Journal of Cancer Therapy, 2012, 3, 1066-1073
http://dx.doi.org/10.4236/jct.2012.36139 Published Online December 2012 (http://www.SciRP.org/journal/jct)
Biomarkers Expression in Cervical Cancer and High
Grade Squamous Intraepithelial Lesions*
Ostojich Marcela1, Gimenez Liliana2, Gian n i Ser g i o1, Alvarez Ana3, Marino Lina2, De Dios Diana1,
Jasnis Maria Adela4
1Departament of Gynecology, Institute of Oncology Angel H. Roffo, University of Buenos Aires, Argentina; 2Department of Patho-
logy, Institute of Oncology Angel H. Roffo, University of Buenos Aires, Argentina; 3Department of Clinical Oncology, Institute of
Oncology Angel H. Roffo, University of Buenos Aires, Argentina; 4Department of Immunobiology, Institute of Oncology Angel H.
Roffo, University of Buenos Aires, Argentina.
Received August 17th, 2012; revised September 19th, 2012; accepted September 30th, 2012
ABSTRACT
Objectives: The finding of new prognostic factors in human cervix cancer is necessary to improve present conventional
treatments. The aim of the present study was to determine the expression and evaluate the prognostic value of hy-
poxia-inducible factor-1(HIF-1α), vascular endothelial growth factor (VEGF) and eritropoyetin receptor (EpoR) in cer-
vix cancer stages IIA-IIB and in preinvasive high grade squamous intraepithelial lesions (HSIL) Methods: The study
included 70 patients with cervix cancer, FIGO stages IIA-IIB, 28 patients with HSIL and normal cervix (n = 28).
HIF-1α, VEGF and EpoR expression were analyzed in tissue samples by immunohistochemistry using commercial
antibodies. Expression and overexpression of the tumor markers were quantified according to German Immunoreactive
Score. Results: HIF-1α, EpoR and VEGF overexpression was detected in 30%, 37% and 51% of cancer patients re-
spectively. Patients with HSIL showed enhanced expression only of EpoR and VEGF (39.2% and 71.4%) while VEGF
was overexpressed in 21% of the specimen. No correlation was found between VEGF and EpoR with disease-free over-
all survival (OS), tumor recurrences or prognostic factors. Only overexpression of HIF-1 was associated with less me-
dian survival measured up to 24 months, unless it was not maintained a long time. Conclusion: Although any of the
markers could be considered as independent prognostic factor for cervix cancer patients, our data showed a significant
increase in their expression from the premalignant lesion up to the invasive stages of tumor progression.
Keywords: Prognostic Marker; Cervical Cancer; Intraepithelial Lesion; HIF-1α; VEGF; EpoR
1. Introduction
Squamous cell carcinoma of cervix is the most frequent
tumor within female genital tract and one of the leading
causes of female cancer-related death. It accounts for
approximately two-thirds of all cervical cancer cases,
whereas adenocarcinomas and adenosquamous carcino-
mas account for 10% - 25% [1]. In our country, cervical
cancer shows an incidence of 17.5 per 100.000 women
and 7.4 deaths per 100.000 women [2]. Invasive cervical
cancer and the precursor lesions are in casual relationship
with infection by oncogen human papillomavirus (HPV).
In the squamous epithelium, HPV mediates cellular dys-
plasia and neoplasia which represent oncogenic progres-
sion [3]. Persistance of HPV infection is not sufficient to
transform the epithelial host cells. Transformation by
HPV depends on the oncoproteins E6 and E7, whose
transcription is modulated by numerous transcription
factors and by epigenetic mechanisms. Thus, genetic and
epigenetic alterations are involved to acquire an immortal
phenotype and to further progress to an overt malignant
and invasive phenotype [4,5]. The E6 and E7 proteins
initiate dysregulation of cell proliferation and of apo-
ptotic mechanisms at defined targets such as p53 and
retinoblastoma (Rb) tumor suppressor proteins, respec-
tively. The E6 protein switches off the apoptotic protec-
tion activated by genomic instability arising as a conse-
quence of E7 induced cell cycle activation [6]. The dra-
matic change in the methylation pattern of several tumors
is characterized by methylation of CpG islands in several
promoter regions. The hypomethylation (extent of in-
corporation of 3H-methyl groups) increased progres-
sively with the grade of cervical neoplasia [7].
Angiogenesis is fundamental for tumor growth since it
requieres an adequate blood supply for its oxygenation
and nutrition demands [6]. Early at the onset, tumor an-
*Declaration of Interest: The authors report no conflict of interest. The
authors alone are responsible for the content and writing of the paper.

Biomarkers Expression in Cervical Cancer and High Squamous Intraepithelial Lesions 1067
giogenesis is mediated not only by angiogenic factors but
also directly by hypoxia [7-9]. As tumors grow larger,
sustained tissue hypoxia may also cause molecular
changes associated with a more malignant phenotype less
sensitive to cytotoxic and/or radiotherapy treatments [10,
11]. Lack of oxygen is a hallmark of solid tumor forma-
tion and constitutes an independent prognostic factor in
diverse malignant tumors [12] Hypoxia-inducible factor-
1 (HIF-1) is the transcriptional factor that mediates cel-
lular response to hypoxia [13]. HIF-1 protein is a het-
erodimer composed of HIF-1α and HIF-1  (ARNT)
subunits that bind to specific hypoxia-responsive ele-
ments (HREs) and it is protected from ubiquitination and
proteosomal degradation under hypoxic conditions [14,
15]. HIF1 influences the expression of a diverse set of
genes associated with tumor progression, e.g. eritropoye-
tin (Epo), transferrin, endothelin-1, iNOS, Glut-1 trans-
porter, VEGF and inflammatory molecules as well as
pro- and antiapoptotic genes [16-18]. Cancer cervix hy-
poxia has been described to be independent of clinical
size, grade and FIGO stage [19,20] and is nowadays con-
sidered a powerful independent prognostic factor for the
outcome of the disease [11,21].
Vascular endothelial growth factor (VEGF) is an im-
portant mediator of tumor angiogenesis. Tumor cells
within tumor microenvironment secrete VEGF under
stressed conditions as hypoxia [22], radiotherapy [23]
and chemotherapy [24], resulting in low response and
thus poor prognosis.
Besides VEGF, erythropoietin (Epo) is another of the
best known hypoxia-regulated genes mediated by HIF-1
[25]. Epo is a glycoprotein hormone that stimulates
erythropoiesis, which exerts its effect by stimulating
growth, preventing apoptosis and inducing differentiation
of red blood cell precursors. Epo receptor (EpoR) be-
longs to the superfamily of cytokine receptors; Epo and
EpoR signaling stimulates not only hematopoietic tissues
and cells, but also a variety of solid tumors including
breast and cervical cancer [26,27].
Our aim was to analyze the expression of HIF-1α,
VEGF and EpoR in locally advanced cervical cancer and
in premalignant HSIL to evaluate their value as prognos-
tic markers of cervix neoplasias. The identification of
prognostic and/or predictive biological markers in cervix
cancer patients and their potential application in new and
improved therapeutics would be of great importance for
survival improvement.
2. Material and Methods
2.1. Patients and Tissue Samples
The retrospective study included 70 patients with cervi-
cal cancer FIGO stages IIA and IIB, treated and followed
up at the Department of Gynecology, Institute of Oncol-
ogy A. H. Roffo (University of Buenos Aires). Patients
were randomly selected from our data base between
years 1987-1999. Inclusion criteria were patients with
diagnosis of uterine cervix carcinoma confirmed by an
expert pathologist and further classified as stage II ac-
cording to FIGO guideline; complete radiotherapy treat-
ment at our Institute (TCT 50 Gy and BT 35 - 40 Gy);
access to histological samples included in paraffin were
also required. Patients of the database with no post-
treatment follow up and with other oncological diseases
(except basocellular tumors) were excluded. Women
with high grade squamous intraepithelial lesions (HSIL)
were also included in the study (n = 28). Normal cervical
tissues (n = 28) were obtained from hysterectomies due
to benign uterine diseases. Clinico-pathological charac-
teristics of patients are shown in Table 1. The study was
approved by the Ethical Committee at the Institute of
Oncology A. H. Roffo.
Table 1. Clinicopathological characteristics of patients.
Median age (range) 46 y (30 - 76)
Tissue Samples n
Normal cervix 28
HSIL 28
Cervical cancer 70
FIGO stage
IIA 13 (18.6)
IIB 57 (81.4)
Histology
Squamous 55 (78.5)
Adenoca 14 (20)
A/s 1 (1.4)
Differentiation grade
G1 14 (20)
G2 20 (28.5)
G3 21 (30)
n.d 15 (21.4)
Platelet count
400.000 33 (47.2)
>400.000 10 (14.3)
n.d 27 (38.5)
Clinicopathological charateristics of cervical cancer patients and samples
size are shown. Adenoc: adenocarcinoma; A/s: adenosquamous; n: indicate
number of patients; numbers between parenthesis indicate % of patients; n.d.:
not determined.

Biomarkers Expression in Cervical Cancer and High Squamous Intraepithelial Lesions
1068
2.2. Immunohistochemistry
Formalin-fixed, paraffin-embedded 4-m tissue sections
were stained with standard immunohistochemical meth-
ods. Sections were deparaffinized and endogenous per-
oxidase was blocked in 3% H2O2 in TBS buffer, pH 7.4
for 10 min. Antigen retrieval was performed using a mi-
crowave. After heating, slides were incubated with anti-
bodies anti-HIF-1α (dilution 1:50, Santa Cruz, sc-53546,)
anti-VEGF (dilution 1:250, Biogenex, PV-483,) and anti-
EpoR (dilution 1:200, Santa Cruz, sc-695) and detected
with secondary biotinilated antibodies. Diaminobenzidi-
ne was used as substrate cromogen and all slides were
then counterstained with hematoxilyn. Negative controls
were performed without primary antibodies.
2.3. Immunoreactivity Score
All sections were evaluated in a blinded manner by two
pathologists. Regions of greatest immunostaining for
each antibody were selected and the percentage of immu-
noreactive cells and staining intensity were scored ac-
cording to German Immunoreactive Score. The percent-
age of immunoreactive tumor cells was rated as follows:
no staining = 0; up to 10% = 1; 11% - 50% = 2; 51% -
80% = 3; >81% = 4; intensity: weak (1+); moderate (2+);
strong (3+). The grade of score expression was obtained
multiplying percentage by intensity: 0, negative; 1 - 4,
weak; 5 - 8, moderate, 9 - 12, strong. Overexpression
was considered for scores 5 to 12 (moderate + strong).
2.4. Statistical Analysis
Correlation between markers (HIF-1, VEGF and EpoR)
and clinicopathological variables was analyzed by Chi-
square or Fisher’s test. Cox regression was used to de-
termine independent prognostic values. Overall survival
analysis was performed using the Kaplan Meyer method
and log-rank test was used to compare survival curves.
Statistical significance was defined as p < 0.05.
3. Results
HIF-1α was mainly identified in tumor cell cytoplasm of
39/70 (55.7%) of cancer specimens, being overexpressed
in nearly a half of them (21/39, 53.8%). On the other
hand, barely 2/28 HSIL and 1/28 normal cervix showed
positive weak staining (p < 0.05 vs cervix cancer) (Table
2, Figures 1(a)-(c)).
EpoR was not only detected in significantly higher
percentage of cancer patients compared to HSIL (72.8%
vs 39.2% respectively, p < 0.05), but half of the positive
cancer samples (26/50) showed overexpression of EpoR
(Figures 2(a)-(c)). Only a small number of normal cervix
(17.8%) expressed EpoR (Table 2).
A very similar high percentage of cancer and HSIL
samples expressed VEGF (70% and 71.4% respectively);
however overexpression was significantly more frequent
in cervix than in HSIL specimens (73.4% vs 30% respec-
tively, p < 0.001). Unexpectedly, positive VEGF was
detected in 35% of normal cervix (p < 0.05 vs HSIL).
Figures 3(a)-(c) show VEGF expression in carcinoma,
preinvasive and normal cervix respectively.
When we evaluated the level of expression of the com-
bination of the three markers in cancer patients, we only
found that a significant number of patients co-expressed
EpoRc with HIF-1 or with VEGF (52.8% and 57% re-
spectively, p < 0.05) (Table 3).
The median survival time among the whole cohort of
Table 2. Expression of HIF-1α, EpoRc and VEGF.
HIF1α EpoRc VEGF
(+) (++) (+) (++) (+) (++) Tissue samples
n (%) n (%) n (%) n (%) n (%) n (%)
N (n = 28) 1 (3.5) - 5 (17.8) - 10 (35.7) -
HSIL (n = 28) 2 (7.2) - 11 (39.2)• - 20 (71.4) 6 (30)
CC (n = 70) 39 (55.7)* 21 (53.8) 51 (72.8)•• 26 (50.9) 49 (70) 36 (73.4)
Number (n) of patients with marker expression (+) and overexpression (++); numbers between parenthesis indicate % of patients; percentage
of overexpression was calculated in positive samples. N: normal cervix; CC: cervical cancer; Significance between groups compared with
Fisher test (p < 0.05). HIF-1: *CC vs HSIL (p < 0.05); EPORc: •HSIL vs N (p<0.05); ••CC vs HSIL (p < 0.05); ººVEGF: HSIL vs N; CC
overexpression vs HSIL (p < 0.05).
Table 3. Coexpression of EpoRc with HIF-1α and with VEGF in cancer patients.
HIF1α (−) HIF1α (+) VEGF (−) VEGF (+)
Biomarkers n (%) n (%) n (%) n (%)
EpoRc (+) 14/70 (20) 37/70 (52.8)* 10/7 (14.2) 40/70 (57)*
Epo Rc (−) 17/70 (24.3) 2/70 (2.8) 11/70 (15.7) 9/70 (13)
Table 3 shows cancer patients with co-expression of EpoR with HIF-1 and VEGF. n (%) = number of patients (percentage). Significance
with Crosstabs, Fisher’s test *p < 0.001).

Biomarkers Expression in Cervical Cancer and High Squamous Intraepithelial Lesions 1069
(a) (b) (c)
Figure 1. Immune staining for HIF-1α in Cancer, HSIL and normal cervix. Immunohistochemistry of cancer, HSIL and
normal tissues using specific monoclonal antibody anti HIF-1α. (a) Representative invasive cervix cancer showing positive
strong cytoplasmatic HIF-1α immunoreactivity. Some positive nucleus are also seen (400×); (b) HIF-1α nuclear expression in
HSIL (100×); (c) negative normal cervix (200×).
(a) (b) (c)
Figure 2. Immune staining for EpoRc in cancer, HSIL and normal cervix. Immunohistochemistry of cancer, HSIL and nor-
mal tissues using specific monoclonal antibody anti EpoRc. (a) Invasive cervix cancer shows intense immunostaining for
EpoR (500×); (b) Representative cytoplasmatic immmunoreactivity in HSIL (400×); (c) Positive immunoreaction in normal
squamous epithelial cells (100×).
(a) (b) (c)
Figure 3. Inmmune staining for VEGF in cancer, HSIL and normal cervix. Immunohistochemistry of cancer, HSIL and
normal tissues using specific monoclonal antibody anti VEGF. (a) Intense VEGF cytoplasmatic immunoreactivity in tumor
cells from invasive cervix cancer (400×); (b) VEGF in HSIL (200×); (c) Positive immunoreactivity in normal epithelial cells
200×). (

Current Distortion Evaluation in Traction 4Q Constant Switching Frequency Converters 1070
cancer patients was 47 months (range 8 - 195 months).
We did not find any significative correlation between
markers expression neither with known established prog-
nostic factors nor with disease-free survival (DFS). Only
overexpression of HIF-1 was significantly associated with
median survival measured at 24 months of evolution
(Kaplan-Meier, p < 0.05); however, at long-term, this
difference was not sustained (Figures 4(a)-(c)). As ex-
pected, no recurrences were detected in patients with
HSIL.
4. Discussion
HIF-1, VEGF and EpoRc expression in cervix cancer was
compared not only with normal cervix but mainly with
premalignant HSIL lesions. Our data showed positive
HIF-1 nuclear and cytoplasmatic immunoreactivity of
tumor cells in 39/70 cancer patients, being overexpressed
in more than a half (53.8%) (Figure 1). HSIL and normal
cervix barely showed weak positive staining (p < 0.05).
Our results are in accordance with recent data reporting
increased HIF-1α and VHL proteins in advanced uterine
cervical carcinomas compared to the corresponding nor-
mal samples and cervical intraepithelial neoplasia [16,28].
Overexpression of HIF-1α was described in many other
solid tumors such as colon, breast, skin, lung, gastric,
pancreatic, prostate and renal carcinoma [11,14,29-31].
Tumor aggressiveness is associated with enhanced ex-
pression of hypoxic associated markers such as HIF-1,
VEGF, GLUT-1, nitric oxide [32]. Since markers of mi-
crovessel density increased from normal epithelium to
squamous cell carcinoma [33], it has been suggested that
HIF-1α should not be used as an endogenous marker of
tumor hypoxia in locally advanced uterine squamous cell
carcinomas with significant prognostic impact [34]. It is
noteworthy that in our cohort of normal cervix tissue,
although 35% were positive for VEGF, HIF-expression
was not detected.
Analysis of overall survival (OS) followed up to 24
months (mo), showed that only HIF overexpression was
significantly associated with lower median survival (12
mo vs 22 mo) in patients with persistent disease (Kaplan
Meier p < 0.05). However, in long term, this difference
was not sustained. Thus, HIF-1 failed to be a prognostic
factor in our cohort of cervix cancer patients (Figure
4(a)).
Angiogenesis plays a key role in tumour growth and
metastasis and angiogenic factors may help to identify
patients with a poor prognosis [34]. Aberrant new blood
vessels lead to tumour hypoxia limiting cell growth.
However, tumor cells can adapt to hypoxic conditions,
switching a more malignant phenotype and/or poor re-
sponse to radio and chemotherapy [35]. VEGF is one of
the main factors involved in tumor vascularization and
(a)
(b)
(c)
Figure 4. Overall Survival (OS) of Patients with Cervix
Cancer according Biomarkers Expression. Figure 4 shows
overall survival (OS) curves of cervix cancer patients fol-
lowed during 120 months and according to the level of ex-
pression of HIF-1α (a), EpoR (b) and VEGF (c). Patientes
with overexpression (- - - over); patients with low or nega-
tive expression (— neg + low).

Biomarkers Expression in Cervical Cancer and High Squamous Intraepithelial Lesions 1071
can be upregulated in response to hypoxia, inducing the
angiogenic switch [36]. We find almost equal expression
of VEGF in cancer and HSIL samples (70% and 71.4%
respectively); however, higher number of cancer patients
showed VEGF overespression (73.4% and 30% respec-
tively, p < 0.05) (Table 2). According to our results we
can assume that although VEGF is expressed in both
cervix cancer and preneoplastic HSIL lesions, advanced
stages of the disease are associated with overexpression
of VEGF. Probably this overexpression might be linked
with a more angiogenic tumor. It has been recently re-
ported that higher endostatin and VEGF expression are
indicative of advanced cervical cancer disease, and that
VEGF allowed for a distinction between patients with
non-invasive and those with recurrent disease [37]. In
patients with cervical carcinoma, the expression of
VEGF-C was also related to lymph node metastasis rep-
resenting a prognostic indicator of cervical cancer [38].
Moreover, among different biological markers of angio-
genesis, only VEGFR2 expression was reported as pre-
dictor of response to radio-chemotherapy in cervical can-
cer [39,40]. It has been recently reported that EGFRc is
an independent prognostic factor in cervix cancer FIGO
stages IIb-IV [41]. Although we have not looked for
EGFRc expression, is has been described a relation be-
tween EGF system and VEGF.
Recently, expression and function of Epo and EpoR in
a variety of human cancers have been reported, including
solid tumors and tumor cell lines [28]. Epo and EpoR are
downstream targets of HIF and can promote acquisition
of malignant phenotype and exert antiapoptotic effects on
certain tumor cell lines [41]. As such, treatment with
rhEpo could have harmful consequences. Thus, it is of
great value to determine the presence of EpoR in cancer
tissues of patients using rhEpo for radio and chemo-
therapy-associated anemia
In our study, EpoR expression not only occurs more
often in cancer patients than in HSILs (72.8% vs 39.2%, p
< 0.05), but is overexpressed in 50.9% of cancer samples.
Less than 20% normal epithelia showed weak staining (p
< 0.05 vs HSIL). According to these results, we can con-
clude that enhanced EpoR expression seemed to be asso-
ciated with tumor malignancy, since it progressively
augmented from normal cervix towards premalignant
lesions with a further significant increase in malignant
cervix cancer.
Contradictory results have been reported about the role
of Epo/EpoR in tumor biology. While the involvement of
Epo in angiogenesis of human gliomas has been reported
[42], other authors have not detected elevated levels of
EpoR in other human tumor cells [43]. Patients with
uterine cervix tumors expressing high levels of Epo, had
reduced overall survival, with no correlation with intra-
tumoral hypoxia [27].
When we analyzed co-expression between the combi-
nation of the different biomarkers, a significant associa-
tion was detected only between the combination of Epo
expression with HIF and with VEGF, demonstrating that
in cervix cancer there is a tendency to the coexpression of
Epo with any of the other two biomarkers. However,
decrease in median survival (at 24 mo) associated with
markers expresison was only detected in patients over-
expressing HIF-1.
In conclusion, the present study suggests that, although
HIF-1 is weakly expressed in preinvasive lesions, VEGF
and EpoR are already strongly expressed (are they HIF-
independent?) with further enhanced expression in IIA-
IIB cancer samples. Thus, the three biomarkers can be
associated with cervix tumor progression along the vari-
ous stages of the disease. In cervical screening, it would
be useful to determine biomarkers turning up already in
the premalignant stage, while in invasive stages it could
be necessary biomarkers of prognostic value or for spe-
cific response to therapy. Studies with larger number of
patients would be necessary to determine if these mo-
lecular targets enable to be taken into account as markers
of cervix tumor prognosis.
5. Acknowledgements
This study was supported by grant CM13-2011 from
Universidad de Buenos Aires, Argentina.
REFERENCES
[1] S. H. Liyanage, C. A. Roberts and A. G. Rockall, “MRI
and PET Scans for Primary Staging and Detection of
Cervical Cancer Recurrence,” Women’s Health, Vol. 6,
No. 2, 2010, pp. 251-267. doi:10.2217/whe.10.7
[2] D. De Dios, S. Gianni, M. Ostojich and C. Kremer,
“Pautas en Oncología. Diagnóstico, Tratamiento y Se-
guimiento del Cáncer,” Instituto de Oncología A H Roffo,
Universidad de Buenos Aires, Buenos Aires, 2010, pp.
137-143.
[3] J. Cuzick, M. Arbyn, R. Sankaranarayanan, V. Tsu, G.
Ronco, M. H. Mayrand, J. Dillner and C. J. Meijer,
“Overview of Human Papillomavirus-Based and Other
Novel Options for Cervical Cancer Screening in Devel-
oped and Developing Countries,” Vaccine, Vol. 26, Suppl.
10, 2008, pp. K29-K41.
[4] F. Kisseljov, O. Sakharova and T. Kondratjeva, “Cellular
and Molecular Biological Aspects of Cervical Intraepi-
thelial Neoplasia,” International Review of Cell and Mo-
lecular Biology, Vol. 271, 2008, pp. 35-95.
doi:10.1016/S1937-6448(08)01202-1
[5] H. Von Keyserling, T. Bergmann, M. Schuetz, U. Schiller,
J. Stanke, C. Hoffmann, A. Schneider, H. Lehrach, A.
Dahl and A. M. Kaufmann, “Analysis of 4 Single-Nu-
cleotide Polymorphisms in Relation to Cervical Dysplasia
and Cancer Development Using a High-Throughput Liga-
tion-Detection Reaction Procedure,” International Jour-

Biomarkers Expression in Cervical Cancer and High Squamous Intraepithelial Lesions
1072
nal of Gynecological Cancer, Vol. 21, No. 9, 2011, pp.
1664-1671. doi:10.1097/IGC.0b013e31822b6299
[6] A. Szalmás and J. Kónya, “Epigenetic Alterations in Cer-
vical Carcinogenesis,” Seminars in Cancer Biology, Vol.
19, No. 3, 2009, pp. 144-152.
doi:10.1016/j.semcancer.2009.02.011
[7] P. M. Das and R. Singal, “DNA Methylation and Can-
cer,” Journal of Clinical Oncology, Vol. 22, No. 22, 2004,
pp. 4632-4642. doi:10.1200/JCO.2004.07.151
[8] K. L. Bennewith and S. Dedhar, “Targeting Hypoxic Tu-
mour Cells to Overcome Metastasis,” BMC Cancer, Vol.
11, 2011, pp. 504-509.
doi:10.1186/1471-2407-11-504
[9] Z. K. Otrock, H. A. Hatoum, A. H. Awada, R. S. Ishak
and A. I. Shamseddine, “Hypoxia-Inducible Factor in
Cancer Angiogenesis: Structure, Regulation and Clinical
Perspectives,” Critical Reviews Oncology/Hematology,
Vol. 70, No. 2, 2009, pp. 93-102.
doi:10.1016/j.critrevonc.2009.01.001
[10] C. S. F. Wong, J. Sceneay, C. M. House, H. M. Halse, M.
C. P. Liu, J. George, T. C. Potdevin Hunnam, B. S. Parker,
I. Haviv, Z. Ronai, C. Cullinane, D. Bowtell and A.
Möller, “Vascular Normalization by Loss of Siah2 Re-
sults in Increased Chemotherapeutic Efficacy,” Cancer
Research, Vol. 72, No. 7, 2012, pp. 1694-1705.
doi:10.1158/0008-5472.CAN-11-3310
[11] P. Vaupel, “Tumor Microenvironmental Physiology and
Its Implication for Radiation Oncology,” Seminars in Ra-
diation Oncology, Vol. 14, No. 3, 2004, pp. 198-206.
doi:10.1016/j.semradonc.2004.04.008
[12] B. Bachtiary, M. Schindl, R. Pötter, B. Dreier, T. H.
Knocke, J. A. Hainfellner, R. Horvat and P. Birner,
“Overexpression of Hypoxia-Inducible Factor-1α Indi-
cates Diminished Response to Radiotherapy and Unfa-
vorable Prognosis in Patients Receiving Radical Radio-
therapy for Cervical Cancer,” Clinical Cancer Research,
Vol. 9, No. 6, 2003, pp. 2234-2240.
[13] G. J. Hutchison, H. R Valentine, J. A. Loncaster, S. E.
Davidson, R. D. Hunter, S. A. Roberts, A. L. Harris, I. J.
Stratford, P. M. Price and C. M. West, “Hypoxia-Induc-
ible Factor 1 Alpha Expression as an Intrinsic Marker of
Hypoxia: Correlation with Tumor Oxygen, Pimonidazole
Measurements, and Outcome in Locally Advanced Car-
cinoma of the Cervix,” Clinical Cancer Research, Vol. 10,
2004, pp. 8405-8412.
doi:10.1158/1078-0432.CCR-03-0135
[14] P. Vaupel, “Metabolic Microenvironment of Tumor Cells:
A Key Factor in Malignant Progression,” Experimental
Oncology, Vol. 32, No. 3, 2010, pp. 125-127.
[15] A. Unruh, A. Ressl, H. C. Mohamed, R. S. Johnson, R.
Nadrowitz, E. Richter, D. M. Katschinski and R. H.
Wenger, “The Hypoxia-Inducible Factor-1 Alpha Is a
Negative Factor for Tumor Therapy,” Oncogene, Vol. 22,
No. 21, 2003, pp. 3213-3220. doi:10.1038/sj.onc.1206385
[16] G. L. Semenza, “Hypoxia-Inducible Factors: Mediators of
Cancer Progression and Targets for Cancer Therapy,”
Trends in Pharmacol Sciences, Vol. 33, No. 4, 2012, pp.
207-214. doi:10.1016/j.tips.2012.01.005
[17] P. Vaupel, “Hypoxia and Aggressive Tumor Phenotype:
Implications for Therapy and Prognosis,” The Oncologis,
Vol. 13, Suppl. 3, 2008, pp. 21-26.
doi:10.1634/theoncologist.13-S3-21
[18] M. W. Luczak., A. Roszak, P. Pawlik, H. Kedzia, M.
Lianeri and P. P. Jagodzinski, “Increased Expression of
HIF-1α and Its Implication in the Hypoxia Pathway in
Parimary Advanced Uterine Cervical Carcinoma,” On-
cology Reports, Vol. 26, No. 5, 2011, pp. 1259-1264.
[19] M. Guppy. “The Hypoxic Core: A Possible Answer to the
Cancer Paradox,” Biochemical and Biophysical Research
Communications, Vol. 299, No. 4, 2002, pp. 676-680.
doi:10.1016/S0006-291X(02)02710-9
[20] W. Y. Lee, S. C. Huang, K. F. Hsu, C. C. Tzeng and W.
Shen, “Roles for Hypoxia-Regulated Genes during Cer-
vical Carcinogenesis: Somatic Evolution during the Hy-
poxia-Glycolysis-Acidosis Sequence,” Gynecologic On-
cology, Vol. 108, No. 2, 2008, pp. 377-384.
doi:10.1016/j.ygyno.2007.10.034
[21] M. Höckel and P. Vaupel, “Oxygenation of Cervix Can-
cers: Impact of Clinical and Pathological Parameters,”
Advances in Experimental Medicine and Biology, Vol.
510, 2003, pp. 31-35. doi:10.1007/978-1-4615-0205-0_6
[22] C. Leo, L. C. Horn, J. Einenkel, B. Hentschel and M.
Höckel, “Tumor Hypoxia and Expression of c-Met in
Cervical Cancer,” Gynecologic Oncology, Vol. 104, No. 1,
2007, pp. 181-185. doi:10.1016/j.ygyno.2006.07.040
[23] M. G. Noordhuis, J. J. Eijsink, F. Roossink , P. de Graeff,
E. Pras, E. Schuuring, G. B. Wisman, G. H. de Bock, A.
G. van der Zee, “Prognostic Cell Biological Markers in
Cervical Cancer Patientes Primarily Treated with (Che-
mo)radiation: A Systemic Review,” International Journal
of Radiation Oncology, Vol. 79, No. 2, 2011, pp. 325-
334. doi:10.1016/j.ijrobp.2010.09.043
[24] D. H. Gorski, M. A. Beckett, N. T. Jaskowiak, D. P. Cal-
vin, H. J. Mauceri, R. M. Salloum, S. Seetharam, A.
Koons, D. M. Hari, D. W. Kufe and R. R. Weichselbaum,
“Blockage of the Vascular Endothelial Growth Factor
Stress Response Increases the Antitumor Effect of Ion-
izing Radiation,” Cancer Research, Vol. 59, No. 14, 1999,
pp. 3374-3378.
[25] P. Gallet, B. Phulpin, J. L. Merlin, A. Leroux, P. Bravetti,
H. Mecellem, N. Tran and G. Dolivet, “Long-Term Al-
terations of Cytokines and Growth Factors Expression in
Irradiated Tissues and Relation with Histological Severity
Scoring,” PLoS One, Vol. 6, 2011, Article ID: e29399.
doi:10.1371/journal.pone.0029399
[26] P. Albertsson, B. Lennernäs and K. Norrby, “Low-Dos-
age Metronomic Chemotherapy and Angiogenesis: Topoi-
somerase Inhibitors Irinotecan and Mitoxantrone Sti-
mulate VEGF-A-Mediated Angiogenesis,” Acta Patho-
logica, Microbiologica et Immunologica Scandinavica,
Vol. 120, No. 2, 2012, pp. 147-156.
doi:10.1111/j.1600-0463.2011.02830.x
[27] C. Leo, L. C., Horn, C. Rauscher , B. Hentschel, A. Lie-
bermann, G. Hildebrandt and M. Höckel, “Expresion of
Erythropoietin and Erytropoietin Receptor in Cervical
Cancer and Relationship to Survival, Hypoxia and Apop-
tosis,” Clinical Cancer Research, Vol. 12 , No. 23, 2006,

Biomarkers Expression in Cervical Cancer and High Squamous Intraepithelial Lesions
1073
pp. 6894-6900. doi:10.1158/1078-0432.CCR-06-1285
[28] G. Acs, P. Acs, S. M. Beckwith, R. L. Pitts, E. Clements,
K. Wong and A. Verma, “Erythropoietin and Erythropoi-
etin Receptor Expression in Human Cancer,” Cancer Re-
search, Vol. 61, 2001, pp. 3561-3565.
[29] H. H. Jo, S. H. Kim, I. A. Park, D. Kang, S. S. Han, J. W.
Kim, N. H. Park, S. B. Kang and Y. S. Song, “Expression
of Vascular Endothelial Growth Factor and Hypoxia In-
ducible Factor-1α in Cervical Neoplasia,” Annals of the
NY Academy of Science, Vol. 1171, 2009, pp. 105-110.
doi:10.1111/j.1749-6632.2009.04891.x
[30] K. Ruan, G. Song and G. Ouyang, “Role of Hypoxia in
the Hallmarks of Human Cancer,” Journal of Cellular
Biochemistry, Vol. 107, No. 6, 2009, pp. 1053-1062.
doi:10.1002/jcb.22214
[31] J.-W. Kim, P. Gao and C. V. Dang, “Effects of Hypoxia
on Tumor Metabolism,” Cancer Metastasis Review, Vol.
26, No. 2, 2007, pp. 291-298.
doi:10.1007/s10555-007-9060-4
[32] J. Mazibrada, M. Rittà, M. Mondini, M. De Andrea, B.
Azzimonti, C. Bogogna, M. Ciotti, A. Orlando, N. Surico,
L. Chiusa, S. Landolfo and M. Gariglio, “Interaction be-
tween Inflammation and Angiogenesis during Different
Stages of Cervical Carcinogenesis,” Gynecologic Oncol-
ogy, Vol. 108, No. 1, 2008, pp. 112-120.
doi:10.1016/j.ygyno.2007.08.095
[33] A. Mayer, A. Wree, M. Höckel, C. Leo, H. Pilch and P.
Vaupel, “Lack of Correlation between Expression of
HIF-1alpha Protein and Oxygenation Status in Identical
Tissue Areas of Squamous Cell Carcinomas of the Uter-
ine Cervix,” Cancer Research, Vol. 64, No. 16, 2004, pp.
5876-5881. doi:10.1158/0008-5472.CAN-03-3566
[34] S. Chouaib, Y. Messai, S. Couve, B. Escudier, M. Has-
min and M. Z. Noman, “Hypoxia Promotes Tumor Growth
in Linking Angiogenesis and Immune Escape,” Frontiers
in Immunology, Vol. 3, No. 21, 2012, pp. 1-10.
[35] E. B. Rankin and A. J. Gaiaccia, “The Role of Hy-
poxia-Inducible Factors in Tumorigenesis,” Cell Death
and Differentiation, Vol. 15, No. 4, 2008, pp. 678-685.
doi:10.1038/cdd.2008.21
[36] D. Liao and R. S. Johnson, “Hypoxia: A Key Regulator
of Angiogenesis in Cancer,” Cancer Metastasis Review,
Vol. 26, No. 2, 2007, pp. 281-290.
doi:10.1007/s10555-007-9066-y
[37] S. Landt, H. Heidecke, C. Reuter, S. Korlach, J. U.
Blohmer, W. Lichtenegger, T. Heusner, F. Stoblen, M.
Thill, J. Barinoff, J. Sehouli and S. Kümmel, “The Utility
of an in Vitro Angiogenesis Score for Prognosis Assess-
ment in Patients with Cervical Cancer,” Anticancer Re-
search, Vol. 31, No. 8, 2011, pp. 2645-2649.
[38] D. M. Ma, Y. P. Xu and L. Zhu, “Expression of Vascular
Endothelial Growth Factor C Correlates with a Poor
Prognosis Based on Analysis of Prognostic Factors in Pa-
tients with Cervical Carcinomas,” Journal of Obstetrics
Gynaecology Research, Vol. 37, No. 11, 2011, pp. 1519-
1524. doi:10.1111/j.1447-0756.2011.01566.x
[39] V. M. Nagy, R. Buiga, I. Brie, N. Todor, O. Tudoran, C.
Ordeanu, P. Virág, O. Tarta, M. Rus and O. Balacescu,
“Expression of VEGF, VEGFR, EGFR, COX-2 and
MVD in Cervical Carcinoma, in Relation with the Re-
sponse to Radio-Chemotherapy,” Romanian Journal of
Morphology and Embryology, Vol. 52, No. 1, 2011, pp.
53-59.
[40] P. Adimi, K. D. Steffensen, D. Schledermann, E. R.
Rasmussen and A. Jakobsen, “The Prognostic Importance
of EGFR and COX-2 Expression in Cervix Cancer Stages
IIb-IVa,” Journal of Cancer Therapy, Vol. 2, No. 1, 2011,
pp 9-15. doi:10.4236/jct.2011.21002
[41] E. Abhold, E. Rahimy, J. Wang-Rodriguez, K. J. Blair, M.
A. Yu, K. T. Brumund, R. A. Weisman and W. M. Ong-
keko, “Recombinant Human Erythropoietin Promotes the
Acquisition of a Malignant Phenotype in Head and Neck
Squamous Cell Carcinoma Cell Lines in Vitro,” BMC
Research Notes, Vol. 4, 2011, p. 553.
doi:10.1186/1756-0500-4-553
[42] B. Nico, T. Annese, D. Guidolin, N. Finato, E. Crivellato
and D. Ribatti, “Epo Is Involved in Angiogenesis in Hu-
man Glioma,” Journal of Neurooncology, Vol. 102, No. 1,
2011, pp. 51-58. doi:10.1007/s11060-010-0294-6
[43] A. Sinclair, N. Rogers, L. Busse, I. Archibeque, W.
Brown, P. Kassner, J. E. V. Watson, G. E. Arnold, K. C.
Q. Nguyen, S. Powers and S. Elliott, “Erythropoietin Re-
ceptor Transcription Is neither Elevated nor Predictive of
Surface Expression in Human Tumour Cells,” British
Journal of Cancer, Vol. 98, No. 6, 2008, pp. 1059-1067.
doi:10.1038/sj.bjc.6604220