37]. Moreover, the infiltrating
macrophages and enhanced angiogenesis were primarily distributed
at the periphery of HCC, which is the predominant place for tumour
cells to invade into surrounding normal tissue and/or blood vessels,
thereby fostering tumour progression and metastatic spread. Zhang
et al. [43] found that depletion of macrophages in combination with
the administration of the anti-angiogenic agent, sorafenib significantly
inhibited tumour progression, tumour angiogenesis, and lung
metastasis compared with sorafenib monotherapy. Moreover, when
TAM infiltration into tumours was prevented by depleting circulating
monocytes, tumour angiogenesis and growth were suppressed [44].
Macrophages in the tumour microenvironment play a pivotal role
in the angiogenic switch. They are an important source of various
angiogenic factors including VEGF and MMP-9 [45,46]. Thus, tumourderived MIP-1α may indirectly induce neovascularization through
recruitment of macrophages into HCC microenvironment.
All together, the present study shed the light on the role of cancerassociated inflammation in HCC progression. The findings that mark
MIP-1α and infiltrating TAMs as key players in tumour growth and
angiogenesis in HCV-related HCC might be translated into clinical
implications. Pharmacological inhibition of MIP-1α by neutralizing
antibodies or blocking its binding to CCR1 may result in disruption
of macrophage recruitment into tumour site and reduction in
tumour growth and angiogenesis. Moreover, selectively depleting
or modulating tumour-promoting characteristics and/or activity of
TAMs, might be another potential therapeutic target.
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Citation: El Aggan HA, Helmy MA, El Deeb NMF, Zeid AE, Yehia MFA (2012) Macrophage Inflammatory Protein-1α (MIP-1 α) in Hepatitis C VirusRelated Hepatocellular Carcinoma: Relation to Clinical Staging and Tumour Angiogenesis. J Clin Exp Pathol 3:133. doi:10.4172/21610681.1000133
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