Histopathology 2010, 57, 796805. DOI: 10.1111/j.1365-2559.2010.03721.

Association of mast cell, eosinophil leucocyte and microvessel

densities in actinic cheilitis and lip squamous cell carcinoma
Ludmilla R Souza, Thiago Fonseca-Silva, Carolina C O Santos, Marcos V M Oliveira, Rodrigo
Correa-Oliveira,1 Andre L S Guimaraes & Alfredo M B De Paula
Health Science Programme, State University of Montes Claros, Montes Claros, and 1Laboratory of Molecular and Cellular
Immunology, Research Center Renee Rachou Fiocruz, Belo Horizonte, MG, Brazil
Date of submission 30 October 2009
Accepted for publication 23 March 2010

Souza L R, Fonseca-Silva T, Santos C C O, Oliveira M V M, Correa-Oliveira R, Guimaraes A L S & De Paula A M B

(2010) Histopathology 57, 796805

Association of mast cell, eosinophil leucocyte and microvessel densities in actinic cheilitis
and lip squamous cell carcinoma
Aims: To determine the contributions of mast cells
(MC), eosinophil leucocytes (EL) and microvessel density (MVD) in lip carcinogenesis, and to establish the
relationships between these biomarkers and their
possible prognostic value in lip squamous cell carcinoma (LSCC).
Methods and results: Archived specimens of lip mucosa
(n = 13), actinic cheilitis (n = 29) and LSCC (n = 29)
were formalin-fixed, paraffin-embedded, sectioned and
stained with toluidine blue and haematoxylin and eosin
(H&E) in order to identify MC and EL and to measure
their densities. Tumour angiogenesis was estimated by
determining, with the use of CD31 antibody MVD in

areas with the highest number of stained microvessels

(hot spots). Progressive increases of MC, EL and MVD
were observed during lip tumour development. Correlation analysis revealed positive associations between the
biomarkers during tumour progression. In LSCC samples, significant associations were found between MVD
values and metastatic disease. On multivariate analysis,
MVD was a predictor of risk of cervical metastasis.
Conclusions: The densities of MC, EL and microvessels
increase during lip carcinogenesis, and for MC and EL
this may be related to the stimulation of tumour
angiogenesis. MVD could be a useful predictor of
cervical metastasis in LSCC.

Keywords: actinic cheilitis, eosinophil leucocyte, histochemistry, immunohistochemistry, lip carcinogenesis,

lip squamous cell carcinoma, mast cell, microvessel density
Abbreviations: AC, actinic cheilitis; EL, eosinophil leucocytes; H&E, haematoxylin and eosin; LSCC, lip squamous
cell carcinoma; MC, mast cells; MVD, microvessel density; TATE, tumour-associated tissue eosinophilia

Introduction
Lip squamous cell carcinoma (LSCC) and its premalignant lesion, actinic cheilitis (AC), are caused principally
by chronic exposure to ultraviolet (UV) B radiation
(280315 nm). The major source of UV B is solar
Address for correspondence: A M B De Paula, Laboratorio de Pesquisa
em Saude, Hospital Universitario Clemente de Faria, Universidade
Estadual de Montes Claros, Av. Cula Mangabeira, 562 Santo
Expedito, Montes Claros 39401-001, Minas Gerais, Brazil.
e-mail: ambpatologi@gmail.com
 2010 Blackwell Publishing Limited.

radiation, but the effect on human health of exposure

to radiation from artificial sources in the workplace and
in tanning salons is becoming increasingly important.1,2 Additionally, a range of factors have been
identified as having an association with the development of both diseases, and these include environmental
(i.e. prolonged exposure to sunlight and rural domicile), behavioural (i.e. use of tobacco), occupational and
genetic influences, as well as socioeconomic status and
viral infections.3,4
The rate of development of a tumour is regulated by a
delicate balance between the pro- and antitumourigenic

MC, EL and MVD in lip carcinogenesis

activities promoted by the neoplastic cells themselves, as

well as by cells in the surrounding microenvironment.
Local inflammation at the site of tumour growth results
in the accumulation of a variety of cell types that are
usually linked to the kinetics of oncogenesis.5 Moreover,
it has been demonstrated that UV radiation not only
contributes to the initiation and promotion of oncogenesis, via effects on cellular DNA and intracellular signal
transduction, but also interferes with the immunity of
the host against cancer cells.1,2,6,7
Several studies have shown that tumour angiogenesis,
i.e. the formation of new blood vessels from pre-existing
vascular structures in the tumour microenvironment,8,9 plays a crucial role in tumour progression
and as a prognostic indicator of human solid cancers.1013 The process of vessel formation is a complex
event comprising degradation of the extracellular
matrix, migration and proliferation of endothelial cells,
formation of new vessels and synthesis of extracellular
matrix. Tumour angiogenesis is mediated in both
tumoral and tissue-resident inflammatory cells through
the release of numerous signalling and growth factors,
but the exact mechanism is still not understood fully,
despite intense investigation.8 Moreover, with regard to
carcinomas of the head and neck, the role of tumour
angiogenesis has not been clearly established.1422
Inflammatory cells act cooperatively and synergistically with both stromal cells and malignant cells by
stimulating endothelial cell proliferation and blood
vessel formation.13,23,24 Much research attention has
been focused on the effects of mast cells (MC) and
eosinophil leucocytes (EL) on tumours in consideration
of their possible roles in the development of tumour
angiogenesis. MC are long-lived cells that are distributed widely within an organism, particularly in connective tissues, and are generally located beneath
epithelial surfaces.2527 Such cells are now recognized
as an early and persistent inflammatory cell type in
many human tumours, and the accumulation of MC is
reportedly associated with tumour progression and
prognosis.2730 MC may act directly by stimulating the
migration and proliferation of endothelial cells or
indirectly by degrading the connective tissue matrix
and activating collagenases in order to provide space
for the formation of neovascular sprouts.31,32 EL are a
distinct lineage of granulocytes that arise in the bone
marrow, circulate in the blood and emigrate into
peripheral tissues.33,34 EL can stimulate tumour angiogenesis through synthesis and release of potent angiogenic factors.23,3537 Additionally, tumour-associated
tissue eosinophilia (TATE)38 has been claimed to be
involved in the biological behaviour and prognosis of
various human malignancies.3942
 2010 Blackwell Publishing Ltd, Histopathology, 57, 796805.

797

It is presumed that the histological identification and

quantification of specific inflammatory cells and microvessel densities (MVD) in tumours could provide an
estimate of the interaction between these biomarkers
and tumour angiogenesis, and of their consequences in
oncogenesis and tumour prognosis. Therefore, the aim
of the present study was to evaluate the densities of MC,
EL and microvessels, and the relationships between
these biomarkers, at different stages of lip carcinogenesis. Additionally, associations between the biomarkers
and the sociodemographic and clinicopathological
parameters of LSCC patients were investigated.

Materials and methods

Ethical approval for this study was obtained from the
relevant local ethics committees (Unimontes COEP1187 2008).
t i s s u e s p e c i m e n s an d p a t i e n t s
Data relating to patients and samples derived therefrom were obtained from the Department of Dentistry
at the State University of Montes Claros, Minas Gerais,
Brazil. The health records of patients were retrieved
and sociodemographic and clinical data were analysed. Archived tissue blocks from surgically resected
samples of LSCC (n = 29; group male:female ratio =
6.2:1; group mean age = 63.34 15.48 years) and
AC (n = 29; group male:female ratio = 4.8:1; group
mean age = 54.39 18.17 years) and of biopsies of
lip mucosa (LM; n = 13; group male:female ratio
1.2:1; group mean age 20.62 10.73 years), used as
controls, were analysed. The diagnoses of patients
presenting AC or LSCC lesions were confirmed by
clinical examination and histopathological analysis.
All LSCC patients received surgical treatment alone.
In the LSCC group, the prognostic analysis was
carried out on the basis of epidemiological and
clinicopathological parameters. Young patients were
considered as subjects who were aged 45 years or less.
All patients were asked about the occurrence of cancer
in a first relative. The term cancer was defined using
the World Health Organization (WHO) definition as
an uncontrolled growth and spread of cells that may
affect almost any tissue of the body. Alcohol consumption and smoking habits of patients were also
investigated. Patients who never drank alcohol and exdrinkers (at least for the last 6 months prior to
diagnosis) were grouped as null for alcohol drinking
habit. Similarly, non-smokers and ex-smokers (similarly to ex-drinkers) were grouped as null for tobacco
consumption.

798

L R Souza et al.

c l i n i c a l a n d m o rp h o l o g i c a l cl a s s i f i c a t i o n
All LSCC patients were classified according to the
International Union against Cancer (UICC)TNM
Classification of Malignant Tumours43 on the basis
of the primary site, as described in the International
Classification of Diseases for Oncology (C00).44 In the
LSCC group, 20.7% of cases were in stage I, 48.3%
were in stage II, 13.8% were in stage III and 17.2%
were in stage IV. For statistical reasons, the clinical
variables of disease recurrence were dichotomized (no
and yes), cervical metastasis (absent: N0 and present:
N1, N2, N3) and tumour size (T1 T2 and T3 T4).
Morphological grading of the LSCC group was based
on WHO criteria, and the morphological analysis was
carried out by an oral pathologist (A.M.B. De Paula)
with no prior knowledge of the demographic or
clinical characteristics relating to the samples. The
final grades established were: well differentiated,
n = 19; moderately differentiated, n = 2; and poorly
differentiated, n = 8. The microscopic features of the
AC samples were analysed and graded as low-risk
(n = 12) and high-risk (n = 17).45 LM group were
from mucoceles of lip, a disease caused by local
trauma in the ducts of minor salivary glands, which
normally affects young people. However, all the
biomarkers studied here were evaluated in morphologically normal areas of tissue that were distant from
the affected site.
h i s t o l o g i c a l an d h i s tochemical staining
For the purposes of morphological and histochemical
analysis, samples were fixed in formalin, embedded in
paraffin, serially sectioned at 5 lm and evaluated
under a conventional light microscope. Sections were
stained with haematoxylin and eosin (H&E) for EL
density analysis. In order to visualize MC, two
sections of each sample were stained with 1%
toluidine blue and counterstained with 5% methanol
yellow for 5 min, following which the sections were
dehydrated, cleared and mounted with synthetic
balsam.
i mm u no h i sto c h em i s t r y f or cd 3 1 an tigen
For the immunohistochemical evaluation of MVD,
sections were mounted on organosilane-coated slides.
The primary mouse monoclonal antibody against
CD31 antigen (Novocastra Laboratories, Newcastle
upon Tyne, UK) was detected with the aid of an
LSAB visualization kit (product # K0690; Dako,
Glostrup, Denmark), employing chromogen diam-

inobenzidine for colour development. Slides were then

counterstained with Mayers haematoxylin and
mounted. Negative controls were obtained by substituting normal whole rabbit serum (product # X0902;
Dako) for the primary antibodies. A sample of oral
haemangioma (shown previously to be strongly CD31positive) was used as a positive control, while smalland intermediate-sized vessels distant from the lesions
served as an internal positive control. Only cells of
capillary-sized vessels, the cytoplasm of which was
stained brown, were considered positive.
d e t e rm i n a t i o n o f mc , el a n d m v d
MC were identified on the basis of cytoplasm with
intense purple granules and nuclei presenting a bluish
morphological aspect. EL were characterized as cells
exhibiting lobulated nuclei and intensely eosinophilic
cytoplasmic granules. For quantitative analysis, an
optical microscope Olympus BH2 (model CX31, RTSF,
Miami, FL, USA), with 10 ocular and 40 objective
lenses, was employed, and an ocular lattice (area
0.092 mm2) with 100 points composed of 10 horizontal
and 10 vertical test lines was superimposed on the test
field to be measured. A total area of 1.84 mm2 was
evaluated for each of the samples, and this corresponded
to 20 randomly selected high-power microscopic fields
located in regions of high MC and EL densities. In the LM
and AC samples, MC and EL densities were determined
in the subepithelial (including epithelium connective
tissue junctions), connective and submucosal zones. In
the case of LSCC samples, densities were assessed in the
connective tissue underlying the invasive front areas of
tumour parenchyma.
Estimation of MVD was performed using a method in
which hot spots with high vessel densities were
selected by immunohistochemical staining for CD-31
antigen.29 Individual microvessels were identified on
the basis of stained endothelial cells or transversally
sectioned tubes with a single layer of endothelial cells
either with or without a thin base membrane. A single
continuous vessel that appeared to involve two or more
CD31 positive foci was counted as one microvessel.
Initially, the sample was inspected at low magnification
in order to become familiar with its size and shape and
to identify the most appropriate fields for counting.
MVD was then performed on three fields in the tumour
sample presenting the highest vascular densities.
Within each hot spot, five high-power microscopic
fields were studied providing a total of 15 analysed
fields per sample. MVD counting was performed by an
investigator who had no knowledge of the clinical data
associated with the sample.
 2010 Blackwell Publishing Ltd, Histopathology, 57, 796805.

MC, EL and MVD in lip carcinogenesis

sta ti st ic al a na ly s i s
Statistical analyses were performed using the SPSS
statistical pack (version 17.0 for Windows; SPSS,
Chicago, IL, USA). Differences between populations
were considered to be significant when the confidence
level was >95% (P < 0.05). The kappa statistic (j) was
applied in order to assess intra-examiner reproducibility relating to the morphological grading of AC and
LSCC samples. Archived samples (n = 15 for each
group) were graded by the same examiner on two
separate occasions, with an interval of 2 weeks,
according to the same morphological criteria, and the
results were submitted to statistical analysis. The
concordance values obtained (AC samples, j = 0.857,
P < 0.05; LSCC samples, j = 0.877, P < 0.05) revealed good agreement between the two assessments.
All the continuous variables studied were distributed
non-parametrically according to the Kolmogorov
Smirnov test. Between-group differences in mean
densities of MC, EL and microvessels were evaluated
using KruskalWallis statistical tests. Spearmans correlation test was employed to assess relations between
biomarkers. Associations between the densities of MC,
EL and microvessels, and the sociodemographic and
clinicopathological findings for the AC and LSCC
groups, were investigated using MannWhitney and
KruskalWallis statistical tests. The association between clinicopathological parameters and risk of the
presence of cervical metastasis was assessed by the
binary logistic regression models, fitted for the best
significance. Bivariate analyses of the independent
variables were performed by the parametric and nonparametric statistical tests. Only independent variables
with association in which P 0.25 on bivariate
analyses were included in the binary logistic regression
models.

Results
Figure 1 presents examples of MC, EL and microvessels
identified in LM, AC and LSCC samples. MC were often
observed in small groups around normal mucosal
tissue and lesions. In the LM samples, MC were not
distributed randomly in submucosal zones but were
localized close to nerves and blood vessels (Figure 1A).
In AC and LSCC samples, MC were generally scattered
throughout the stroma adjacent to dysplastic neoplastic tissues, and were also located near to or around the
blood capillaries and nerves (Figure 1B,C). EL were
identified in LM samples at low but detectable levels
located primarily in subepithelial tissue (Figure 1D). In
dysplastic and neoplastic samples, EL were observed in
 2010 Blackwell Publishing Ltd, Histopathology, 57, 796805.

799

the form of a sequential and progressive infiltration

into the stroma of lesions, attaining the highest density
in the submucosa. In AC lesions, EL were frequently
noted adjacent to basophilic amorphous areas that
were characteristic of solar elastosis (Figure 1E).
Microvessels positive for CD31 antigen were scarce
and distributed uniformly in the stroma of LM samples
(Figure 1G). In AC lesions these structures were
observed preferentially in the lamina propria (Figure 1H), while in LSCC a higher expression of CD31
antigen was detected in the tumour stroma adjacent to
the invasive front (Figure 1).
The mean density values for MC, EL and microvessels
present in the LM, AC and LSCC samples are shown in
Table 1. The density scores for MC, EL and microvessels
exhibited statistically significant increases with the
pathological progression of lip carcinogenesis (i.e.
according to the transition from LM to AC and
ultimately to LSCC). With respect to MC densities,
significant differences were observed between the LM
and LSCC groups and between the AC and LSCC
groups, while for EL densities and MVD there were
significant differences between all groups of samples
(P < 0.05) (Figure 2). Additionally, correlation analysis revealed positive associations between all biomarkers; namely, MC EL (r = 0.584, P < 0.05), MC MVD
(r = 0.445, P < 0.05) and EL MVD (r = 0.528,
P < 0.05) (Figure 2).
In contrast, densities of MC, EL and microvessels
were not associated with morphological gradings for
AC epithelial dysplasia. Moreover, no relationships
could be established in the LSCC group between MC or
EL densities and sociodemographic and clinicopathological parameters. However, significant associations
were found between MVD values and the occurrence of
cervical metastasis (Table 2). Binary logistic regression
analysis showed that high values of MVD (above of
median count value) were associated exclusively with
high risk of cervical metastasis [odds ratio (OR): 1.560;
confidence interval (CI) 95%: 1.0852.241].

Discussion
Tumour-infiltrating cells at the periphery of a tumour,
and especially at sites of obvious invasion, have been
considered to play an important role in tumour
progression and the biological behaviour of various
human malignancies. However, these cells can induce
contradictory actions that may contribute to, inhibit, or
have no influence on, tumour progression and prognosis.5,4648 Information relating to the influence of MC
and EL on cancer progression and, consequently, their
potential prognostic value, is somewhat ambiguous.

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L R Souza et al.

LM

AC

LSCC

Figure 1. Mast cells, eosinophil leucocytes and immunohistochemical expression of CD31 antigen (microvessels) in lip mucosa (LM; A, D, G),
actinic cheilitis (AC; B, E, H) and lip squamous cell carcinoma (LSCC; C, F, I) samples. A, B, C, Slides stained with toluidine blue,
arrowheads indicate mast cells; D, E, F, slides stained with haematoxylin and eosin, arrowheads indicate eosinophil leucocytes;
G, H, I, colour developed with diaminobenzidine and counterstained with Mayers haematoxylin.

Thus MC, which are naturally cytotoxic, are known to

suppress tumour growth and to release anti-tumour
compounds,28 while EL play a role in antibody-dependent cell-mediated cytotoxicity, and in the synthesis
and release of cytokines that could contribute, either
direct or indirectly, to tumour cytotoxicity.40,49,50
In contrast, some studies have demonstrated that MC
and EL can also exert important pro-tumourigenic
effects.51,52 It appears, therefore, that MC and EL
develop diverse activities according to the different
conditions found in the tumour microenvironment, but
the nature of the factors that determine whether such
cells will support anti- or pro-tumourigenic processes
are currently unclear.
In the present study, increases in the densities of MC,
EL and microvessels were related to progression of lip
tumours. The types and quantities of cells that constitute the immune infiltrates in precancer and solid

tumours are related to the local production of chemotactic cytokines synthesized and released by tumour,
immune and stromal cells.5,5355 The sequential
migration of MC and the presence of high densities of
these cells in progressive oral mucosal dysplasia
and subsequent development of squamous cell carcinoma point to the influence of tumour-favouring
effects.27,30,56 The cytokine stem cell factor (SCF) and
its c-kit receptor stimulate the directional motility of
both mucosal and connective tissue-type MC.57 In the
case of EL, chemotactic factors such as eotaxin, which
exert a selective action on EL, have also been claimed to
play a role in the mechanism of in situ EL infiltration
and maintenance. A number of other factors appear
to regulate the expression of vascular cell adhesion
molecule-1 (VCAM-1), which is an important receptor
in EL transmigration through the endothelium.39,50,58
In this manner, the results reported herein could be
 2010 Blackwell Publishing Ltd, Histopathology, 57, 796805.

MC, EL and MVD in lip carcinogenesis

801

Table 1. Densities of mast cells, eosinophil leucocytes and microvessels in lip mucosa (LM), actinic cheilitis (AC) lesions and lip
squamous cell carcinoma (LSCC) samples
Mast cell density
Groups

Mean SD

Range

Eosinophil leucocyte density

Microvessel density

Mean SD

Mean SD

Range

Range

LM (n = 13)

24.80 19.60

051.6

12.7 4.25

5.4317.9

5.49 1.15

3.877.5

AC (n = 29)

53.33 53.81

0216.3

28.3 17.07

4.8971.2

7.43 3.55

0.3314.4

47.83190.8

11.54 4.91

2.0721.1

LSCC (n = 29)

118.44 42.4

5.43203.8

117.6 37.4

SD, Standard deviation.

associated with the migration processes and the accumulation sites of MC and EL during lip carcinogenesis
because, once located in the tumour microenvironment, these cells could exert pro-tumourigenic activities that would serve to stimulate the progression of lip
tumour. Interestingly, UV radiation is known to
suppress host immunity against the development of
tumour cells.1,2,7 It is possible, therefore, that photo
damage of lip tissue could promote changes in the
microenvironment causing the region to become more
conducive to the migration and maintenance of MC
and EL during solar carcinogenesis. These hypotheses,
however, require confirmation by further study.
Microvessel density is reportedly increased significantly in a relatively large spectrum of premalignant
squamous cell lesions, including oral mucosa, and
apparently performs an important role in the transition
of normal tissue to the precancerous state and,
eventually, to full-blown cancer.11,13 The results of
the present study are in agreement with earlier
reports of increased MVD during oral carcinogenesis
leading to intense vascularization in malignant
tumours.14,19,59,60 Preinvasive malignant cells associated with oral squamous cell carcinoma are known to
remain dormant until they become angiogenic, and
this is followed by a phase of rapid tumour growth. It is
possible that increased MVD during lip tumour progression reflects the increasing nutrient requirements
of actively growing transforming cells.
A further interesting finding of the present study was
the significant and positive relationship between MC
and EL densities and angiogenesis. Inflammatory cells
communicate via a complex network of intercellular
signalling pathways mediated typically by surface
adhesion molecules, cytokines and cytokine receptors.
Recent studies have shown that MC and EL participate
in a complex self-perpetuating cycle, such that EL
produces mediators responsible for MC differentiation,
activation, proliferation and survival,32 while activated
MC release mediators that favour EL recruitment and
 2010 Blackwell Publishing Ltd, Histopathology, 57, 796805.

activation.59 The present results support the hypothesis

that both MC and EL upregulate angiogenesis from the
initial to the end stages of lip carcinogenesis by acting
as modulators of the microenvironment, expressing
a range of potent pro-angiogenic factors, and thus
making these regions more permissive of cancer
progression. However, the exact mechanisms by which
MC and EL promote angiogenesis remain unclear. MC
accumulate at the boundary between healthy tissues
and malignancies and are often found in close association with blood vessels prior to the onset of
angiogenesis in tumours.15,31,32,61 MC are rich in
metalloproteases, such as tryptase and plasminogen
activator, that contribute to the degradation of the
extracellular matrix, which is the first step in neoangiogenesis and tumour invasiveness.61 Moreover, MC
synthesize and release potent angiogenic cytokines,
such as vascular endothelial growth factor (VEGF),
fibroblast growth factor-b (FGF-b), transforming
growth factor-b (TGF-b), nerve growth factor (NGF),
tumour necrosis factor-a (TNF-a) and interleukin 8
(IL-8), and the serine proteases tryptase and chymase.
On this basis, it has been suggested that MC act as the
angiogenic switch during the early stages of tumour
development.62 Similarly, it has been proposed that EL
play a regulatory role in pro-angiogenesis by stimulating matrix remodelling and endothelial cell migration,
proliferation and subsequent sprout formation. The
most important selective mediator of angiogenesis
released or induced by EL is VEGF, but the cells also
release granulocytemacrophage colony-stimulating
factor, FGF-b, NGF, TNF-a, IL-8, and eotaxin.3537,63
A number of reports have suggested that high MC
and EL densities may be associated with tumour
behaviour in head and neck squamous cell carcinoma.30,40,51,6466 In the present study, however,
neither MC nor EL appeared to exert any influence on
the biological behaviour of LSCC. It would seem,
therefore, that the determination of increased MC
and EL densities in tumour tissue from pathological

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L R Souza et al.

c
MC density (cells/mm2)

125.0
100.0
b

75.0
50.0

25.0
0.0
LM
P = 0.184

LSCC
P = 0.000

AC
P = 0.000

ab

bc

ac

c
EL density (cells/mm2)

125.0
100.0
75.0
50.0
b
25.0

0.0
LM
P = 0.002

ab

LSCC
P = 0.000

AC
P = 0.000

bc

ac

c
12.5
10.0
MVD

b
7.5
a
5.0
2.5
0.0
LM
P = 0.034

ab

AC
P = 0.001

bc

LSCC
P = 0.001

ac

Figure 2. Mean densities (standard deviations) of mast cells (MC)

and eosinophil leucocytes (EL), and microvessel densities (MVD) in lip
mucosa (LM), actinic cheilitis (AC) lesions and lip squamous cell
carcinoma (LSCC) samples. Different lower-case letters above the bars
indicate significant differences at P < 0.05.

specimens associated with good or bad prognosis is

insufficient to explain the actual role of these cells in
neoplastic tissues. An accurate insight into the role of
inflammatory cells in tumours may be obtained from
the use of activation or inhibition markers, together
with a consideration of the local microenvironmental
stimuli in the regulation of the functions of these cells.
Blood vessels play a crucial role in malignant tumour
prognosis.10,13 Having developed an intrinsic vascular
network, the neoplastic mass is apparently able to grow
indefinitely both in situ and at distant sites, thus
enabling its cells to enter the vascular bed and colonize
other organs.9 It has been verified in the present study
that high values of MVD were linked to a high risk of
cervical metastasis. A few studies in head and neck
squamous cell carcinomas have also revealed that the
high density of tumour microvessels is associated with
metastatic disease.15,16 It has been shown that angiogenesis can stimulate tumour development and metastasis by providing an efficient vascular supply, as well
as facilitating the intravascular transportation of metastatic malignant cells.15 In two independent studies
of MVD in LSCC samples, no statistically significant
correlations between MVD and metastatic disease were
observed.21,22 Nevertheless, different monoclonal antibodies were used to identify the blood vessels in the
LSCC samples, and this makes it difficult to make
comparisons between studies.
A number of explanations are available to account
for the conflicting results obtained in this and in
earlier studies. Thus, it may be that MC and EL
initially infiltrate cancerous tissue in order to suppress tumour activity, but that the cancer cells
subsequently stimulate the angiogenic properties of
MC and EL while suppressing their cytotoxic functions. Furthermore, cells are generally not functional
unless activated, but the quantitative analysis of
inflammatory cells per se is not capable of discriminating between activated and resting cells. In this
context, the methodologies employed to identify and
categorizes blood vessels and inflammatory cells in
published studies have varied widely. There are
several important limitations to the current study,
such as sample size, particularly for statistical
reasons, and also the typical difficulties of making
causal inferences in cross-sectional study design. In
the LM group, the increased densities of mast cells
and eosinophils could reflect age-related changes
rather than change associated with tumour lip
progression. However, with this study design, such
analysis is difficult to evaluate.
Because tumour angiogenesis is generally accepted
as being beneficial to tumour growth, and as both MC
 2010 Blackwell Publishing Ltd, Histopathology, 57, 796805.

MC, EL and MVD in lip carcinogenesis

803

Table 2. Association between densities of mast cells (MC) and eosinophil leucocytes (EL) and microvessel densities (MVD), and
the epidemiological, clinical and morphological parameters of the lip squamous cell carcinoma group
MC

EL

Parameters

Mean values

P-values

Age
Young (n = 5)

108.37

0.707

Old (n = 24)

120.54

Family history of cancer

No (n = 20)
Yes (n = 9)

118.26

117.03

Absent (n = 3)

130.61

Alcohol drinking habit

Present (n = 18)

114.52

Absent (n = 11)

124.85

Recurrence
No (n = 22)
Yes (n = 7)

124.92

108.43

T3 T4 (n = 8)

144.70

Cervical metastasis
Absent (n = 20)

108.18

Present (n = 9)

141.24

WHO grading
Well differentiated (n = 19)

0.724

119.85

0.862

116.74

0.616

121.55

0.706

121.80

0.115

121.07

0.500

113.22

0.541

110.92

0.283

120.71

11.73

0.474

10.85

0.406

12.10

0.262

10.59

0.071

14.04
0.144

132.37
0.765

0.832

9.80

129.00
0.070

11.53

12.67

106.60
0.067

0.175

9.87

110.67
0.146

4.99

P-values

11.56

83.15
0.574

Mean values

12.09

119.44

98.06

Tumour size
T1 T2 (n = 21)

36.80

P-values

116.94

118.84

Smoking habit
Present (n = 26)

Mean values

MVD

10.03

0.005*

14.89
0.875

12.52

Intermediate (n = 2)

129.62

118.48

8.55

Undifferentiated (n = 8)

112.29

109.92

9.97

0.225

*Values bearing asterisks are statistically significant (P < 0.05) according to MannWhitney and KruskalWallis tests.

and EL seem to have an indirect effect on accelerating

lip tumour progression, it is proposed that the reversal
of this process, i.e. enhancing the cytotoxic functions of
these cells and suppressing their angiogenic activities,
could afford a novel strategy for developing treatments
for this type of lip cancer. In our LSCC samples, high
counts of MVD were associated with a high risk of
cervical metastasis.

Conflicts of interest
None to declare.
 2010 Blackwell Publishing Ltd, Histopathology, 57, 796805.

Acknowledgement
The authors are grateful for support from Fundacao
de Amparo a` Pesquisa do Estado de Minas Gerais
(FAPEMIG).

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