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Rom J Morphol Embryol
2019, 60(1):59–68
ISSN (print) 1220–0522 ISSN (online) 2066–8279
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The prognostic value of CXCR4, MMP-2 and MMP-9 in tongue
squamous carcinoma
FELICIA ILEANA CIUCĂ1), PETRE-COSTIN MĂRĂŞESCU2), MARIUS MATEI3), ALMA-MARIA FLORESCU2),
CLAUDIU MĂRGĂRITESCU1), ALIN DRAGOŞ DEMETRIAN4), IRINA DRĂGAN4), CRISTIANA IULIA DUMITRESCU5)
1)
Department of Pathology, Faculty of Dentistry, University of Medicine and Pharmacy of Craiova, Romania
2)
Department of Dental Materials, Faculty of Dentistry, University of Medicine and Pharmacy of Craiova,
Romania
3)
Department of Histology, University of Medicine and Pharmacy of Craiova, Romania
4)
Department of Thoracic Surgery, University of Medicine and Pharmacy of Craiova, Romania
5)
Department of Clinical Pharmacology, University of Medicine and Pharmacy of Craiova, Romania
Abstract
Currently, tongue squamous cancer appears to be more frequent, especially among adults under the age of 45. Approximately 50% of these
patients are diagnosed late, with clinically detectable metastases; the five-year survival rate of patients with loco-regional metastases is less than
60%. In order to explain this behavior, many investigations have been conducted in recent years, most of them focusing on identification of
potential prognostic and therapeutic markers involved in the pathogenesis of tongue cancers. Our research follows the same trend, which
aims to study the prognostic implications of immunohistochemical (IHC) expression of markers C-X-C chemokine receptor type 4 (CXCR4),
matrix metalloproteinase (MMP)-2 and MMP-9 in 54 cases of tongue squamous carcinoma. The cases were selected from the archives of
the Laboratory of Pathology, Emergency County Hospital, Craiova, Romania, from the 2015–2017 period. They were immunohistochemically
processed using the labeled Streptavidin–Biotin (LSAB) enzyme detection technique, and as a method of evaluating reactions, the IHC score
developed by Remmele & Stegner. Reactivity for the investigated markers was recorded in both primary tumors, parenchymal and stromal,
and in lymph node metastases, and also in normal or dysplastic mucosa adjacent to tumor lesions. The maximum tumor reactivity was
recorded for CXCR4, followed by MMP-9 and MMP-2. In addition, all of these markers were expressed stronger in the invasion front and
especially in the lymph node metastatic forms. This immunoprofile would suggest their implication in loco-regional invasion and dissemination
processes, allowing the selection of the most aggressive forms of tongue squamous carcinoma.
Keywords: tongue, squamous, prognostic, CXCR4, MMP-2, MMP-9.
Introduction
Although the global trend is a decrease of oral
squamous cell carcinoma (SCC) incidence, there has been
an increase in tongue localization, especially among adults
less than 45 years old [1, 2]. This can be explained in part by
changing the exposure profile of risk factors associated with
classical risk factors (smoking and alcohol consumption) or
solitary action of human papillomavirus (HPV) infections
(16, 18 and possibly other), other infections with oncogenic
viruses, genetic abnormalities and/or as a consequence of
exposure to other environmental agents more or less known
to have carcinogenic action [3–5]. The most prominent
prognostic factors for tongue cancer seem to remain the
tumor, node, metastasis (TNM) stage and topographic
location. Thus, at the time of diagnosis, at least 50% of
patients with tongue cancer have either clinical detectable
metastases or undetectable metastases, which greatly reduce
the survival rate [6, 7]. According to data provided by the
American Cancer Society (ACS), the five-year survival
rate of patients with local metastases is 78%, decreasing
to 63% in patients with regional metastases, and in the
case of those with remote metastases to 36% [8]. On the
other hand, cases located of the tongue base appear to
have the lowest survival rate at five years, respectively
42.6% [9].
Most studies indicate oral tumor genesis as a multi-
stage process, in which multiple genes alterations would
occur and as a result, a disruption of oncogenes and
suppressor genes function. Also, an abnormal increase
in growth factor secretion, overexpression of surface
receptors, hyperactivity of intracellular signaling pathways
and transcription factors, all compete with carcinogenesis
[10]. Many of these events are scarcely elucidated, and
a lot of research is currently underway on the main
prognostic and therapeutic factors involved in the
pathogenesis of tongue cancers.
Aim
Our investigation also aims to study the prognostic
implications of immunohistochemical (IHC) expression
of C-X-C chemokine receptor type 4 (CXCR4), matrix
metalloproteinase (MMP)-2 and MMP-9 markers in 54
cases of squamous carcinoma of the tongue.
Materials and Methods
A number of 54 cases of squamous carcinoma localized
to the tongue, diagnosed and operated between 2015–
2017, were investigated in the Department of Oral and
Maxillo-Facial Surgery and in the Department of Surgery,
Emergency County Hospital, Craiova, Romania. For the
R J M
E
Romanian Jo urnal of
Morphology & Embryology
http://www.rjme.ro/
Felicia Ileana Ciucă et al.
60
IHC processing, the corresponding histopathological
blocks were used from the archives of the Laboratory
of Pathology of the same Hospital.
After reviewing of the histopathological specimens,
4 μm serial sections were made from the selected paraffin
blocks, which were applied to electrostatically-charged
glass slides. They were subjected to the classic IHC
processing protocol using the labeled Streptavidin–Biotin 2
(LSAB2) enzyme detection system and the Dako kit
(Redox, Romania – K0675).
Table 1 presents the primary antibodies used in the
study along with their main characteristics.
Table 1 – Antibodies used in the study and their main
characteristics
Antibody Clone /
Producer Dilution Antigen
retrieval
External
positive
control
CXCR4
Rabbit, polyclonal /
Thermo Scientific
(PA3-305)
1:500 Citrate,
pH 6
Squamous
carcinoma
MMP-2
Rabbit, polyclonal /
Santa Cruz
Biotechnology
(sc-8835-R)
1:50
0.1 M
Citrate,
pH 6
Granulation
tissue
MMP-9
Mouse, monoclonal
7-11C / Santa Cruz
Biotechnology
(sc-13520)
1:50
0.1 M
Citrate,
pH 6
Granulation
tissue
CXCR4: C-X-C chemokine receptor type 4; MMP: Matrix metallo-
proteinase.
The visualization of the reactions was done with
3,3’-Diaminobenzidine (DAB, from Redox, Romania –
DAKO, K3468) chromogen and the counterstaining was
done with the Mayer’s Hematoxylin (from Tunic, Bio-
Optica, Romania – M06002). To validate the reactions, we
used positive external controls by omitting the primary
antibody.
As a method of quantification of IHC reactions, we used
the immunoreactivity score (IRS) given by Remmele &
Stegner, consisting in examining at ×40 objective at least
five tumor areas with maximum tumor reactivity (set at
×10 objective), by determining the percentage of immuno-
reactive tumor cells multiplied by the intensity of immuno-
reactions [11]. The percentages of the immunostained
tumor cells were: 1 (25% marked cells), 2 (26–49%
marked cells), 3 (50–74% marked cells), and 4 (over 75%)
and the immunoreactions intensity was rated as: 1 (weak),
2 (moderate), 3 (strong). Finally, the IRS varied between
1–12. The presence of stromal immunoreactivity in this
study was evaluated only qualitatively by notifying
the presence or absence of reactivity for these markers
and identifying their subcellular locations.
The images were captured using the Nikon Eclipse
55i microscope, equipped with a 5-megapixel cooling
camera and Image-Pro Plus software. For the statistical
analysis, Student’s t, analysis of variance (ANOVA),
χ2 (chi-square) and Pearson tests were used from the
Statistical Package for the Social Sciences (SPSS) 10
software. For statistically testing the probability of
association between the different descriptive categories
in this study, we made contingency tables with those data,
and subsequently used chi-square test.
The results were considered statistically significant
when p<0.05. For comparisons on several variables of
interest, we used the ANOVA test.
Results
The main clinical and morphological characteristics of
the investigated cases are shown in the Table 2. Analyzing
the data presented in Table 2, we noticed the prevalence
of tongue SCC in people over 60 years (55.56%), male
(57.41%), affecting mainly the mobile part of the tongue
(66.66%). Histopathologically, moderately differentiated
cases (44.44%) predominate and, as well as the pTNM
stage, the majority were diagnosed in stage III (37.04%)
and stage II, respectively (33.33%).
In the Table 2 are shown the average IRS for each
antibody analyzed in relation to the main clinical-
morphological variables.
Table 2 – The main clinical-morphological variables
of our casuistry and IRS distribution according to these
variables
IRS (average ± SD)
Clinical-
morphological
variables
No.
of
cases
[%]
CXCR4 MMP-2 MMP-9
Age [years]
<60 24 44.44
1.91±
2.47
0.44±
0.6
1.67±
2.35
>60 30 55.56
2.63±
2.78
0.74±
0.99
2.35±
2.67
Gender
F 23 42.59
2.02±
2.68
0.41±
0.76
1.96±
2.75
M 31 57.41
2.52±
2.6
0.78±
0.86
2.11±
2.29
Topography
Mobile portions
(including margins) 36 66.66 3.1±
2.75
0.76±
0.87
2.61±
2.57
Fixed portions 18 33.33 1.44±
2.26
0.44±
0.77
1.4±
2.35
Degree of differentiation
Well
differentiated 18 33.33 1.37±
2.24
0.31±
0.51
1.18±
2.02
Moderate
differentiated 24 44.44 2.13±
2.77
0.61±
0.96
1.96±
2.78
Low
differentiated 12 22.22 1.04±
2.06
0.3±
0.74
0.85±
1.69
pTNM
I 7 12.96
0.44±
1.26
0.07±
0.26
0.42±
1.42
II 18 33.33
1.34±
2.1
0.31±
0.58
1.18±
1.98
III 20 37.04
1.93±
2.71
0.42±
0.66
1.35±
2.02
IV 9 16.67
0.93±
2.16
0.37±
0.92
1.05±
2.54
IRS: Immunoreactivity score; SD: Standard deviation; CXCR4: C-X-C
chemokine receptor type 4; MMP: Matrix metalloproteinase; F: Female;
M: Male; pTNM: Pathological tumor, node, metastasis.
IHC study with the CXCR4 antibody
In normal or dysplastic tongue mucosa, the reactivity
for CXCR4 was present predominantly in the intermediate
layer cells with mainly cytoplasmic and less membranous
The prognostic value of CXCR4, MMP-2 and MMP-9 in tongue squamous carcinoma
61
immunostaining pattern (Figure 1A). Reactivity was
also noted in the associated inflammatory infiltrates, the
striated muscle fibers, the epithelium of the extralobular
excretory ducts of the minor salivary glands, and the
endothelial cells of the blood vessels in the chorion or
tumor stroma.
In tumor tissue, we recorded reactivity for CXCR4
in all our investigated cases (100%), but IRS ranged
from 1 to 9, the medium value of IRS was 4.54±1.9.
The maximum reactivity (IRS=9) was observed in two
cases of G1 and G2 tongue SCCs, developed at the level
of the mobile tongue, in a woman older than 60 years
and in the stage III pTNM, respectively in a man over
60 years of age and stage IV pTNM. The tumor reactivity
pattern was predominantly cytoplasmic and membranous
(Figure 1B). We did not notice nuclear reactivity in
the tumor cells. Regarding the degree of differentiation,
the maximum reactivity was recorded in the moderate
and well-differentiated variant; the cells with spinous
morphology had the highest reactivity (Figure 1, C and
D). In addition, we noticed a slightly higher reactivity
in the invasion front, compared to the superficial area
of the tumors (Figure 1E). Reactivity for CXCR4 was
more evident in lymph node metastatic forms, where
reactivity was more evident especially in squamous areas
(Figure 1F).
Figure 1 – Tongue SCC: (A) The CXCR4 membranar and cytoplasmic reactivity of the basal and parabasal layer
cells from the adjacent tongue tumor epithelium; (B) The CXCR4 predominant cytoplasmic reactivity of tumor cells;
(C and D) The CXCR4 cytoplasmic reactivity of tumor cells, in a moderate-differentiated SCC, inside tumor versus
invasion front; (E) The CXCR4 cytoplasmic reactivity of tumor cells, from invasive front of moderate differentiated
SCC; (F) The CXCR4 predominant cytoplasmic reactivity of tumor cells with squamous cell morphology from the tongue
SCC lymph node metastases. Anti-CXCR4 antibody immunostaining: (A and D) ×40; (B and C) ×200; (E and F) ×100.
SCC: Squamous cell carcinoma; CXCR4: C-X-C chemokine receptor type 4.
Felicia Ileana Ciucă et al.
62
IHC study with MMP-2 antibody
At the level of the tongue epithelium, adjacent to
neoplastic lesions, the reactivity for MMP-2 was present
in almost its entire thickness, the reaction pattern was
predominant at the membrane level and, especially, in
the intermediate layer cells. The dysplastic epithelium
also showed cytoplasmic responsiveness in the atypical
basal cells within the lesion (Figure 2A). Cytoplasmic
reactivity for MMP-2 has also been noted in endothelial
cells of blood vessels, striated muscle fibers, stromal
fibroblasts, macrophages, and epithelium of small salivary
gland excretory channels.
Tumor MMP-2 reactivity was recorded in 46 (85.18%)
cases, the IRS average being 1.18±1.41, with values ranging
from 0 to 4 (recorded in a female over 60 years, in the
mobile portion of the tongue, with G2 differentiation degree
and in stage IV pTNM). The pattern of MMP-2 tumor
reactivity was heterogeneous, ranging from membrane
pattern, present especially in well-differentiated forms
(Figure 2B), to a cytoplasmic visible one, especially in
moderately differentiated forms (Figure 2C) and respectively
to a nuclear present only in low differentiated tumor forms
(Figure 2D). We did not notice differences in tumor
reactivity between the invasion front (Figure 2E) and the
superficial regions of the tumors.
The MMP-2 tumor reactivity was much more pronounced
in tumors with lymph node metastases compared to non-
metastatic tumors. Reactivity in metastases was predo-
minantly membranous and less cytoplasmic (Figure 2F).
Figure 2 – Tongue SCC: (A) The MMP-2 membranous reactivity of the intermediate layer cells from adjacent tongue SCC
epithelium; (B) The predominant MMP-2 membranous reactivity of tumor cells in a well-differentiated tongue SCC;
(C) Predominant cytoplasmic reactivity of tumor cells to MMP-2, in a moderately differentiated SCC; (D) Cytoplasmic and
nuclear reactivity of tumor cells to MMP-2, in a poorly differentiated SCC; (E) Cytoplasmic and nuclear reactivity of tumor
cells to MMP-2, from invasive front of moderately differentiated SCC; (F) Membranous and cytoplasmic MMP-2 reactivity
especially of the tumor cells with squamous morphology from the tongue SCC lymph node metastases. Anti-MMP-2
antibody immunostaining: (A and F) ×100; (B–E) ×200. SCC: Squamous cell carcinoma; MMP-2: Matrix metalloproteinase-2.
The prognostic value of CXCR4, MMP-2 and MMP-9 in tongue squamous carcinoma
63
IHC study with the MMP-9 antibody
In the tongue epithelium, adjacent to neoplastic lesions,
the reactivity for MMP-9 was present in the intermediate
and superficial layer, the pattern of the reaction being
membranous and cytoplasmic (Figure 3A). The cytoplasmic
pattern of the reaction was more evident in the tumor-
associated hyperplasic lesions. In addition, the cytoplasmic
reactivity for MMP-9 has been highlighted in endothelial
cells of blood vessels, stromal fibroblasts, inflammatory
cells, striated muscle fibers, and epithelial salivary glandular
channels.
At tumor level, the reactivity was higher compared
to that recorded for MMP-2, the IRS average for the
investigated casuistry being 4.02±2.16. The IRS varied
between 0 and 9. Tumor reactivity was higher in moderate
and poorly differentiated forms compared to well-differ-
entiated ones, with cytoplasmic pattern prevailing in
moderate cases and the membranous pattern in the poorly
differentiated cases (Figure 3, B–D). Also, a higher tumor
reactivity was noted especially at the invasion front,
prevailing in moderate and low differentiated forms
(Figure 3E). Reactivity also occurred in the lymph node
metastases (Figure 3F), the reactivity seems to be greater
in the metastatic primitive tumors.
Figure 3 – Tongue SCC: (A) The MMP-9 membranous and cytoplasmic reactivity of the intermediate layer cells from
adjacent tongue SCC epithelium; (B) The MMP-9 predominantly membranous and cytoplasmic reactivity of tumor
cells, in a well-differentiated SCC; (C) Predominant cytoplasmic reactivity of tumor cells to MMP-9, in a moderately
differentiated SCC; (D) Cytoplasmic reactivity of tumor cells to MMP-9, in a poorly differentiated SCC; (E) Cytoplasmic
reactivity of tumor cells to MMP-9, from the invasion front of moderately differentiated SCC; (F) The MMP-9
membranous and cytoplasmic reactivity of tumor cells with squamous morphology from the tongue SCC lymph node
metastases. Anti-MMP-9 antibody immunostaining: (A, C and F) ×100; (B, D and E) ×200. SCC: Squamous cell
carcinoma; MMP-9: Matrix metalloproteinase-9.
Felicia Ileana Ciucă et al.
64
Statistical study on reactivity
for the three markers
Statistically comparing the IRS obtained for CXCR4
versus MMP-2, we noticed the existence of a poor direct
correlation (r=0.356, p<0.05) between these two markers
(Figure 4). Instead, we found a moderate direct correlation
between CXCR4 and MMP-9 (r=0.65, p<0.05) (Figure 5)
and between MMP-2 and MMP-9 (r=0.685, p<0.05)
(Figure 6).
Figure 4 – Statistical analysis of the obtained IRS.
A weak direct correlation between CXCR4 and MMP-2
(r=0.356, p<0.05). IRS: Immunoreactivity score;
CXCR4: C-X-C chemokine receptor type 4; MMP-2:
Matrix metalloproteinase-2.
Figure 5 – Statistical analysis of the obtained IRS.
Moderated direct correlation between CXCR4 and
MMP-9 (r=0.65, p<0.05). IRS: Immunoreactivity score;
CXCR4: C-X-C chemokine receptor type 4; MMP-9:
Matrix metalloproteinase-9.
Figure 6 – Statistical analysis of the obtained IRS.
A moderate direct correlation between MMP-2 and
MMP-9 (r=0.685, p<0.05). IRS: Immunoreactivity score;
MMP-2: Matrix metalloproteinase-2; MMP-9: Matrix
metalloproteinase-9.
Statistically analyzing the expression of the three
markers in relation to the main morphoclinical variables
followed in the study, we noticed a higher expression
tendency for each of the three markers, especially in those
less than 60 years of age compared to those over 60,
for MMP-2 the differences were statistically significant
(p<0.05). For the pTNM parameter, the ANOVA test
revealed significant differences between the four stages
only for MMP-2 [F(3.5)=2.79, p<0.001] and MMP-9
[F(3.5)=5.17, p<0.01]. In contrast, regarding the tumor-
differentiation parameter, the ANOVA test did not
reveal statistically significant differences for any of the
investigated markers.
Discussions
Tongue cancer has a particular place in oral cancers,
considering epidemiology, prognosis and survival. A series
of studies show that in tongue cancer at least 50% of the
diagnosed cases already present metastasis [6], lymph
node disseminations being considered the most important
prognostic factor [7]. Moreover, cases with cancers at
the base of the tongue, at the time of diagnosis, develops
at least 31.4% of contralateral lymph node metastases [6],
and the survival rate of such patients is less than 43%
[9]. The process of oral carcinogenesis is complex and
multistage, following a sequence of events, from normal
epithelium is reaching to dysplasia and finally an invasive
carcinoma develops. In order to elucidate the molecular
profile of oral cancer, in recent decades, a series of genomic
and proteomic studies have been carried out, attempting
to identify the genetic alterations occurring in oncogenes
and other tumor suppressor genes, to determine the degree
of involvement of genomic instability and epigenetic
changes and to establish a profile of gene expressions
that take place during oral oncogenesis [12]. Behind the
aggression of tongue cancers is extracellular matrix (ECM)
degradation, due to the MMPs secreted by both tumor cells
and some of the associated stromal tumor cells [13]. In
addition, CXCR4 chemokines have been shown to regulate
the secretion of these enzymes, and there have been
numerous studies that have shown that tumor expression
levels of CXCR4, MMP-2, MMP-9 and MMP-13 in oral
squamous carcinomas are coupled [13–15]. In this regard,
our study also attempted to investigate the correlations
between the expression levels of CXCR4, MMP-2 and
MMP-9 in tongue squamous carcinomas, in relation to
their main clinical and morphological characteristics and
also to investigate their possible prognostic role in the
evaluation to such patients.
Chemokines represent a class of small cytokine-like
proteins that can bind and activate the family of seven
transmembrane receptors coupled to G protein (chemokine
receptors) [16]. These chemokines are expressed by a series
of tumors and play important roles in initiating mitosis,
modulating apoptosis, survival and angiogenesis [17]. The
interaction between stromal cell-derived factor-1 (SDF-1)
and the CXCR4 chemokine receptor has been shown to
play a major role in tumorigenesis, proliferation, metastasis
and angiogenesis in a number of human tumors, such
as: pulmonary cancer [18], malignant melanoma [19],
esophageal cancer [20], ovarian cancer [21], glioblastoma
The prognostic value of CXCR4, MMP-2 and MMP-9 in tongue squamous carcinoma
65
[22], cholangiocarcinoma [23], and basal cell carcinoma
[24]. In oral squamous cancers, CXCR4 has been shown
to promote the migration and invasion of cancer cells by
regulating the expression of MMP-9 and MMP-13, most
likely via the activation of the extracellular signal-
regulated kinase (ERK) signaling pathway [13].
In our study, we recorded the presence of an immuno-
reactivity in the intermediate layer of the normal or
dysplastic epithelium adjacent to the tumors, as well as in
the chorion and tumor stroma at the level of inflammatory
cells, vascular endothelial cells, striated muscle fibers and
in the epithelium of the extralobular excretory ducts of the
minor salivary glands. At tumor level, the reactivity for
CXCR4 was superior to the MMP-2 and MMP-9 markers.
The registered IRSs varied between 1 and 9. Relative to
the degree of differentiation, IRSs were higher in moderate
and low differentiated forms, tumor cells with spinous
morphology having the highest reactivity. The immuno-
reactivity pattern was a predominant cytoplasmic and
membranous one. In addition, the reactivity appears to
be greater at the invasion front and especially in the
metastatic forms, the reactivity being also noted in the
lymph node metastases.
Literature studies indicate variations in CXCR4
expression in oral squamous carcinomas in percentages
ranging from 28.6% to 100% [15, 25–27], differences
that can be explained by the use of various expression
quantification systems for this marker and/or the use of
different CXCR4 clones [28]. In the study undertaken
by Xia et al., expression of C-X-C motif chemokine
ligand 12 (CXCL12)/CXCR4 was present in both tumor
specimens and premalignant oral lesions, suggesting that
the CXCL12/CXCR4 axis would play important roles in
the premalignant stages of the oral mucosa, contributing
to the progression of carcinomas within this localization
[29]. A series of studies indicated a direct correlation
between CXCR4 expression in primary tumors and the
expression of this marker in lymph node metastases [15,
28, 30, 31]. Thus, activation of the CXCR4 receptor would
play a major role in the lymph node metastasis of oral
squamous carcinomas, the receptor being sensitive to
CXCL12 chemoattraction secreted in distant primary tumor
assays, targeting tissues for metastatic carcinoma cells
expressing high levels of the CXCR4 receptor [15, 25, 30,
32]. In addition, there has been evidence of correlations
between the level of CXCR4 tumor expression and the
tumor stage (levels being higher in the most advanced
stages) [30, 31], respectively correlations with perineal
invasion and vascular invasion [30]. On the other hand,
carcinoma cell motility, tumor invasion and metastasis
in oral squamous carcinomas can be explained by the
involvement of the CXCR4 receptor in regulating MMP-9
and MMP-13 expression, as well as by promoting the
epithelial–mesenchymal transition (EMT) process [13,
33, 34], demonstrated by correlation of expression of this
receptor with vimentin expression level in oral primary
tumors [30]. At the same time, literature data indicated
that this receptor may be considered as an independent
prognostic marker for patients with oral squamous
carcinomas [28, 35]. In this regard, a study of SCCs
developed at the level of the mobile portion of the tongue
showed that patients with tumors that expressed a high
level of CXCR4 had a more limited prognosis [30].
MMP-2 is a type IV collagenase of 72 kDa molecular
weight, also known as gelatinase A [36], which is involved
in the degradation of the ECM under both physiological
conditions (embryonic development, endometrial cycle,
reproduction, wound healing, bone remodeling) and in
various pathological conditions (arthritis, invasion and
cancer metastasis) [37]. MMP-2 being involved in the
degradation of the collagen IV from the basal membrane
component, has a major role in the cancer metastasis
process [38]. In addition, invadopodia structures involved
in the process of tumor invasion also concentrate the
MMPs, including MMP-2, and the degradation products
of these enzymes promote the formation of these pro-
invasive structures [39]. Also, MMP-2, as other MMPs,
determine the proteolytic activation of transforming
growth factor-beta (TGF-β), a factor that has been shown
to promote EMT process, that has a major role in cancer
metastasis [40].
In our study, we documented the presence of MMP-2
immunoreactivity in the normal, hyperplastic or dysplastic
epithelium adjacent to tumor lesions, with a particular
membranous reaction pattern evident in the intermediate
layer cells. A cytoplasmic reactivity was noted in the
dysplastic areas. In addition, reactivity for MMP-2 was
also noted in the endothelial cells of blood vessels, in the
striated muscle fibers, stromal fibroblasts, macrophages
and epithelium of the small salivary gland excretory
channels. At the tumor level, the reactivity for MMP-2
was inferior to that for CXCR4 and MMP-9. IRS scores
for MMP-2 varied between 0 and 4. We did not notice
IRS differences of reactivity depending on the degree of
differentiation, but differences related to the preponderant
pattern of reagents were present. Thus, we recorded
preponderant membranous reactivity in the differentiated
forms, one of the cytoplasmic types at the level of the
moderate forms and a pattern of nuclear reactivity for the
poorly differentiated forms. Lymph node metastatic forms
appear to be much more reactive than non-metastatic,
with metastases accounting for somewhat lower reactivity
compared to primary tumors.
Literature studies indicate a higher concentration of
MMP-2 (both latent and active) in squamous carcinoma
of the head and neck, compared to adjacent normal tissues
[41, 42]. Furthermore, it appears that the active form of
MMP-2 is in double concentration compared to MMP-9,
in the oral squamous carcinoma [41]. Most authors did not
find statistically significant correlations between MMP-2
levels and different clinical-morphological parameters,
including: gender, tumor stages, nuclear grading, tumor
differentiation, smoking exposure [41, 43, 44]. However,
most studies indicate a positive correlation between MMP-2
expression and lymph node metastasis and prognosis of
patients with squamous carcinoma of the head and neck
[41, 45–48]. Pu et al. showed that elevated levels of MMP-2
and vascular endothelial growth factor-C (VEGF-C)
expression in both primary tumors and corresponding
lymph node metastases correlated statistically significantly
with the general survival rate of patients with oral
carcinomas [48]. However, Mishev et al. believes that the
level of MMP-2 expression in primary tumors should not
be considered as a reliable predictive marker of tumor
invasiveness in oral squamous carcinomas [43], while
Felicia Ileana Ciucă et al.
66
Katayama et al. have failed to establish a correlation
between MMP-2 tumor expression and metastatic potential,
or prognosis in oral squamous carcinomas [49].
Strictly related to tongue squamous carcinomas, MMP-2
expression level was much higher in tumor tissue compared
to normal tongue mucosa or dysplastic lesions in this
localization [50]. MMP-2 was expressed not only in tumor
cells but also at stromal level, in macrophages and vascular
endothelial cells. Thus, the secretion from the MMP tumor
stroma is equally important, since their stromal expression
level correlated with lymph node metastases and with a
worse prognosis [50]. Moreover, several studies of tongue
squamous carcinomas have shown that the level of MMP-2
expression in these tumors can be used as a prognostic
factor [50–52].
MMP-9, also known as type IV collagenase or
gelatinase B, is a matrixin belonging to the large family
of dependent zinc metalloproteinases, enzymes involved
in the ECM degradation in both physiological processes
(embryonic development, reproduction, angiogenesis, bone
development, wound healing, cell migration, learning
and memory processes) and in pathological processes
(arthritis, intracerebral hemorrhage and metastasis) [53,
54]. MMP-9 has been shown to play a major role in
neovascularization by proteolytic degradation of basal
membrane blood vessels and release of the active form
of VEGF [55].
The vast majority of authors note elevated levels of
MMP-9 expression in squamous carcinoma of the head
and neck, including at oral level, but the prognostic
significance of this increased expression remains a
contradictory subject. Thus, Guttman et al. could not
establish a correlation between MMP-9 tumor expression
and primary tumor size or laterocervical lymph node
metastasis in laryngeal cancers [56]. On the other hand,
Katayama et al. reported a correlation between the MMP-9
expression rate and the loco-regional lymph node metastasis
rate and/or distant metastasis, as well as a poor prognosis
[49]. Also, de Vicente et al. showed that MMP-9 expression
did not correlate with clinical variables, namely tumor
stage and relapse rate [57], while Ikebe et al. have noted
that expression of MMP-2 and MMP-9 has been associated
with invasiveness but not with metastatic potential in oral
squamous carcinomas [58]. On the other hand, Riedel et al.
have not found a correlation between MMP-9 expression
and T-stage and N-tumor, respectively, but correlated with
a worse prognosis in patients with squamous carcinoma
of the head and neck [59] while O-Charoenrat et al.
showed that increased MMP-9 messenger ribonucleic
acid (mRNA) levels correlated with advanced T and N
stages in head and neck cancers [60]. Kato et al. found
an increased expression of total MMP-9, its active form
was much less expressed compared to MMP-2, and the
latter correlated with advanced disease stages [47]. All
these contradictory data come to support the idea that
MMP-9 is not the only factor involved in the tumor-
invasive process of the head and neck and that it can
play fluctuating roles in this process [61].
During our study, we observed reactivity for MMP-9
at normal or dysplastic epithelium almost similar to that
of MMP-2, but with a higher and obvious intensity at
the intermediate and superficial layer. Reactivity for
MMP-9 was also noted in the cytoplasm of endothelial
cells of blood vessels, stromal fibroblasts, inflammatory
cells, striated muscle fibers, and epithelial salivary gland
ducts. At tumor level, the immunoreactivity for MMP-9
was superior to that of MMP-2, IRSs ranging between
0 and 9. Moderate and low differentiated forms were
associated with the highest reactivity, the pattern being
predominantly cytoplasmic. Tumor reactivity appeared
to be higher at the invasion front, especially in low
differentiated forms and particularly in metastatic forms
compared to non-metastasizing. Reactivity for MMP-9
at the level of lymph node metastases was less comparing
with primary tumors.
Statistically, we recorded the existence of moderate
direct correlations between CXCR4 and MMP-9, respectively
MMP-2 and MMP-9, and between CXCR4 and MMP-2,
we observed a poor direct correlation. In addition, signifi-
cant differences were noted for all three markers analyzed
with age and the highest IRS being obtained in individuals
aged up to 60 years. At the same time, for the MMP-2 and
MMP-9 markers the only statistically significant differences
were found for the four-stage pTNM. For the rest of the
investigated morpho-clinical parameters we did not observe
the existence of statistically significant differences.
Conclusions
Reactivity for the three investigated markers was
present in both in parenchyma and tumor stroma, but
also in normal mucosa, or dysplasia adjacent to tumor
lesions. The biggest tumor reactivity was recorded for
CXCR4, followed by MMP-9 and MMP-2. Lymph node
metastatic forms have the highest reactivity, suggesting
the involvement of these markers in locoregional lymph
node dissemination of tongue squamous carcinomas.
Tumor reactivity was also higher at the invasion front,
suggesting their involvement in invasiveness. Therefore,
the three investigated markers can be used as prognostic
markers by selecting cases with the most severe prognosis.
Conflict of interests
The authors declare that they have no conflict of
interests.
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Corresponding authors
Claudiu Mărgăritescu, Professor, MD, PhD, Department of Pathology, Faculty of Dentistry, University of Medicine
and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania; Phone +40740–152 550, e-mail:
c_margaritescu2000@yahoo.com
Alin Dragoş Demetrian, Lecturer, MD, PhD, Department of Thoracic Surgery, University of Medicine and Pharmacy of
Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania; Phone +40722–668 853, e-mail: alin_demetrian@yahoo.com
Received: July 1, 2018
Accepted: April 30, 2019
