Chromosomal damage as prognosis marker in cervical carcinogenesis
Cancer of the uterine cervix is the third most common cancer in women worldwide and the most common cancer among Mexican and Latin American women. Risk factors that have been associated with the development of cervical intraepithelial neoplasia suggest that Human Papillomavirus (HPV) types 16, 18, 3...
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Cortés-Gutiérrez, E. Dávila-Rodríguez, M. Cerda-Flores, R.M. 2017-11-29T19:55:50Z 2017-11-29T19:55:50Z 2014 Chromosomal damage as prognosis marker in cervical carcinogenesis / E. Cortés-Gutiérrez, M. Dávila-Rodríguez, R.M. Cerda-Flores // Цитология и генетика. — 2014. — Т. 48, № 3. — С. 54-63. — Бібліогр.: 86 назв. — англ. 0564-3783 DOI: 10.3103/S0095452714030049 https://nasplib.isofts.kiev.ua/handle/123456789/126654 Cancer of the uterine cervix is the third most common cancer in women worldwide and the most common cancer among Mexican and Latin American women. Risk factors that have been associated with the development of cervical intraepithelial neoplasia suggest that Human Papillomavirus (HPV) types 16, 18, 31, and 33 entail a high risk of developing a malignancy of this type. The accumulation of genetic alterations allows the growth of neoplastic cells; chromosomal instability is an event that occurs in the precancerous stages. The candidate cancer risk biomarkers include cytogenetic endpoints, such as chromosomal aberrations, sister chromatid exchange, micronuclei, and the outcomes of comet assay and DNA breakage detection-fluorescence in situ hybridization. The patterns identified in these cytogenetic studies indicate that chromosomal instability is a transient and chromosomally unstable intermediate in the development of cervical lesions. In this context, the mechanisms that may underlie the progressive increase in genetic instability in these patients seem to be related directly to HPV infection. The studies discussed in this paper show that chromosomal instability may serve as a biomarker by predicting the progression of cervical intraepithelial neoplasia. Nevertheless, these results should be validated in larger, prospective studies. Рак шейки матки является третьим по распространенности в мире типов рака у женщин и наиболее часто встречающимся у женщин Мексики и Латинской Америки. Факторы риска, связанные с развитием интраэпителиальной цервикальной неоплазии, предполагают, что папилломавирус человека (HPV) типов 16, 18, 31 и 33 влечет за собой высокий риск развития опухолей этого типа. Накопление генетических изменений делает возможным рост опухолевых клеток, хромосомная нестабильность является событием, которое предшествует предраковым стадиям. Возможные биомаркеры риска опухоли включают цитогенетические критерии, такие как хромосомные аберрации, обмен сестринских хроматид, микроядра, и заканчиваются Comet-анализом и детекцией поломок ДНК с помощью флюоресцентной гибридизации in situ. Образцы, идентифицированные в таких цитогенетических исследованиях, показывают, что хромосомная нестабильность является транзиентным промежуточным звеном в развитии цервикальных нарушений. В этой связи механизмы, которые могут лежать в основе прогрессирующей генетической нестабильности у таких пациентов, кажутся непосредственно связанными с инфекцией HPV. Настоящее исследование показывает, что хромосомная нестабильность может служить биомаркером для предсказания развития интраэпителиальной цервикальной неоплазии, тем не менее эти результаты должны быть оценены в более масштабных исследованиях. en Інститут клітинної біології та генетичної інженерії НАН України Цитология и генетика Обзорные статьи Chromosomal damage as prognosis marker in cervical carcinogenesis Хромосомные повреждения как прогнозные маркеры карценогенеза шейки матки Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Chromosomal damage as prognosis marker in cervical carcinogenesis |
| spellingShingle |
Chromosomal damage as prognosis marker in cervical carcinogenesis Cortés-Gutiérrez, E. Dávila-Rodríguez, M. Cerda-Flores, R.M. Обзорные статьи |
| title_short |
Chromosomal damage as prognosis marker in cervical carcinogenesis |
| title_full |
Chromosomal damage as prognosis marker in cervical carcinogenesis |
| title_fullStr |
Chromosomal damage as prognosis marker in cervical carcinogenesis |
| title_full_unstemmed |
Chromosomal damage as prognosis marker in cervical carcinogenesis |
| title_sort |
chromosomal damage as prognosis marker in cervical carcinogenesis |
| author |
Cortés-Gutiérrez, E. Dávila-Rodríguez, M. Cerda-Flores, R.M. |
| author_facet |
Cortés-Gutiérrez, E. Dávila-Rodríguez, M. Cerda-Flores, R.M. |
| topic |
Обзорные статьи |
| topic_facet |
Обзорные статьи |
| publishDate |
2014 |
| language |
English |
| container_title |
Цитология и генетика |
| publisher |
Інститут клітинної біології та генетичної інженерії НАН України |
| format |
Article |
| title_alt |
Хромосомные повреждения как прогнозные маркеры карценогенеза шейки матки |
| description |
Cancer of the uterine cervix is the third most common cancer in women worldwide and the most common cancer among Mexican and Latin American women. Risk factors that have been associated with the development of cervical intraepithelial neoplasia suggest that Human Papillomavirus (HPV) types 16, 18, 31, and 33 entail a high risk of developing a malignancy of this type. The accumulation of genetic alterations allows the growth of neoplastic cells; chromosomal instability is an event that occurs in the precancerous stages. The candidate cancer risk biomarkers include cytogenetic endpoints, such as chromosomal aberrations, sister chromatid exchange, micronuclei, and the outcomes of comet assay and DNA breakage detection-fluorescence in situ hybridization. The patterns identified in these cytogenetic studies indicate that chromosomal instability is a transient and chromosomally unstable intermediate in the development of cervical lesions. In this context, the mechanisms that may underlie the progressive increase in genetic instability in these patients seem to be related directly to HPV infection. The studies discussed in this paper show that chromosomal instability may serve as a biomarker by predicting the progression of cervical intraepithelial neoplasia. Nevertheless, these results should be validated in larger, prospective studies.
Рак шейки матки является третьим по распространенности в мире типов рака у женщин и наиболее часто встречающимся у женщин Мексики и Латинской Америки. Факторы риска, связанные с развитием интраэпителиальной цервикальной неоплазии, предполагают, что папилломавирус человека (HPV) типов 16, 18, 31 и 33 влечет за собой высокий риск развития опухолей этого типа. Накопление генетических изменений делает возможным рост опухолевых клеток, хромосомная нестабильность является событием, которое предшествует предраковым стадиям. Возможные биомаркеры риска опухоли включают цитогенетические критерии, такие как хромосомные аберрации, обмен сестринских хроматид, микроядра, и заканчиваются Comet-анализом и детекцией поломок ДНК с помощью флюоресцентной гибридизации in situ. Образцы, идентифицированные в таких цитогенетических исследованиях, показывают, что хромосомная нестабильность является транзиентным промежуточным звеном в развитии цервикальных нарушений. В этой связи механизмы, которые могут лежать в основе прогрессирующей генетической нестабильности у таких пациентов, кажутся непосредственно связанными с инфекцией HPV. Настоящее исследование показывает, что хромосомная нестабильность может служить биомаркером для предсказания развития интраэпителиальной цервикальной неоплазии, тем не менее эти результаты должны быть оценены в более масштабных исследованиях.
|
| issn |
0564-3783 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/126654 |
| citation_txt |
Chromosomal damage as prognosis marker in cervical carcinogenesis / E. Cortés-Gutiérrez, M. Dávila-Rodríguez, R.M. Cerda-Flores // Цитология и генетика. — 2014. — Т. 48, № 3. — С. 54-63. — Бібліогр.: 86 назв. — англ. |
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| fulltext |
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 354
Cancer of the uterine cervix is the third most common cancer
in women worldwide and the most common cancer among
Mexican and Latin American women. Risk factors that have
been associated with the development of cervical intraepi-
thelial neoplasia suggest that Human Papillomavirus (HPV)
types 16, 18, 31, and 33 entail a high risk of developing
a malignancy of this type. The accumulation of genetic
alterations allows the growth of neoplastic cells; chromo-
somal instability is an event that occurs in the precancer-
ous stages. The candidate cancer risk biomarkers include
cytogenetic endpoints, such as chromo somal aberrations,
sister chromatid exchange, micronuclei, and the outcomes
of comet assay and DNA breakage detection-fluorescence
in situ hybridization. The patterns identified in these cy-
togenetic studies indicate that chromosomal instability is a
transient and chromosomally unstable intermediate in the
development of cervical lesions. In this context, the mecha-
nisms that may underlie the progressive increase in genetic
instability in these patients seem to be related directly to
HPV infection. The studies discussed in this paper show that
chromosomal instability may serve as a biomarker by pre-
dicting the progression of cervical intraepithelial neoplasia.
Nevertheless, these results should be validated in larger,
prospective studies.
Key words: chromosomal instability, cervical cancer, bio-
marker.
Introduction. Cancer of the uterine cervix is the third
most common cancer in women worldwide and the most
common cancer among Mexican and Latin American
women [1]. Precancerous lesions of the cervix, com-
monly designated «dysplasia», present a complex problem
of progression and regression because of their biological
behavior. Dysplastic lesions of the cervix are morpho-
logically classified into the following stages: mild cervical
intraepithelial neoplasia (CIN 1), moderate (CIN 2),
and severe (CIN 3). According to the Bethesda System
for reporting cervical/vaginal cytological diagnoses [2],
the lesions are classified as squamous intraepithelial le-
sions (SIL), which are either low-grade (LG-SIL), corre-
sponding to human papillomavirus (HPV) infection and
CIN 1, or high-grade (HG-SIL), corresponding to CIN
2 and CIN 3. Risk factors that have been associated with
the development of CIN suggest that HPV types 16, 18,
31, and 33 entail a high risk of developing a malignancy
of this type.
Considering that carcinogenesis is a complex stepwise
process and that the accumulation of genetic alterations
allows the growth of neoplastic cells, chromosomal insta-
bility is an event that occurs in the precancerous stages.
The development and validation of biomarkers that
can anticipate the clinical diagnosis and suggest cancer
prevention interventions in populations at risk are among
the most promising strategies for cancer prevention. The
candidate cancer risk biomarkers include cytogenetic
endpoints, such as chromosomal aberrations [3], sister
chromatid exchange (SCE) [4], micronuclei (MN) [5],
and the outcomes of comet assay [6, 7] and DNA break-
age detection-fluorescence in situ hybridization (DBD-
FISH) [8].
Chromosomal aberrations. Numerical changes in spe-
cific chromosomes (aneusomy) can involve a gain (e.g.,
trisomy) or loss (e.g., monosomy) with respect to the
normal condition (disomy). A misdivision gives rise to
an amplification of the whole genome (polyploidy) [9]
and structural anomalies (e.g., deletions, translocations,
and isochromosomes).
Numerical or structural chromosomal anomalies, or
a combination of the two, are related events that oc-
cur during the early stages and progression of cervical
carcinogenesis [10, 11]. The numerical and/or structural
deviations of some chromosomes (i.e., monosomy and
polysomy of chromosomes 1, 3, and X) are routinely
used as positive genetic biomarkers in the diagnosis of
cervical cancer and prediction of disease progression
ОБЗОРНЫЕ СТАТЬИ
E.I. CORTÉS-GUTIÉRREZ 1, M.I. DÁVILA-RODRÍGUEZ 1, R.M. CERDA-FLORES 2
1 Department of Genetics. Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social. Monterrey, México
2 Facultad de Enfermería, Universidad Autónoma de Nuevo León. Monterrey, México
E-mail: elvacortes@cibinmty.net
CHROMOSOMAL DAMAGE AS PROGNOSIS MARKER
IN CERVICAL CARCINOGENESIS
© E.I. CORTÉS-GUTIÉRREZ, M.I. DÁVILA-RODRÍGUEZ,
R.M. CERDA-FLORES, 2014
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 3 55
Chromosomal damage as prognosis marker in cervical carcinogenesis
[12–14]. Structural anomalies and numerical anoma-
lies (aneusomy) of chromosome 1 have been described
as the most frequent karyotypic changes in cervical
cancer. It is possible that one or more of the tumor
suppressor genes (PAX7, FBG3, ARH1, NEK2, RGL,
and ARCH) located on chromosome 1 are involved
in the development or progression of cervical cancer
[15]. Aneusomy in chromosomes 1, 7, 8, 11, 17, and X
have also been reported based on fluorescence in situ
hybridization (FISH) and interphase cytogenetic find-
ings [16–21]. In addition to the primary structural and
numerical aberrations that are responsible for the initia-
tion of carcinogenesis, new or secondary chromosomal
abnormalities in the karyotype (e.g., stickiness, pulveri-
zation, chromatin extraction, chromatid gap, chromatid
constriction, isochromatid break, endomitosis, and ring
chromosomes) of a patient may appear as signaling a
change usually to a more aggressive disorder [22]. An
increased frequency of spontaneous chromosome aber-
rations was observed among patients with cervical pre-
cancerous lesions [23].
Sister chromatid exchange (SCE) is a reciprocal ex-
change between sister chromatids. SCEs are generally
visualized by exposing cells (in vitro or in vivo) to 5-bro-
modeoxyuridine for two cell cycles and allowing subse-
quent differential staining of sister chromatids. Exchanges
are detected at switches in stained regions between sister
chromatids. The molecular mechanisms underlying SCE
are not known fully, but they occur after exposure to vari-
ous genotoxic agents and are believed to indicate DNA
damage [24]. The SCE phenomenon is widely used as a
reliable indicator of chromosome (DNA) instability [25]
and has been suggested as a preclinical marker for the
breast cancer gene [26].
The performance of the SCE test to evaluate the
genomic damage in patients with cervical carcinoma
is more practical in cultured lymphocytes than in solid
tumors because of the difficulties in obtaining surgical
specimens. The use of lymphocytes is based on the as-
sumption that there should be an association between the
extent of chromosomal damage in lymphocytes and in
tumor cells [27]. A significant increase of the number of
SCEs was reported previously in patients with cancer of
the uterine cervix [28–33]; however, other studies have
not shown significant differences [34]. These results sug-
gest that the frequency of SCE is higher in patients with
cervical carcinoma than in controls, in studies with a
statistical power of 0.80. It could be assumed that these
patients show a certain amount of chromosome instabil-
ity. This finding is in agreement with those reported by
other authors [27–33]. High values of statistical power
(0.99 to 1.00) were achieved in all references listed in Ta-
ble I (computed by the authors using the Stata software)
[34]. Conversely, Adhvaryu et al. [35] did not find that
SCE was a significant marker in cervical cancer patients,
possibly because of the low statistical power (1 – =
= 0.57) in the experimental design.
SCEs are associated with cervical cancer. The incon-
sistency of the results of other studies might be attribut-
able to the low statistical power in the experimental de-
sign of those studies; however, the usefulness of increased
SCE levels as a preclinical marker to identify women at
a high risk of developing cancer needs to be ascertained
in follow-up studies of precancerous lesions with high
levels of SCE.
Micronuclei. A micronucleus (MN) is formed by
chromosomes or chromosome segments that fail to be in-
corporated in the nuclei during cell division. MN can be
generated through various processes, i.e., chromosomal
damage and chromosome loss (aneuploidy). During the
past few decades, MN has generally been used as a bio-
marker of chromosomal damage, genome instability, and
cancer risk, integrating acquired mutations and genetic
susceptibility toward mutations [36]. Therefore, increased
MN frequency is expected in preneoplastic conditions,
which has been demonstrated [37–41]. The role of MN
in various steps of carcinogenesis has been substantiated
by investigators and it has clearly been shown that the
level of baseline chromosome damage is much higher in
untreated cancer patients than in cancer-free controls
[42]. Therefore, MN scoring could be used as a biomarker
to identify various preneoplastic conditions much earlier
Table 1. Sister chromatid exchange frequencies
in women with carcinoma of the uterine cervix
in studies with various statistical powers [34]
Indications. N – number of patients studied; P < 0.05;
C – control, P – patients, X ± SD – average ± standard
deviation.
Author N
SCE
(X ± SD)
Statis-
tical
power
Mitra et al. [28]
Murty et al. [29]
Adhvaryu et al.
[35]
Yokota et al.
[32]
Lukovic et al.
[30]
Dhillon et al.
[31]
Capalash et al.
[33]
Cortés-Gutiérrez
et al. [34]
13
11
46
43
13
13
35
18
21
19
14
20
30
15
28
28
10.05 ± 2.35 (C)*
6.95 ± 1.53 (P)
10.15 ± 2.49 (C)*
7.55 ± 2.24 (P)
9.68 ± 0.97 (C)
8.91 ± 1.15 (P)
10.00 ± 1.80 (C)*
7.60 ± 0.80 (P)
8.92 ± 1.47 (C)*
6.94 ± 1.00 (P)
9.44 ± 1.27 (C)*
6.09 ± 1.07 (P)
7.18 ± 1.23 (C)*
4.68 ± 1.82 (P)
7.80 ± 1.05 (C)*
6.98 ± 1.13 (P)
0.99
1.00
0.57
1.00
0.99
1.00
1.00
0.80
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 356
E.I. Cortés-Gutiérrez, M.I. Dávila-Rodríguez, R.M. Cerda-Flores
than the manifestations of clinical features and might
specifically be exploited in the screening of high-risk
populations for a specific type of cancer [43, 44]. For
these reasons, the prevalence of MN in epithelial cells
has been considered a potential tissue-specific indicator
of cancer risk [43, 44]. Occasional studies have shown
increased MN frequency in invasive cervical cancer and
researchers have suggested that the MN score in exfo-
liated cervical cells may be an additional criterion for
establishing cervical cancer risk [37, 45]. However, there
are only a limited number of studies on MN scoring
for the assessment of cervical cancer risk [37, 45] and
on MN scoring in cervical preneoplastic and neoplastic
conditions [11, 46]. Accordingly, an analysis of MN in
Table 2. Micronuclei frequencies in women with cervical neoplasic lesions as reported in the literature
Author N Diagnostic
No. cells
studied
MN Conclusion
Samanta et al.
[55]
Samanta et al.
[54]
Aires et al.
[53]
Campos et al.
[52]
Leal-Garza et
al. [47]
Cerqueira et
al. [51]
Chakrabarti et
al. [50]
30
23
40
40
30
38
22
10
12
10
27
35
10
25
16
15
10
10
10
10
45
113
24
14
4
48
33
32
6
ASCUS
CIN
Control
Inflammatory
ASC-US
LG-SIL
HG-SIL
Control
Inflammatory
LG-SIL
HG-SIL
Control
Inflammatory
CIN 1
CIN 2
CIN 3
Control
LG-SIL
HG-SIL
Invasive
Control
Inflammatory
LG-SIL
HG-SIL
Cancer
Control
Inflammatory
LG-SIL
HG-SIL
30,000
23,000
40,000
40,000
30,000
38,000
20,000
24,000
20,000
54.000
35,000
10,000
25,000
16,000
15,000
10,000
10,000
10,000
10,000
145,388
345,235
65,171
43,086
10589
24,000
16,500
16,00
300
2.87 ± 2.21*
8.35 ± 6.45
1.05 ± 1.59*
0.42 ± 0.71
2.87 ± 2.21
4.74 ± 5.62
19.73 ± 17.18
3**
7
8
61
1.3 ± 1.4*
7.2 ± 9.6
4.3 ± 4.3
10.6 ± 5.3
22.7 ± 11.9
3.05*
7.1
9.7
14.0
27**
166
38
47
14
1.91**
3.80
3.5
4.0
MN score may be helpful in identifying the
true CIN cases that are mislabeled as ASCUS
on cervical smear. In future, MN score can
be used as an additional biomarker in cervical
cancer screening
MN scoring on the epithelial cells of cervix
could be used as a biomarker in cancer scre-
ening. This is an easy, simple, reliable, repro-
ducible and objective test and can be done on
routinely stained smears
MN test and Papanicolaou test may be both
utilized for screening women who are at risk
of developing cervical cancer
The prevalence of MN in exfoliated uterine
cervical cells was greater in the patients with
one or more risk factors for cancer than in the
patients without risk factors
A positive linear trend between the MN fre-
quency and increased cervical cancer risk.
After being validated, MN could be used as
screening and annual PAP test and as part of
cancer staging
MN testing would be helpful in monitoring
smokers with cervical intraepithelial lesions
The rising frequency of MN in exfoliated ce-
rvical cells reflects a sustained mutagenesis in
cervical epithelium
Indications. N – number of cells studied; ASCUS – Atypical Cells of Undetermined Significance; CIN – Cervical
Intraepithelial Neoplasia; LG-SIL – low-grade squamous intraepithelial lesion; HG-SIL – high-grade squamous
intraepithelial lesion. * Frequencies of MN/1000 cells. ** Frequencies of MN/100 cells.
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 3 57
Chromosomal damage as prognosis marker in cervical carcinogenesis
peripheral blood lymphocytes (PBLs) revealed the pres-
ence of a correlation between MN frequency and grade
of cervical lesions [47] and provided evidence that MN
frequency in PBLs is a predictive biomarker of cancer risk
within a population of healthy subjects [48].
The similarity between the level of chromosome dam-
age in surrogate tissues, such as oral mucosa or PBLs,
and the corresponding damage in cancer-prone tissues
provided the rationale for the use of these biomarkers as
markers of cancer risk [49].
Several studies reported an association between MN
frequency and progression of precursor lesions of cervi-
cal cancer [46, 47, 50–54]; however, the elevation in
the number of these structures in women with HG-SIL
did not reach significance. Samanta et al. [55] reported a
significant increase of MN in CIN lesions compared with
Atypical Cells of Undetermined Significance (Table 2).
A recent study [56] provided strong evidence that MN
frequency assessed in the PBLs of disease-free subjects
is a good predictor of cancer death risk, as evaluated in
a nested case-control study performed 14 years after the
original recruitment.
In particular, the high reliability and low cost of the
micronucleus assay has contributed to the worldwide suc-
cess and adoption of this biomarker for in vitro and in
vivo studies of genome damage in cervical neoplasia [57].
Comet assay. The comet or single-cell gel electropho-
resis (SCGE) assay is now widely accepted as a standard
method for the assessment of DNA damage in individual
cells. It has been used in a wide variety of applications,
including human biomonitoring, genotoxicology, and
ecological monitoring, and as a tool to investigate DNA
damage and repair in various cell types. It can be used to
detect the DNA damage in individual cells and to assess
the presence of double-stranded breaks, single-stranded
breaks, and alkali-labile sites [6].The sensitivity of the
SCGE assay and its ability to measure DNA damage in
individual cells have rendered it a rapid tool that is use-
ful in addressing a wide range of questions in biology,
medicine, and genetic toxicology.
Several studies have shown that basal DNA damage
is increased in PBLs of patients suffering from a variety
of cancers, including head and neck, breast, renal, es-
ophageal, bladder, ovarian, and lung cancer. The authors
of many of these studies, as well as other studies, have
also extracted PBLs from cancer patients (usually prior
to radiotherapy or chemotherapy) and exposed them to
DNA-damaging agents in vitro, to assess whether the
susceptibility to DNA damage and subsequent repair ca-
pacity in these cells are significantly different from those
observed in control samples [6].
A direct association between genomic damage in cer-
vical epithelial cells and the progression of LG-SIL to
HG-SIL has been demonstrated in two studies [58, 59].
The SCGE assay may serve as a novel tool to predict the
fate of cervical dysplasia; however, further standardiza-
tion and experimental validation studies are needed.
DNA breakage detection-fluorescence in situ hybridi-
zation (DBD-FISH). This technique is a new procedure
that allows the cell-by-cell detection and quantification
of DNA breakage in the whole genome or within spe-
cific DNA sequences. Cells embedded in an inert agarose
matrix on a slide are lysed to remove membranes and
proteins and the remaining nucleoids are subjected to
controlled denaturation with an alkali. The alkali trans-
forms DNA breaks into restricted single-stranded DNA
(ssDNA) motifs, which can be detected via hybridization
with specific or whole-genome fluorescent DNA probes.
As the number of DNA breaks increases in a target region,
so do the amounts of ssDNA produced and probes hy-
bridized, resulting in a more intense FISH signal, which
can be quantified using image analysis systems [60–62].
Moreover, the alkaline treatment may break the sugar-
phosphate backbone at basic sites or at sites with deoxyri-
bose damage, transforming these lesions into DNA breaks
that are also converted into ssDNA. DNA damage levels
may be a consequence of the torsional stress on DNA
loops associated with tight chromatin packing, may vary
among cell types in conventionally conformed genomes
(e.g., sperm and lymphocytes) [63], and may change if
the cell is under stress, such as in the presence of a viral
infection.
DNA damage is a product of external stressors, which
also act on the genome [64]. It may also be a consequence
of the torsional stress on DNA loops that is associated
with tight chromatin packing. Abundant damage has been
found in the chromatin of condensed mitotic chromo-
somes [64]. In addition, the exposure of DNA to con-
stant tension above a critical level leads to its unwinding
[65]. Although the molecular biology and significance of
constitutive DNA damage are not well understood, some
observations support the idea that these genomic regions
escape the normal DNA configuration and may be tran-
sient structural features of cells. Even under homeostatic
cell conditions, the presence of DNA breakage may vary
among cell types [63]. It is noteworthy that the applica-
Table 3. Comparison of the integrated density (ID) after
fluorescence densitometry, in cervical epithelial cells
of control women, women with LG-SIL, and women
with HG-SIL, as assessed using DBD–FISH [70]
Indications. N – number of cells studied; ID – fluo-
rescence area × fluorescence intensity; a Different to cont-
rol (p = 0.0001); b Different to LG-SIL (p = 0001);
c Different to HG-SIL (p = 0.0001).
Group N
Number of
studied cells
ID (X ± SD)
Control
LG-SIL
HG-SIL
10
10
10
500
500
500
38 E7 ± 70 E7 b,c
926 E7 ± 1926 E7 a,c
4339 E7 ± 3161 E7 a,b
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 358
E.I. Cortés-Gutiérrez, M.I. Dávila-Rodríguez, R.M. Cerda-Flores
tion of a whole-genome probe to other somatic cells does
not yield a homogeneous background DBD-FISH signal,
as certain chromatin regions are more strongly labeled. In
human leukocytes, the DBD-FISH areas with a more in-
tense background visualized using a whole-genome probe
corresponded to areas containing 5 bp satellite DNA
sequences [66]. In mouse splenocytes, the background
areas corresponded to repetitive DNA satellite sequences
located in pericentromeric regions [67], whereas in Chi-
nese hamster cells, they corresponded to pericentromeric
interstitial telomeric-like DNA sequences [68]. Their
presence is not limited to mammalian species, as they
have also been found in insects [69].
Cortés-Gutiérrez et al. [70] reported that LG-SIL
patients exhibited a hybridization signal that was 20 times
greater than the signal observed in control individuals,
which reflected the basal level of DNA damage detected,
and that HG-SIL patients exhibited a hybridization signal
that was 100 times greater than the basal control signal
(Table 3).
The DBD-FISH technique is easily applicable to cer-
vical scrapings and provides prompt results that are easy
to interpret; however, these results need to be validated
in larger prospective studies.
Genomic instability in cervical cancer. The possible
mechanisms that may explain the progressive increase of
DNA damage in patients with cervical neoplasia include:
1) metabolic stress due to tumor growth; 2) a «clasto-
genic» product released by tumor cells; 3) micronutrient
deficiencies, such as folate and vitamin B12 deficiencies
[71, 72], and HPV infection. HPV DNA can be detected
in 95–100 % of cervical cancer specimens and it has been
called a «necessary cause» of cervical cancer [4, 73]. Alva-
rez-Rosero et al. [74] observed a correlation between the
presence of high-risk HPV infection in cervical cells and
the induction of genomic instability (chromosomal aber-
rations) in lymphocytes. Similarly, Cortés-Gutiérrez et al.
[75] reported that women with HPV infection had a higher
MN frequency in cervical cells. A chromosomal profile of
high-grade cervical intraepithelial neoplasia was related to
duration of preceding high-risk HPV infection [76].
Mechanistically, chromosome breakage in HPV-on-
coprotein-expressing cells probably increases the cellular
susceptibility to DNA damage or the defective repair
Table 4. Classic cytogenetic tests as candidate biomarkers of cervical cancer risk
Cytogenetic
test
Sample (tissue) Advantages Limitations Validations
Chromosomal
abnormalities
SCE
MN
Comet assay
Lymphocytes
Lymphocytes
Lymphocytes
Cervical epithelium
Lymphocytes
Cervical epithelium
Indicator chromosomal in-
stability
Sensible indicator chromo-
somal instability
Easy, simple, reliable, repro-
ducible, objective, and low
cost
No required cellular culture
Easy, simple, reliable, repro-
ducible, objective, and low
cost
Sensitive, rapid and versatile
Sensitive, rapid and versatile.
No required cellular culture
Required cellular cul-
ture
Laborious results in-
terpretation
Required cellular cul-
ture
Laborious results in-
terpretation
Required cellular cul-
ture
Limited number of stu-
dies in cervical pre-
neoplastic lesions [11,
37, 45, 46]
Required fluorescence
equipment.
Few studied in dys-
plasias [58, 59]
Inconsistency of the re-
sults [10–23]
The inconsistency of the
results [28–34]
Strong evidence of good
predictor of cancer death
risk [56]
Taking into account the
role of possible confoun-
ders and effect modifiers
Consistency of results
Validation and detailed
follow up studies are re-
quired
Experimental validation,
standardization and in-
terpretation are needed
Validation and detailed
follow up studies are re-
quired
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 3 59
Chromosomal damage as prognosis marker in cervical carcinogenesis
of DNA damage as a consequence of reduced p53 or
pRB function [77]. The HPV-16 E7 oncoprotein induces
centrosome abnormalities, thereby disrupting mitotic fi-
delity and increasing the risk for chromosome misseg-
regation and aneuploidy. In addition, expression of the
high-risk HPV E7 oncoprotein stimulates DNA replica-
tion stress, which is a potential source of DNA breakage
and structural chromosomal instability [78, 79]. Unre-
paired, broken DNA can promote gene translocations
or gene amplifications/deletions, which may provide a
growth advantage to cells through gain of oncogenes or
loss of tumor suppressors. This chromosomal instability
may promote the development of cells with numerical
and structural aberrations in chromosomes 1, 3, 5, 11,
and 17 [12, 74, 80], which are critical factor for cervi-
cal carcinoma development and malignant progression
[12, 13].
Several lines of evidence show that expression of
HPV-16 E6 and E7 can independently induce structural
chromosomal instability using the comet assay [77].
The induction of chromosomal instability is an emer-
ging topic in viral tumorigenesis in humans and is as-
sociated with high-risk HPV [81], hepatitis B virus [82],
Kaposi’s sarcoma herpesvirus [83], and human T-cell
leukemia virus type 1 (HTLV-1) [84]. The level of DNA
damage in the genome as a structural feature of the chro-
matin may be unbalanced after exposure of cells to stress,
such as a viral infection. Several studies have reported
changes in chromatin organization during carcinogen-
esis and the subsequent association of distorted DNA-
binding proteins with the nuclear matrix, which may have
a functional role in chromatin organization and gene
regulation [85]. An increase in the incidence of DNA
single breakage (dsb), which is mediated by Ku70 deple-
tion, is associated with HPV-16 episomal loss in cervical
keratinocytes and with a new integration event. Normal
levels of host DNA repair proteins, including Ku70, may
protect against such events by preventing the genera-
tion of host dsb and linearized viruses. Interestingly, the
HPV-16 E7 protein may play a direct role in inducing
integration by interference with the nonhomologous end
joining (NHEJ) pathway. Expression of HPV-16 E7 in
the HPV-negative cervical keratinocyte cell line C33A
resulted in the upregulation of the Ku70-binding pro-
tein [86], which may interfere with normal NHEJ and
increase the frequency of dsb. Despite the well known
function of HPV-16 E7 to induce DNA damage, the
precise source of DNA double strand breaks remains
poorly understood.
Clinical significance. From a clinical perspective, the
presence of chromosomal instability may help distinguish
patients with clinically significant cervical lesions from
those who have insignificant lesions, thus discriminating
lesions (Table 4). The ease and low cost of the detection
of MN may allow its use as a prognostic indicator dur-
ing the planning and validation of programs for cancer
monitoring and prevention. In brief, MN scoring on the
epithelial cells of the cervix could be used as a biomarker
in cancer screening. This is an easy, simple, reliable, re-
producible, and objective test that can be performed on
routine stained smears.
Further investigations are required to confirm and
validate the results of chromosomal abnormalities, SCE,
and comet assay. Moreover, the results of DBD-FISH
are preliminary.
Conclusions and future directions. Cervical cancer is
the corollary of a long process that has its onset in LG-
SIL and HG-SIL precursor lesions. Vaccination against
HPV infection and periodical Papanicolaou cervical cy-
tological screening are effective measures for preventing
cervical cancer. Despite the effectiveness of Papanicolaou
examinations, the detection of chromosomal instability
as an early biomarker of cervical cancer risk should be
improved, to reduce the incidence of cervical cancer. Ad-
ditional research is needed, not only to gain better insight
into the association between the frequency of MN and
cancer, but also to evaluate the benefits of including bio-
markers of cancer risk in the surveillance of populations
at increased environmental or genetic risk. The former
goal is easier to achieve, and plans already exist within
the framework of the HUMN project for increasing the
size of the study group, both by including new national
cohorts and by extending the length of the follow-up
period for those cohorts currently included in the study.
The latter goal implies an improved understanding of the
association between chromosome instability and cervical
cancer. In particular, the consideration of the role of
possible confounders and effect modifiers, such as diet,
oxidative stress, and genetic polymorphisms, would be
desirable before the routine application of these biomark-
ers in population studies aimed at estimating the risk of
cancer.
E.I. Cortés-Gutiérrez, M.I. Dávila-Rodríguez,
R.M. Cerda-Flores
ÕÐÎÌÎÑÎÌÍÛÅ ÏÎÂÐÅÆÄÅÍÈß
ÊÀÊ ÏÐÎÃÍÎÇÍÛÅ ÌÀÐÊÅÐÛ
ÊÀÐÖÅÍÎÃÅÍÅÇÀ ØÅÉÊÈ ÌÀÒÊÈ
Ðàê øåéêè ìàòêè ÿâëÿåòñÿ òðåòüèì ïî ðàñïðîñò-
ðàíåííîñòè â ìèðå òèïîâ ðàêà ó æåíùèí è íàè-
áîëåå ÷àñòî âñòðå÷àþùèìñÿ ó æåíùèí Ìåêñèêè è
Ëàòèíñêîé Àìåðèêè. Ôàêòîðû ðèñêà, ñâÿçàííûå ñ
ðàçâèòèåì èíòðàýïèòåëèàëüíîé öåðâèêàëüíîé íåî-
ïëàçèè, ïðåäïîëàãàþò, ÷òî ïàïèëëîìàâèðóñ ÷åëîâåêà
(HPV) òèïîâ 16, 18, 31 è 33 âëå÷åò çà ñîáîé âûñîêèé
ðèñê ðàçâèòèÿ îïóõîëåé ýòîãî òèïà. Íàêîïëåíèå ãå-
íåòè÷åñêèõ èçìåíåíèé äåëàåò âîçìîæíûì ðîñò îïó-
õîëåâûõ êëåòîê; õðîìîñîìíàÿ íåñòàáèëüíîñòü ÿâ-
ëÿåòñÿ ñîáûòèåì, êîòîðîå ïðåäøåñòâóåò ïðåäðàêî-
âûì ñòàäèÿì. Âîçìîæíûå áèîìàðêåðû ðèñêà îïóõî-
ëè âêëþ÷àþò öèòîãåíåòè÷åñêèå êðèòåðèè, òàêèå êàê
õðîìîñîìíûå àáåððàöèè, îáìåí ñåñòðèíñêèõ õðîìà-
òèä, ìèêðîÿäðà, è çàêàí÷èâàþòñÿ Comet-àíàëèçîì è
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 360
E.I. Cortés-Gutiérrez, M.I. Dávila-Rodríguez, R.M. Cerda-Flores
äåòåêöèåé ïîëîìîê ÄÍÊ ñ ïîìîùüþ ôëþîðåñöåíò-
íîé ãèáðèäèçàöèè in situ. Îáðàçöû, èäåíòèôèöèðî-
âàííûå â òàêèõ öèòîãåíåòè÷åñêèõ èññëåäîâàíèÿõ, ïî-
êàçûâàþò, ÷òî õðîìîñîìíàÿ íåñòàáèëüíîñòü ÿâëÿåòñÿ
òðàíçèåíòíûì ïðîìåæóòî÷íûì çâåíîì â ðàçâèòèè
öåðâèêàëüíûõ íàðóøåíèé. Â ýòîé ñâÿçè ìåõàíèçìû,
êîòîðûå ìîãóò ëåæàòü â îñíîâå ïðîãðåññèðóþùåé
ãåíåòè÷åñêîé íåñòàáèëüíîñòè ó òàêèõ ïàöèåíòîâ, êà-
æóòñÿ íåïîñðåäñòâåííî ñâÿçàííûìè ñ èíôåêöèåé
HPV. Íàñòîÿùåå èññëåäîâàíèå ïîêàçûâàåò, ÷òî õðî-
ìîñîìíàÿ íåñòàáèëüíîñòü ìîæåò ñëóæèòü áèîìàð-
êåðîì äëÿ ïðåäñêàçàíèÿ ðàçâèòèÿ èíòðàýïèòåëè-
àëüíîé öåðâèêàëüíîé íåîïëàçèè, òåì íå ìåíåå ýòè
ðåçóëüòàòû äîëæíû áûòü îöåíåíû â áîëåå ìàñøòàá-
íûõ èññëåäîâàíèÿõ.
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Received 01.06.12
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]
/OmitPlacedBitmaps false
/OmitPlacedEPS false
/OmitPlacedPDF false
/SimulateOverprint /Legacy
>>
<<
/AddBleedMarks false
/AddColorBars false
/AddCropMarks false
/AddPageInfo false
/AddRegMarks false
/ConvertColors /ConvertToCMYK
/DestinationProfileName ()
/DestinationProfileSelector /DocumentCMYK
/Downsample16BitImages true
/FlattenerPreset <<
/PresetSelector /MediumResolution
>>
/FormElements false
/GenerateStructure false
/IncludeBookmarks false
/IncludeHyperlinks false
/IncludeInteractive false
/IncludeLayers false
/IncludeProfiles false
/MultimediaHandling /UseObjectSettings
/Namespace [
(Adobe)
(CreativeSuite)
(2.0)
]
/PDFXOutputIntentProfileSelector /DocumentCMYK
/PreserveEditing true
/UntaggedCMYKHandling /LeaveUntagged
/UntaggedRGBHandling /UseDocumentProfile
/UseDocumentBleed false
>>
]
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice
|