Group Title: BMC Cancer
Title: Microarray comparative genomic hybridization detection of chromosomal imbalances in uterine cervix carcinoma
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Title: Microarray comparative genomic hybridization detection of chromosomal imbalances in uterine cervix carcinoma
Physical Description: Book
Language: English
Creator: Hidalgo, Alfredo
Baudis, Michael
Petersen, Iver
Arreola, Hugo
Pina, Patricia
Vazquez-Ortiz, Guelaguetza
Hernandez, Dulce
Gonzalez, Jose
Lopez, Ricardo
Perez, Carlos
Garci­a, Jose
Vazquez, Karla
Alatorre, Brenda
Salcedo, Mauricio
Publisher: BMC Cancer
Publication Date: 2005
Abstract: BACKGROUND:Chromosomal Comparative Genomic Hybridization (CGH) has been applied to all stages of cervical carcinoma progression, defining a specific pattern of chromosomal imbalances in this tumor. However, given its limited spatial resolution, chromosomal CGH has offered only general information regarding the possible genetic targets of DNA copy number changes.METHODS:In order to further define specific DNA copy number changes in cervical cancer, we analyzed 20 cervical samples (3 pre-malignant lesions, 10 invasive tumors, and 7 cell lines), using the GenoSensor microarray CGH system to define particular genetic targets that suffer copy number changes.RESULTS:The most common DNA gains detected by array CGH in the invasive samples were located at the RBP1-RBP2 (3q21-q22) genes, the sub-telomeric clone C84C11/T3 (5ptel), D5S23 (5p15.2) and the DAB2 gene (5p13) in 58.8% of the samples. The most common losses were found at the FHIT gene (3p14.2) in 47% of the samples, followed by deletions at D8S504 (8p23.3), CTDP1-SHGC- 145820 (18qtel), KIT (4q11-q12), D1S427-FAF1 (1p32.3), D9S325 (9qtel), EIF4E (eukaryotic translation initiation factor 4E, 4q24), RB1 (13q14), and DXS7132 (Xq12) present in 5/17 (29.4%) of the samples.CONCLUSION:Our results confirm the presence of a specific pattern of chromosomal imbalances in cervical carcinoma and define specific targets that are suffering DNA copy number changes in this neoplasm.
General Note: Periodical Abbreviation:BMC Cancer
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General Note: M3: 10.1186/1471-2407-5-77
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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BMC CancerBioMed Central

Research article

Microarray comparative genomic hybridization detection of
chromosomal imbalances in uterine cervix carcinoma
Alfredo Hidalgo1,8, Michael Baudis2, Iver Petersen3, Hugo Arreola1,
Patricia Pifial, Guelaguetza Vazquez-Ortiz1, Dulce Hernandez4,
Jose Gonzilez5, Minerva Lazos6, Ricardo L6pez', Carlos Perez', Jose Garcia7,
Karla Vizquez', Brenda Alatorrel and Mauricio Salcedo*

Address: 'Laboratorio de Oncologia Gen6mica, Unidad de Investigaci6n Medica en Enfermedades Oncol6gicas, Centro Medico Nacional Siglo
XXI-IMSS, Mexico, 2Division of Pediatric Haematology/Oncology, University of Florida, Gainesville, USA, 3Institute of Pathology, University
Hospital Charite, Berlin, Germany, 4Servicio de Epidemiologia, Hospital de Oncologia, Centro Medico Nacional Siglo XXI-IMSS, Mexico, 5Clinica
de Displasias, Hospital de Gineco-Obstetrica No. 4, Luis Castelazo Ayala-IMSS, Mexico, 6Departamento de Patologia, Facultad de Medicina
UNAM-Hospital General de Mexico, SS, Mexico, 7Laboratorio de Biologia Te6rica, Departamento de Investigaci6n, Universidad La Salle, Mexico
and 8Instituto Nacional de Medicina Genomica, Secretaria de Salud, Mexico
Email: Alfredo Hidalgo; Michael Baudis; Iver Petersen;
Hugo Arreola; Patricia Pifia; Guelaguetza Vazquez-Ortiz;
Dulce Hernandez; Jose Gonzalez; Minerva Lazos;
Ricardo Lopez; Carlos Perez; Jose Garcia;
Karla Vazquez; Brenda Alatorre; Mauricio Salcedo*
* Corresponding author

Published: 09 July 2005 Received: 22 February 2005
BMC Cancer 2005, 5:77 doi:10.1 186/1471-2407-5-77 Accepted: 09 July 2005
This article is available from:
2005 Hidalgo et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Chromosomal Comparative Genomic Hybridization (CGH) has been applied to all
stages of cervical carcinoma progression, defining a specific pattern of chromosomal imbalances in
this tumor. However, given its limited spatial resolution, chromosomal CGH has offered only
general information regarding the possible genetic targets of DNA copy number changes.
Methods: In order to further define specific DNA copy number changes in cervical cancer, we
analyzed 20 cervical samples (3 pre-malignant lesions, 10 invasive tumors, and 7 cell lines), using the
GenoSensor microarray CGH system to define particular genetic targets that suffer copy number
Results: The most common DNA gains detected by array CGH in the invasive samples were
located at the RBPI-RBP2 (3q21 I-q22) genes, the sub-telomeric clone C84CI 1/T3 (5ptel), D5S23
(5pl 5.2) and the DAB2 gene (5p 13) in 58.8% of the samples. The most common losses were found
at the FHIT gene (3p I4.2) in 47% of the samples, followed by deletions at D8S504 (8p23.3), CTDPI-
SHGC- 145820 (18qtel), KIT (4q I I -q 12), DI S427-FAFI (l p32.3), D9S325 (9qtel), EIF4E eukaryoticc
translation initiation factor 4E, 4q24), RBI (13q14), and DXS7132 (Xq 12) present in 5/17 (29.4%)
of the samples.
Conclusion: Our results confirm the presence of a specific pattern of chromosomal imbalances
in cervical carcinoma and define specific targets that are suffering DNA copy number changes in
this neoplasm.

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Uterine cervix carcinoma (UCC) represents the second
cause of death among the female population worldwide.
The fact that more than 99% of all the cervical invasive
tumors are positive for infection with high risk human
papillomavirus (HPV) suggests that this is one of the most
important factors for the development of this neoplasm
[1,2]. These viruses can induce cellular transformation by
several mechanisms; the viral oncoproteins E6 and E7 can
interact with cellular proteins involved in important cellu-
lar functions, such as tumor suppression, apoptosis, cell
cycle control, genomic instability, transcriptional regula-
tion and immune evasion [3].

The induction of genomic instability by HPV seems to be
particularly important for the establishment and develop-
ment of an invasive tumor [4,5] since this increased
genomic plasticity would generate cellular clones with
enhanced transforming and invasive potential [6].

Metaphase comparative genomic hybridization (mCGH)
has been applied to study different stages of this tumor
[4,7-19], detecting specific patterns of chromosomal
imbalances that arises very early during the development
of cervical carcinoma, suggesting that the gain of chromo-
some 3q is one of the most important genetic alteration
that defines the transition from a pre-malignant lesion to
an invasive carcinoma [4]. Some of these imbalances have
been related to specific clinical behaviors, such as the pres-
ence of lymph node metastases [9]. However, given the
spatial resolution ofmCGH [20], little is known about the
identity of specific genes that might be the targets of
regional chromosomal imbalances. Matrix-based CGH or
array CGH overcomes this problem increasing the sensi-
tivity for the detection of DNA copy number changes at
specific loci, through the use of well defined genomic
DNA fragments whose mapping location is known,
arrayed onto a solid surface [21-23], thereby achieving a
resolution of copy number imbalances up to the single
gene level.

In order to refine the patterns of chromosomal imbal-
ances present in cervical carcinoma, and trying to identify
specific genes that might be targets of copy number
changes in this tumor, we applied microarray CGH on 20
uterine cervix-derived samples (three pre-malignant
lesions, 10 invasive tumors and seven UCC derived cell
lines) to detect DNA copy number changes at the single
gene level.

Cervical tissues
All described procedures have been evaluated and
approved by the local committee of ethics of the Mexican
Institute of Social Security (IMSS), and all samples were

taken after informed consent from the patients. The pre-
malignant lesions and the invasive tumors were collected
by colposcopy-directed biopsies at the Gynecology
Department of the Hospital General de Mexico, Mexico
City. The biopsies were divided in three sections. The cen-
tral part was used for genomic DNA extraction using the
Wizard Genomic kit (Promega, Madison, WI, USA), and
the extremes were fixed with 70% ethanol overnight and
paraffin embedded. Hematoxilin-eosin stained sections
from these biopsies were analyzed in order to confirm the
presence of at least 70% tumoral cells in the samples.

Cell lines
The cell lines included in this study were: CasKi, SiHa,
both positive for HPV16, and HeLa (HPV18) The CaLo
and ViBo cell lines were established from stage IIB inva-
sive tumors, while INBL and RoVa from a stage IVA tumor.
These cells are HPV18 positive and were established from
tumor explants at the laboratory of Cell differentiation
and Cancer of the National University of Mexico [24]. The
chromosomal CGH profiles of CaLo, ViBo, INBL and
RoVa have been published recently [19].

HPV detection and typing
HPV detection was carried out by PCR using the consen-
sus primers MY09 and MY11 for the LI region of the viral
genome. After a 5 min. denaturation at 94 C, 100 ng of
DNA were subjected to 35 amplification cycles with the
following parameters: 94 C for 1 min., 55 C for 2 min.
and 73 C for 3 min., with a final extension step of 7 min.
at 72C. The amplicon was labeled using the Big Dye
sequencing kit and sequenced on an ABI371 sequencer
(Applied Biosystems, Foster City, CA, USA). BLAST http-.
/ sequence comparison
was used in order to define the viral type.

Microarray CGH
Microarray CGH was performed using the GenoSensor
Array 300 system, following the manufacturer's instruc-
tions (ABBOT-Vysis, Downers Grove, IL, USA). Each array
contains 861 spots, representing 287 chromosomal
regions that are commonly altered in human cancer, such
as telomeres, regions involved in microdeletions, onco-
genes, and tumor suppressor genes. Briefly, 100 ng of
genomic DNA were labeled by a random primer reaction
during two hours. Tumor DNA was labeled with Cy3 and
the normal female reference DNA with Cy5. After the
labeling reaction, the probes were digested with DNAse at
15 C for one hr., followed by two ethanol-purifications;
finally the probe size was checked by gel electrophoresis.
The hybridization mixture consisted of 2.5 tl of each of
the differentially labeled DNAs plus 25 tl of hybridiza-
tion buffer provided in the kit. This mixture was dena-
tured at 800C for 10 min. at 800C, followed by
incubation at 37 C for one hr. Five tl of this probe were

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BMC Cancer 2005, 5:77

applied onto the spotted area of the array under a cover-
slip and hybridized in a humid chamber containing 50%
formamide (FA)/2XSSC at 37C for 72 hrs. After hybridi-
zation, the arrays were washed 3X in 50%FA/2XSSC at
40 C for 10 min/wash, followed by four 5 min. washes in
1XSSC at room temperature. Finally, the arrays were
briefly rinsed in distilled water, mounted and counter-
stained in the dark for 45 min. with DAPI (4,6-diamino-

Image capture and analysis
Array analysis was performed immediately after counter-
staining using the GenoSensor scanner and software. This
system generates a genomicc analysis report", indicating
which chromosome regions in the array are involved in
copy number changes, as well as a spreadsheet containing
the data generated by a single experiment. In order to
compare all the experiments, a database was created using
the normalized, bias corrected, tumor/normal ratio value
of each experiment [see additional file 1]. Since each spot
in the array is present in triplicates, the median of the
three spots of each probe in the array was calculated and
its log2 transformed value was used for further analysis. A
fluorescence ratio >1.25 (log2 = 0.32) was considered as a
DNA gain, while DNA losses were scored when the ratio
was <0.75 (log2 = -0.41). A ratio >2 (log2 = 1) was consid-
ered as a high copy number amplification.

HPV detection and typing
One of the premalignant lesions was positive for HPV16
infection; one for HPV31 and the other for HPV58. In the
invasive tumors, seven were positive for HPV16 and in
three cases, we were not able to detect HPV sequences
with the oligonucleotides we used for PCR amplification.
As expected, CasKi and SiHa were positive for HPV16,
while HeLa, INBL, CaLo, ViVo and RoVa were positive for

Microarray comparative genomic hybridization
All our samples, except one pre-malignant lesion, pre-
sented alterations, ranking from 1/287 (alterations/total
targets in the array) in a pre-malignant lesion to 175/
287alterations in the cell line RoVa. We found almost
twice the number of DNA gains than DNA losses (571 vs.
298) and the average number of copy number alterations
(ANCA=total number of alterations in the sample collec-
tive/total number of cases) was 43.45 per case.

One of the pre-malignant lesions did not show any alter-
ation, while amplifications at MSH2-KCNK12 (2p22.3-
2p22.1), TCL1A (14q32.1) and TOP1 (20q12) were
found in a second pre-malignant lesion and DMBT1
(10q25.3), ERBB2 (17ql2), and 4qTEL11 (4qtel) amplifi-
cation was found in the third sample from this group.

In the invasive tumors and the cell lines, the most com-
mon amplifications (58.8% of the samples) were found at
the clones RBP1-RBP2 (retinol binding protein 1 and 2,
3q21-q22), present as a high copy number amplification
(HCNA) in two samples; DAB2 (disabled homolog 2,
mitogen-responsive phosphoprotein (Drosophila), 5pl3;
C84C11/T3 (5ptel) and D5S23 (5pl5.2), followed by
gains of Tp63 (3q27-q29, 2 HCNA); EGFR (Epidermal
growth factor receptor, 7pl2.3-p12.1, 4 HCNA) and
D5S2064 (5pl5.2), in 52.9% of the invasive samples and
amplification of INS (Insulin, llptel) in 47% of the

The most common deletion was found at the clone corre-
sponding to the FHIT (Fragile histidine triad) gene
(3p14.2), present in 47% of the invasive samples, fol-
lowed by deletions at D8S504 (8p23.3), CTDP1-SHGC-
145820 (18qtel), KIT (4q11-q12), D1S427-FAF1
(lp32.3), D9S325 (9qtel), EIF4E eukaryoticc translation
initiation factor 4E, 4q24), RB1 (13q14), and DXS7132
(Xql2) present in 29.4% of the samples. A histogram of
the DNA copy number alterations detected in the tumor
samples analyzed by array CGH is presented in figure 1.
The results of these experiments can be accessed through
the Progenetix CGH database[25].

Previous studies using chromosomal CGH have delimited
a specific pattern of chromosomal imbalances in cervical
carcinoma. However, there is little knowledge regarding
the identity of particular genes that might be the targets
for these copy number changes, making microarray CGH
an attractive method in order to define these particular
gene targets.

There was concordance between the alterations detected
by microarray CGH and the pattern of chromosomal
alterations already described by chromosomal CGH.
Although the array that we used did not cover the entire
genome, we were able to detect alterations at particular
genes and genetic markers that might be related to the
transformation process in the cervical epithelium.

It is important to notice that the limited number of pre-
malignant lesions analyzed did not allowed us to detect
any particular region that might be related with this stage
of the disease. However, an interesting candidate gene
amplified in one pre-malignant sample and in 5 invasive
tumors was MSH2-KCNK12 (2p22.3-2p22.1). This gene is
the human homolog of the E. coli mismatch repair gene
mutS, and has been found mutated in hereditary nonpoly-
posis colon cancer. Higher MSH2 expression has been
described in cervical intraepithelial neoplasias and inva-
sive cervical carcinomas than in non-neoplastic cervical

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BMC Cancer 2005, 5:77

. tr4: '14




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7E L

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h t54N'-24


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Figure I

Histogram showing the incidence of alterations in the invasive tumors and cell lines in each of the targets printed on the CGH

array. The incidence value is shown at the bottom of the figure; negative values indicate DNA losses, positive values DNA


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BMC Cancer 2005, 5:77

lesions. An altered expression of this gene has also been
proposed as an important event during cervical carcino-
genesis [26,27]. Interestingly, the invasive samples show-
ing MSH2 amplification presented with a high number of
alterations (>40), suggesting a possible connection
between increased copy number of this gene and chromo-
somal instability in invasive cervical carcinomas.

One of the most important genetic events during cervical
carcinoma progression is the gain of 3q. This alteration
has been detected in early stages of cervical transforma-
tion and in cooperation with other imbalances, seems to
play an important role in tumor development. Microarray
analysis identified among the most prevalent alterations
in cervical tumors and cell lines the amplification of the
RBP1- RBP2 (Retinol binding protein 1 and 2, 58.8%) and
Tp63 (52.9% of the samples) genes, located at 3q21-q22
and 3q27-q29, respectively.

Tp63 is a homolog of the p53 tumor suppressor gene. Its
protein is inactivated by the E6 HPV oncoprotein and
plays a primordial role in the development of squamo-
stratified epithelia. Tp63 is highly expressed in the basal
stratum of these epithelia with diminished expression in
the differentiated strata, suggesting that the presence of
this protein preserves the self-renewal capacity of the epi-
thelial stem cells after an asymmetric division, in which
one of the daughter cell must conserve its epithelial stem-
cell properties and the other daughter cell is committed to
the differentiation process [28]. This protein has been
detected in human cervical tissues in the basal and para-
basal layers of the ectocervical squamous epithelium, and
it is not present in the differentiated layers. In premalig-
nant lesions and invasive squamous tumors, a strong p63
expression has been described [29,30]. The presence of
this protein has also been associated with poor survival
and locoregional failure after radiation and chemotherapy
[31]. Expression of the epidermal growth factor receptor
(EGFR, 7pl2.3-p12.1), which was found amplified in
52.9 % of the invasive tumors that we analyzed, was found
to be a prognostic predictor of extrapelvic failure after
treatment, and the expression of both molecules was
found to be a very good risk factor measurement in
patients with stage IIB squamous cell carcinoma of the
uterine cervix, who had received radiotherapy and concur-
rent chemotherapy [31].

DAB2 on 5pl13 was amplified in 58.8% of the invasive
cases. The DAB2 gene has been identified as a potent
tumor suppressor gene in prostate and ovarian carcinoma
[32], and loss of expression of this gene has been associ-
ated with the transition of ovarian epithelial cells to pre-
malignant states [33]. DAB2 has been implicated in cell
positioning control and seems to mediate the require-
ment for basement membrane attachment of epithelial

cells [34]. To our knowledge, there are no available
reports analyzing the expression of this gene in the uterine
cervix or in cervical carcinoma. The amplification of this
gene seems contrary to its putative role as a tumor sup-
pressor gene. A possible explanation for this observation
might be the loss of one allele followed by the amplifica-
tion of the remaining chromosome. However, since array
CGH does not offer any type of information regarding the
parental origin of the amplified chromosome, this situa-
tion can not be confirmed.

Detection of the TERC gene amplification has been
recently proposed as a potential marker for the evaluation
of cervical carcinoma progression [35]; however, we
detected amplification of the clone representing this gene
in less than 10% of the samples analyzed by CGH arrays.
FISH analysis, as described by Heselmayer et al., detected
a higher prevalence of nuclei with a diploid pattern than
those with a tetraploid pattern, even in the high grade
lesions. Furthermore, the percentage of nuclei with more
than 2 copies of 3q, including the tetraploid cells ranged
between 3.3 to 50% of the CIN3 (cervical intraepithelial
neoplasia grade 3). Dellas et al., [91 used in situ hybridi-
zation to analyze the prevalence of 3q amplifications in
cervical cancer tissue arrays, detecting low level amplifica-
tions in most of the tumors studied. These results suggest
that these low copy number gains might not be ade-
quately detected by chromosome or even array CGH, due
to the contamination with normal cells and/or the pres-
ence of a high number of diploid or tetraploid cells in the

Regarding DNA losses, the FHIT (fragile histidine triad,
3pl4.2) gene suffered losses in 47% of the cases. Aberrant
expression of this gene has been well documented in cer-
vical carcinoma and has been related to lymph node
metastasis, parametrial invasion, and vaginal involve-
ment in invasive tumors [36]. An association between
FHIT gene abnormalities and infection with particular
HPV types has been suggested, since 87% of the cases with
absent FHIT expression were positive for HPV16 infection
[37]. Furthermore, abnormal expression of this gene has
been found in significantly younger patients than those
with normal expression, suggesting that abnormalities in
the regulation of this gene might be accelerating carcino-
genesis in cooperation with HPV [37]. These observations
might be related to the preferential integration of HPV
into fragile sites, particularly FRA3B, where FHIT is
located [38].

In conclusion, microarray CGH allowed the detection of
particular genes located in regions with common DNA
copy number changes in cervical carcinoma. Further stud-
ies using CGH arrays with a higher resolution and the

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Table I: Clinical stage and HPV status of the analyzed samples.


Invasive tumors




Cell lines





possibility to combine LOH with copy number changes,
might be useful for the detection of gene specific targets
that are relevant for the genesis and progression of cervical

CGH: Comparative Genomic Hybridization, UCC: Uter-
ine cervix carcinoma, HPV: Human papilloma virus,
DAPI: 4,6-diamino-2-phenylindole.

Competing interests
The authors) declare that they do not have any compet-
ing interests.

Authors' contributions
AH: Performed the microarray CGH experiments, data
analysis and paper writing; MB: Help with data submis-
sion to the Progenetix database, data analysis; IP: pro-
vided training for the experiments, CGH data analysis; PP:
tissue processing; GV: HPV typing; DH: sample collection;
JG: provided access to the samples; ML: access to samples,
histopathological analysis; RL: sample collection; CP:
DNA extraction; JG: Help with data analysis; KV: DNA
extraction; BA: HPV typing; MS: project coordinator.

Additional material

Additional File 1
Raw data from the CGH microarray experiments. This is text file contain-
ing the raw data from the microarrays, the first column denotes the iden-
tification of the clone, column 2 represents the cytogenetic position of the
clone, sample data begins in column 3.
Click here for file
2407-5-77-S1 txt]

This work was partially funded through the 7114 and 34686 grants from the
Mexican Council of Science and technology (CONACyT) and the Mexican
Institute for Social Security (IMSS-FOFOI FP- 2001-2003). AH, GVO, CP,
RL were recipients of scholarships from the CONACyT, IMSS and DGEP-
UNAM. We would like to thank Abbott-Vysis for providing the CGH array
system for this analysis. This work was submitted in partial fulfillment of the
requirements for the Ph.D. degree of HA at the Ph.D. in Biomedical Sci-
ences, National University of Mexico.

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