Journal of
Oral Pathology & Medicine
doi: 10.1111/j.1600-0714.2009.00858.x
Study of growth factors and receptors in carcinoma
ex pleomorphic adenoma
Cristiane Furuse1, Lucyene Miguita1, Ana Cláudia Garcia Rosa1, Andresa Borges Soares1, Elizabeth Ferreira
Martinez1, Albina Altemani2, Vera Cavalcanti de Araújo1
1
Department of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas, São Paulo, Brazil; 2Department of
Pathology, School of Medicine, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
SUMMARY
Carcinoma ex pleomorphic adenoma (CXPA) is a rare
malignant salivary gland tumor derived from a pre-
existing pleomorphic adenoma. It is a good model to
study the evolution of carcinogenesis, starting with in situ
areas to frankly invasive carcinoma. Growth factors are
associated with several biological and neoplastic pro-
cesses by transmembrane receptors. In order to investi-
gate, by immunohistochemistry, the expression of some
growth factors and its receptors [EGF receptor, fibro-
blast growth factor, fibroblast growth factor receptor 1,
fibroblast growth factor receptor 2, hepatocyte growth
factor, c-Met, transforming growth factor (TGF) b1,
TGFbR-II and insulin-like growth factor receptor 1] in the
progression of CXPA, we have used ten cases of CXPA in
several degrees of invasion- intracapsular, minimally and
frankly invasive carcinoma- with only epithelial compo-
nent. Slides were qualitatively and semi-quantitatively
evaluated according to the percentage of stained tumor
cells from 0 to 3 (0 = less than 10%; 1 = 10–25%; 2 = 25–
50%; 3 = more than 50% of cells). Malignant epithelial
cells starting with in situ areas showed stronger expres-
sion than luminal cells of pleomorphic adenoma for all
antibodies. Most of the intracapsular, minimally and
frankly invasive CXPA presented score 3. However, score
2 was more evident in the frankly invasive one. In small
nests of invasive carcinoma, negative cells were observed
probably indicating that the proliferative process is re-
placed by the invasive mechanism. Altogether this data
infers that these factors may contribute to cell prolifer-
ation during initial phases of the tumor.
J Oral Pathol Med (2010)
Correspondence: Vera Cavalcanti de Araújo, Rua Vicente Leporace,
1220 apt. 91, São Paulo, SP, Brazil, 04619-033. Tel: +55 11 5044 0762,
Fax: +55 11 5041 2992, E-mail: vcaraujo@slmandic.com.br
Accepted for publication September 30, 2009
J Oral Pathol Med
ª 2010 John Wiley & Sons A/S Æ All rights reserved
interscience.wiley.com/journal/jop
Keywords: carcinoma ex pleomorphic adenoma; growth factors
Abbreviations: rRNA, ribosomal ribonucleic acid; ErbB ⁄ HER,
epidermal growth factor receptor family; PDGFR-a, platelet
growth factor receptor type a; FGFR2b, fibroblast growth factor
receptor isoform 2b; FGFR2c, fibroblast growth factor receptor
isoform 2c; GTPase, guanosine triphosphate hydrolase enzyme;
GEP100, guanine-nucleotide exchange protein 100; TGF-a,
transforming growth factor a.
Introduction
According to the World Health Organization the
carcinoma ex pleomorphic adenoma (CXPA) is defined
as an epithelium malignization derived from a pleomor-
phic adenoma (1). The structural diversity of CXPA
enables us to an important template study of the
evolution of carcinogenesis, from benign to malignant
tumor (2–7).
Several growth factors are involved in carcinoma
initiation and progression including fibroblast growth
factor (FGF), hepatocyte growth factor (HGF), epider-
mal growth factor (EGF), insulin-like growth factor
(IGF) and transforming growth factor b (TGFb) (8–17).
Growth factors and their receptors have been intensely
studied in tumorigenesis of mammary, skin, prostate
and bladder carcinomas (18–21), blood cancer (8, 22),
neural astrocytoma (23), but rarely studied in salivary
gland tumors (24–27).
Among them, the FGF-2 is a member of a family of
heparan-biding polypeptides, localized in extracellular
matrix, cytoplasm and nucleus of the cells (28–30). It
acts on cells by intracrine, paracrine and autocrine
mechanisms (28, 31, 32) activating intracelullar path-
ways by tyrosine kinase receptors (FGFR-1 and FGFR-
2) (29, 30, 33, 34), or by internalization to cytoplasm
and translocation to nucleus acting directly on nucleo-
lus where FGF-2 participates in rRNA transcription
(35, 36). In the nucleus, FGF-2 promotes mitogenic
 Study of growth factors and receptors in carcinoma
Furuse et al.

2
function, cell differentiation, angiogenesis, phenotypic according to the presence of epithelial and ⁄ or myoepi-
transformation (35, 37), and survival of tumor and stem thelial cells using immunohistochemistry for cytokera-
cells (8, 38–40). tins (epithelial cells), vimentin and a-smooth muscle
HGF is a cytokine that induces many biological actin (myoepithelial cells) as previously described in
functions not only in hepatocytes but also in many Altemani et al. (2). Tumors with epithelial components
epithelial cells (41, 42) by activating a tyrosine kinase were selected in a total of 10 cases (Table 1) and
signaling cascade after binding to the proto-oncogenic classified according to Brandwein et al. (62) taking into
C-Met receptor (43). In normal tissue, HGF plays a role account the extension of invasion beyond the previous
in the dynamic construction and reconstruction of pleomorphic adenoma capsule as intracapsular (without
tissues during organogenesis and tissue regeneration invasion), minimally invasive (£1.5 mm of invasion) and
(44, 45). In mature tissues, HGF has an organotrophic frankly invasive. In addition, luminal cells from residual
role in regeneration and protection of various tissues pleomorphic adenoma present in the CXPA specimens
(44). HGF also has angiogenic activity for vascular and 4 cases of pleomorphic adenoma without malignant
endothelial cells (46). In tumor tissues, cells utilize the transformation, retrieved from the files of the Depart-
biological actions of HGF for their dissociative, invasive ment of Oral Pathology of São Leopoldo Mandic
and metastatic behavior. Institute and Research Center, Campinas, SP, Brazil,
EGF receptor (EGFR) is a member of ErbB ⁄ HER were also analysed.
transmembrane protein family that performs many Serial sections, 3 lm thick, were obtained from
biological functions in normal cells, as proliferation, paraffin-embedded samples and the dewaxed sections
adhesion and migration (47, 48). Many growth factors were processed for epitope desmasking. Endogenous
of EGF family can activate EGFR on cell surface, peroxidase was blocked by incubation in 3% hydrogen
resulting in a downstream cascade of proteins which peroxide. After washing, the sections were incubated
promotes ⁄ promoting signal transduction (48). In neo- with primary polyclonal antibodies to FGF-2, FGFR-1,
plastic cells, the super-expression of receptor as well as FGFR-2, HGF-A, C-Met, TGFb-1, EGFR and IGFR-
mutations of its cytoplasmatic domain contributes to 1, and primary monoclonal antibody to TGFbR-II
constitutive signalization which appears to be relevant in (Table 2). Proper positive controls were utilized for each
the growth and progression of many cancers (49). antibody. Omission of the primary antibody constituted
IGF plays an important role in normal cellular the negative control. Signal detection was performed
growth and development and it has been implicated using the DAKO EnVision Peroxidase procedure
in the regulation of tumor growth (50–52). One of (DAKO, Carpinteria, CA, USA), followed by a dia-
its receptors, insulin-like growth factor receptor 1 minobenzidine chromogen solution and counterstaining
(IGFR-1), is a transmembrane receptor with tyrosine with Mayer’s hematoxylin.
kinase activity, and regulates cell growth and meta- The labeled sections were qualitatively and semi-
bolism (53, 54). quantitatively evaluated by six independent examiners.
TGFb-1 family encompasses a group of structurally The qualitative analysis was performed evaluating the
related growth and differentiation factors which have positive cells in different areas including in situ and
diverse activities in the regulation of cell growth, peripheral areas of both intracapsular and minimally
differentiation, embryonic induction and morphogenesis invasive tumors, large and small groups of cells of the
in a wide range of cells and tissues (55, 56). It has also frankly invasive carcinoma. For semi-quantitative eval-
been implicated in carcinogenesis progression in differ- uation, we followed the recommendation of the Mem-
ent tumors (57–59). TGFb-1 signals through the type I bers of Ad-Hoc Committee on Immunohistochemistry
receptor (TGFbR-I) and the type II receptor (TGFbR- Standardization (63). The scores for the expression of
II) (9, 12) and it usually stimulates mesenchymal cells each protein were assigned according to the percentage
growth but inhibits epithelial cell proliferation (60). In
cancer, it plays a major role by suppressing tumor
growth in the early phase of neoplasia, while promoting
tumor progression and metastasis in later phases (61). Table 1 Sex, age, localization, and degree of invasion of the
carcinoma ex pleomorphic adenoma
Based on the important role of growth factors and
their receptors in many tumors, the aim of the present
study was to investigate the participation of these Case Age Salivary Degree of
no. Sex (years) gland invasion
growth factors in CXPA with different evolution stages
by immunohistochemistry. 1 Male 58 Parotid Intracapsular
2 Female 50 Parotid Intracapsular
3 Female 37 Submandibular Intracapsular
Materials and methods 4 Female 51 Parotid Intracapsular
5 Female 65 Parotid Minimally invasive
The present study protocol was approved by the Ethics 6 Female 43 Parotid Minimally invasive
Committee of School of Medicine of the State Univer- 7 Male 74 Parotid Minimally invasive
sity of Campinas, SP, Brazil. The CXPA cases were 8 Female 62 Submandibular Frankly invasive
9 Male 66 Parotid Frankly invasive
retrieved from the files of the Pathology Department at 10 a a
Parotid Frankly invasive
the School of Medicine of the State University of
a
Campinas, Campinas, Brazil. The tumors were classified Data not available.

J Oral Pathol Med


Table 2 Treatment for antigen retrieval, dilution and incubation time of primary antibodies
Antibody Product name ⁄ ctalog number
a
EGFR EGFR(1005) ⁄ sc-03
FGF-2a FGF-2(147) ⁄ sc-79
FGFR-1a Flg(C-15) ⁄ sc-121
FGFR-2a Bek(C-17) ⁄ sc-122
IGFRb IGF-I R ⁄ AF-305-NA
HGF-Aa HGFA-S(N-19) ⁄ sc-1372
C-Metc Met antibody ⁄ Ab27492
TGFb-1a TGFb1(V) ⁄ sc-146
TGFbR-IIa TGFb RII(E-6) ⁄ sc-17792
a
Santa Cruz Biotechnogy, Inc., Santa Cruz, CA, USA.
b
R&D Sytems, Inc., Minneapolis, MN, USA.
c
Abcam Inc., Cambridge, MA, USA.
of stained tumor cells from 0 to 3 (0, <10%; 1, 10–25%;
2, 25–50%; 3, staining of more than 50% of cells).
Results
The immunohistochemical expression of the growth
factors and their receptors was evaluated in 10 cases of
CXPA with only epithelial component in different
degrees of invasion– intracapsular, minimally invasive
and frankly invasive.
The expression of all growth factors and receptors
evaluated in this study was present in the malignant
epithelial cells from the beginning of the malignization
process. Examples are shown in Fig. 1.
The qualitative analysis showed almost the same
pattern of staining for all the growth factors and
receptors studied in CXPA cases. Some were present
only in the cytoplasm and others in both cytoplasm and
nucleus.
The malignant epithelial cells showed stronger expres-
sion than luminal cells of pleomorphic adenoma, except
for HGF-A which presented the similar pattern in both
tumors. Invasive tumors showed disappearance of the
staining in some small nests and in isolated cells. This
phenomenon was less visible for EGFR.
The scores attributed by the semi-quantitative ana-
lyzes for the residual pleomorphic adenoma and pleo-
morphic adenoma without transformation as well the
CXPA are present in Table 3, as an average score for
each antibody. Most of the CXPA tumors presented
score 3 for all the studied proteins, despite of the degree
of invasion; meanwhile only HGF-A presented score 3
for the luminal cells of pleomorphic adenoma (residual
and without malignant transformation).
Discussion
Several growth factors families, implicated as autocrine
and paracrine mediators of stromal–epithelial interac-
tions, are involved in carcinoma initiation and progres-
sion. These include the EGF, the FGF, the IGF, HGF,
and the TGFb. Most of these factors are predominantly
stimulators of proliferation and can play a part in
promoting the carcinogenic process (9).
Study of growth factors and receptors in carcinoma
Furuse et al.
3
Antigen retrieval Dilution Incubation time
Citrate 0.01 M; pH 6.0; 95C, 30 min 1:100 18 h
Citrate 0.01 M; pH 6.0; 95C; 30 min 1:100 18 h
Citrate 0.01 M; pH 6.0; 95C; 30 min 1:150 18 h
Citrate 0.01 M; pH 6.0; 95C; 30 min 1:50 40 min
Citrate 0.01 M; pH 6.0; 95C, 30 min 1:7 60 min
Citrate 0.01 M; pH 6.0; 95C, 30 min 1:500 60 min
Citrate 0.01 M; pH 6.0; 95C, 20 min No Dilution 18 h
No treatment 1:200 40 min
Pepsin 0.4% pH1.8, 37C, 30 min 1:50 18 h
It is well known that during development various
tissues navigate to distinctive phases of proliferation,
growth, migration, differentiation and death. This pro-
cess is guided by a plethora of growth factors which are
able to converge to execute complex multigene devel-
opmental programmes. Although there is still some
controversy (64), it is widely accepted that cancer cells
use the same strategies by which the embryo grows and
develops (65, 66). Morphogenesis and metastasis seem
to arise from the same genetic event that instructs cells
to detach from a primary colony, cross tissue bound-
aries, adhere to and migrate through extracellular
matrices, and to escape death caused by an unfamiliar
tissue context. This program has been called invasive
growth, and is regulated by specific extracellular signals
(66, 67).
Carcinomatous areas, from all CXPA, overexpressed
all growth factors (FGF-2, TGFb-I, HGF-A) and also
the receptors (EGFR, FGFR-1, FGFR-2, IGFR,
TGFbR-II and C-MET) used in this study. The
platelet-derived growth factor-A (PDGF-A) and its
receptor (PDGFR-a) were present with the same
pattern of expression in carcinomatous areas of CXPA
(68). Thus, various studies have demonstrated the
participations of these growth factors in many malig-
nant tumors.
FGF-2 (also known as basic-FGF) is expressed in
lung cancer (69), retinoblastoma (70), hemangioperi-
cytoma of the liver (71), multiple myeloma (72), prostate
cancer (10), bladder carcinoma (10, 73) and melanoma
(74). In mammary tumors, FGF-2 is associated to
growth and metastasis via neovascular stability (16). In
salivary gland adenocarcinoma, FGF-2 is observed in
immortalized cell clone, non-metastasizing and also
metastasizing cell clones (24). Its expression in serum
concentration is increased along with the progression of
human salivary gland cancer cells, contributing to tumor
proliferation and metastasis (24).
FGFR-1 has been extensively studied in breast and
prostate cancer (10, 34), squamous cell carcinoma (10),
hematologyc malignancies (10), gliomas and meningio-
mas (74, 75), astrocytoma (30), liver metastasis of
colorectal cancer (15) and also expressed in benign
salivary gland tumor as pleomorphic adenoma (76, 77).
J Oral Pathol Med
 Study of growth factors and receptors in carcinoma
Furuse et al.

A B

C D

E F

Figure 1 Immunohistochemical staining of growth factors and receptors in carcinoma ex pleomorphic adenoma. (A) Positive staining of TGFb in
malignant cells of CXPA. Note negative expression in residual pleomorphic adenoma (200·). (B) expression of HGFA in in situ areas of
intracapsular type (400·). (C, D) Large blocks of carcinoma cells in minimally invasive type of CXPA. In (C), staining for IGFR (200·), and in (D)
for FGFR2 (200·). (E, F) Small nests of carcinoma cells in minimally invasive type of CXPA. In (E), negative carcinoma cells are evident using
FGF2 antibody (400·). In (F), EGFR expression is shown (400·).

Table 3 Semi-quantitative analysis of the growth factors and their receptors in residual pleomorphic adenoma of carcinoma ex pleomorphic
adenomas and in pleomorphic adenoma without malignant transformation

Tumors FGF-2 FGFR-1 FGFR-2 TGFb-1 TGFbR-II IGFR EGFR HGF-A c-Met
PA luminal cells 1 1 1 (nuclei) 0 0 0 1 3 0
CXPA intracapsular
#1 3 2 2 3 3 3 3 3 3
#2 3 3 3 3 3 3 3 3 3
#3 3 3 3 3 3 3 3 3 3
#4 3 3 0 3 3 3 0 3 3
CXPA minimally invasive
#5 3 3 3 3 3 3 3 3 3
#6 3 3 3 3 3 3 3 3 3
#7 3 3 3 3 3 3 3 3 3
CXPA frankly invasive
#8 3 3 2 3 3 2 3 3 3
#9 2 3 0 3 2 3 3 3 3
#10 3 3 2 3 2 3 3 2 3

PA, pleomorphic adenoma; CXPA, carcinoma ex-pleomorphic adenoma.

Scores: 0 = less than 10% of tumor cells; 1 = staining of 10–25% of tumor cells; 2 = staining of 25–50% of cells; 3 = staining of more than 50%
of cells.

J Oral Pathol Med


FGFR-2 was not present in pleomorphic adenoma but
was strongly expressed in CXPA cases. FGFR-2 is
associated with increased risk of breast cancer (14, 74,
78, 79). Gene amplification or missense mutation of
FGFR2 occurs in gastric cancer, lung cancer, breast
cancer, ovarian cancer, and endometrial cancer (80).
This evidences raise the possibility that this receptor,
FGFR-2, might be related with a malignant phenotype
of epithelial cells.
In the present study, TGFb-1 was observed in all
carcinoma cells, but negative in epithelial and myo-
epithelial pleomorphic adenoma cells. TGFb has a
dual role in carcinogenesis, initially it acts as a tumor
suppressor and causes growth arrest of epithelial cells
and in the early stages of cancer, but in an established
tumor, TGF-b exerts an effect which contributes for the
survival, progression and metastasis of the tumor by
promoting epithelial-mesenchymal transition (EMT),
angiogenesis and escape from immune surveillance
(12). TGFb-1 is found in adenocarcinoma (12), carci-
noma-associated fibroblast (81), but negative in pleo-
morphic adenoma (25). In human breast cancer cells,
TGFb has been shown to induce breast tumorigenesis
and metastatic progression (82).
HGF-A and c-Met were strongly expressed in all
CXPA cases. HGF-A (also known as scatter factor) and
its receptor (c-Met, a proto-oncogene also known as
HGFR), play an important role in angiogenesis and
tumor growth (83). HGF is expressed in many malig-
nant tumor as osteosarcoma, rabdomyosarcoma (11),
adenoid cystic carcinoma (84, 85) and breast cancer (86).
While c-Met overexpression is evidenced in colorectal
carcinomas, hepatocarcinomas, gastrinomas, and carci-
nomas of the pancreas, stomach, prostate, ovary and
breast (11), and also found in lung adenocarcinoma,
colorectal, ovarian, head and neck squamous cell
carcinoma, liver glioma, renal clear cell and papillary
cancer, stomach cancer, melanoma (87), papillary car-
cinoma of thyroid (88), sporadic papillary renal cancer,
childhood hepatocellular carcinoma, osteosarcoma and
rabdomyosarcoma (11). In pleomorphic adenoma,
HGF-A was also present, probably playing a role in
cell differentiation as demonstrated by Niranjan et al.
(89).
In the present study, IGFR-1 was observed in all
carcinoma cells. Expression of IGF and IGFR-1 has
been detected in various human malignancies, such as
brain (90), colorectal (17), breast (50) and oral (91)
carcinomas, suggesting that these molecules induce cell
proliferation and inhibition of apoptosis in an autocrine
or paracrine manner (50, 54, 92, 93). In addition, IGFR-
1 has shown to be involved in malignant transformation
and the frequent detection of IGFR-1 expression in
human cancer has fired the efforts to develop IGFR-1
targeted therapy (51, 94, 95).
Invasive tumors showed absence of the staining in
some small nests and in isolated cells. Only EGFR was
present in these nests. The absence of the antibodies
FGF-2, FGFR-1, FGFR-2, HGF-A, c-Met, TGFb-I
and TGFbR-I in these areas is in accordance with other
studies which have pointed out that the cells lose their
Study of growth factors and receptors in carcinoma
Furuse et al.
5
proliferative capacity in order to invade (96, 97). It is
believed that cancer cells cannot move and proliferate
simultaneously, a mechanism known as the go-or grow’
hypothesis (96). When there is no cell motility, tumor
cell exclusively proliferates, and when movement initi-
ates it changes the fitness landscape on which the cell
population evolves (6, 7, 97).
Meanwhile, the expression of studied growth factors
were lost in the small nests of cells, this fact was less
evident for EGFR. The phosphorylation of EGFR
activates multiple biological processes including cell
motility and invasion, by EGFR-GEP100-Arf6 path-
way, activating matrix invasion and deranging E-cadh-
erin (98). Advanced stage colorectal carcinoma presents
significantly higher co-expression of EGFR, TGF-a and
protein S6K, a ribosomal protein that regulates cell
adhesion and invasiveness (99). EGFR is vastly studied
in breast tumors (98, 100, 101), colorectal cancer (17),
pancreatic and oral carcinoma (100), gastric carcinoma
(102) and also salivary gland tumors (103–105). Several
therapies have been developed to inactivate the EGFR
pathway including monoclonal antibodies against the
extracellular domain of EGFR (106).
In conclusion, the obtained results demonstrated that
carcinoma cells from CXPA presented the expression of
all growth factors and receptors in a derisive way. We
observed that EGFR, FGF-2, FGFR-1, FGFR-2,
HGF-A, c-Met, TGFb-1, TGFbR-II and IGFR-1 were
more expressed in carcinomatous cells of CXPA than in
epithelial cells of pleomorphic adenoma ductal areas.
Moreover, this expression was more intense in intra-
capsular and minimally invasive CXPA, than frankly
invasive ones, probably contributing to cell proliferation
during initial phases of the tumor and demonstrating the
self-sufficiency of carcinomatous cells. In frankly inva-
sive tumors we also identified strong EGFR expression
in small nests of peripheral borders of invasive CXPA,
indicating that this receptor may be related to invasive-
ness and cell detachment of CXPA.
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Acknowledgements
The authors wish to thank Audrey Jordão Basso and Jeruza Pinheiro da
Silveira Bossonaro for their excellent technical expertise and assistance.
This work was supported by FAPESP – grant no. 04 ⁄ 07960-0
Conflict of interest statement
None declared.