# Correlation of DNA Ploidy with Progression of Cervical Cancer

Authors: M. Singh; S. Mehrotra; N. Kalra; U. Singh; Y. Shukla
Journal: Journal of Cancer Epidemiology
 (2008)

---

## Abstract

The majority of squamous cell carcinomas of cervix are preceded by visible changes in the cervix, most often detected by cervical smear. As cervical cancer is preceded by long precancerous stages, identification of the high-risk population through detection of DNA ploidy may be of importance in effective management of this disease. Here we attempted to correlate aneuploid DNA patterns and their influence on biological behavior of flow-cytometry analysis of DNA ploidy which was carried out in cytologically diagnosed cases of mild (79), moderate (36), and severe (12) dysplasia, as well as “atypical squamous cells of unknown significance (ASCUS)” (57) along with controls (69), in order to understand its importance in malignant progression of disease. Cytologically diagnosed dysplasias, which were employed for DNA ploidy studies, 39 mild, 28 moderate, and 11 severe dysplasia cases were found to be aneuploid. Out of the 69 control subjects, 6 cases showed aneuploidy pattern and the rest 63 subjects were diploid. An aneuploidy pattern was observed in 8 out of 57 cases of cytologically evaluated ASCUS. The results of the followup studies showed that aberrant DNA content reliably predicts the occurrence of squamous cell carcinoma in cervical smear. Flow cytometric analysis of DNA ploidy may provide a strategic diagnostic tool for early detection of carcinoma cervix. Therefore, it is a concept of an HPV screening with reflex cytology in combination with DNA flow cytometry to detect progressive lesions with the greatest possible sensitivity and specificity.


---

## Body

## 1. Introduction

Cervical cancer is the second most common malignancy in
women worldwide. It is estimated that, globally, 466,000 new cases of cervical cancer are diagnosed
each year. As per estimate 231,000 women in a year
die of cervical cancer and 80% of them are from the developing world [1]. Use of cytological screening technique, Papanicolaou (Pap) test, has significantly increased the detection of cervical
cancer [2]. Cancer of cervix emerges from a defined series of preneoplastic
lesions with increasing cellular dysplasia, referred to as cervical
intraepithelial neoplasia (CIN) grade I, II, and III. However, CIN lesions
frequently regress, and not all CIN III lesions, progress to invasive cancer
[3]. The group of CIN is very heterogeneous, particularly in their
clinical behavior. Persistent infection with high-risk-type human papilloma viruses (HPVs) is the strongest independent
risk factor for cervical cancer [4]. CIN lesions induced by HPV infection and
genomes of these viruses were detected in the vast majority of cervical cancer
specimens [5–7]. During the course of tumor initiation in an HPV-infected cervical epithelial cell, an aneuploid DNA pattern develops [8],
which along with its descendants shows genetic instability. Aneuploidy reflects
a situation of uncontrolled increase of DNA and loss of essential information
[9] and plays an important role in neoplastic transformation. Increased
aneuploid DNA value with the increase in grades of cervical dysplasia has long
been considered to be a specific prognostic marker of malignancy [10]. However,
at present, no well-established technique, which, qualitatively, predicts the
clinical outcome of cervical dysplasia except Pap smear screening, is
available. Approximately 15–30% of all women who developed low-grade squamous intraepithelial lesion (LSIL) have a
probability of developing moderate-to-severe cervical intraepithelial neoplasia
(CIN II and III) identified on a subsequent cervical biopsy [11]. To prevent
cervical malignancy and its precursors, several adjuvant diagnostic methods
have been proposed to strengthen the accuracy of cytological and histological
diagnosis [12]. As chromosomal aneuploidy has been used as an early key event
in tumorigenesis caused by genetic instability [13, 14], the cytometric
equivalence of chromosomal aneuploidy detected by DNA flow-cytometry may serve
as a marker of neoplasia and provide valuable information for the diagnosis and
understanding of pathogenesis of cervical cancer. Since aneuploid pattern of
DNA is a useful parameter for the prediction of progression and persistence of “atypical squamous cells of
unknown significance (ASCUS)”, low-grade squamous intraepithelial
lesion (LSIL/mild dysplasia/CIN I), and high-grade squamous intraepithelial
lesion (HSIL/moderate and severe
dysplasia/CIN II and III) in clinical
practice, the present study was designed to investigate whether
alteration in cellular/nuclear content predicts the occurrence of cancer in
cervical smears. We have thus estimated their DNA content using flow cytometry
and compared it with the cytological findings.


## 2. Materials and Methods

### 2.1. Subjects and Sampling

The cases were derived from the ongoing outpatient cytology screening at Department
Obstetrics and Gynecology, King George’s Medical University (Lucknow, India) for
the present study. Cytologically diagnosed 127 cases of dysplasia (79 mild, 36
moderate, and 12 severe), 57 cases of “atypical
squamous cells of unknown significance (ASCUS)” and 69 controls,
which enrolled during the period of 2004–2006, were recruited from a
prospective followup study to understand the biological behavior and the
natural history of cervical precancerous lesions. None of the patients had any previous history of CIN or had been treated
for CIN. All the control women had normal smears. Age of the subjects
ranged from 30 to 75 years.An experienced gynecologist collected cervical samples
with blunt end of Ayer’s spatula. Two fresh spatulas were used for each
preparation. Smears were fixed in alcohol and stained according to Pap method. The blunt end of the second spatula was
transferred into a cryo tube immediately, containing 3 mL chilled PBS (7.2 pH)
and quick frozen at −20°C until further use for flow-cytometric
analysis. Patients were followed up by cytological as well as DNA flow cytometric
analysis.


### 2.2. Criteria for Cytological Classification

Cytological screening was performed with conventional Pap staining by trained cytologists.
All slides were evaluated according to the criteria of the Bethesda System
[15]. Precancerous lesions of the uterine cervix were classified as normal,
ASCUS, LSIL (mild dysplasia/CIN
grade I), and HSIL (moderate dysplasia/CIN grade
II, severe dysplasia/CIN grade III and carcinoma in situ/CIS).


### 2.3. Criteria for Management of Precancerous Lesions

To understand the biological behavior
of cervical precancerous lesions, the following criteria were followed.(i)
The cases initially diagnosed as LSIL (CIN I) and HSIL
(CIN II and III) were made to undergo a second Pap smear test after 30 days.(ii)
Cytological confirmed dysplasia
cases were enrolled for the followup with the consent of the patients.(iii)
Cases initially diagnosed as CIN
III were subjected to colposcopy directed biopsy on day 30 and were directed to
gynecologist for further management. A cytological control examination was done
after every 6 and 3 months for LSIL and HSIL subjects, respectively, and were
categorized as “returned to normal” (after the morphology normalized);
progressed (when presented with higher grade of dysplasia); and with no change
(when remained at the same grade of dysplasia). The samples were also tested
for the DNA ploidy pattern.(iv)
ASCUS and all aneuploid cases from control group were also followed for a
period of one year.


### 2.4. Flow-Cytometric DNA Analysis

To determine the cellular DNA content, the protocol of Nicoletti
et al. was followed [16]. In brief, cells were fixed with chilled ethanol for
30 minutes at 4°C temperature. After adding propidium
iodide (Sigma-Aldrich, Miss, USA), the mixture was stored in dark for 1 hour at
room temperature. Lymphocytes were used for calibration. The maximum permitted
coefficient of variation (CV) for calibration was 2%. A total of 15,000 cells
were counted in each sample. DNA histogram cell-cycle analysis was performed as
described by Rabinovitch [17], using ModFit LT for Mac V2.0
software
after excluding debris by using electronic gate. The events in G0-G1, S, and G2-M
phases of the cell cycle were counted. Cases were regarded as acceptable for
analysis if the CV of the G0-G1 peak was 7.0. Lesions were classified as
diploid, tetraploid, or aneuploid. If two distinct G0-G1 peaks were present
with a DNA index of ≥1.15 (each containing ≥10% of
total cell population), the histogram was considered as aneuploid (DNA
content). By convention, the first G0-G1 peak represented the diploid peak.
Diploid and tetraploid lesions were classified as nonaneuploid. Histogram
patterns were termed diploid if there was a single G0-G1 peak. A histogram was
classified as tetraploid only if a large peak observed in G2-M (DNA index of
1.80–2.2), containing ≥20% of
the total curve and was also associated with a corresponding 8 N peak.


### 2.5. Statistical Analysis

EPI-INFO software was used for
calculating the odd ratio andχ2 (chi-square) values. A P-value
of .05 or less was
considered statistically significant (95% CI).


## 2.1. Subjects and Sampling

The cases were derived from the ongoing outpatient cytology screening at Department
Obstetrics and Gynecology, King George’s Medical University (Lucknow, India) for
the present study. Cytologically diagnosed 127 cases of dysplasia (79 mild, 36
moderate, and 12 severe), 57 cases of “atypical
squamous cells of unknown significance (ASCUS)” and 69 controls,
which enrolled during the period of 2004–2006, were recruited from a
prospective followup study to understand the biological behavior and the
natural history of cervical precancerous lesions. None of the patients had any previous history of CIN or had been treated
for CIN. All the control women had normal smears. Age of the subjects
ranged from 30 to 75 years.An experienced gynecologist collected cervical samples
with blunt end of Ayer’s spatula. Two fresh spatulas were used for each
preparation. Smears were fixed in alcohol and stained according to Pap method. The blunt end of the second spatula was
transferred into a cryo tube immediately, containing 3 mL chilled PBS (7.2 pH)
and quick frozen at −20°C until further use for flow-cytometric
analysis. Patients were followed up by cytological as well as DNA flow cytometric
analysis.


## 2.2. Criteria for Cytological Classification

Cytological screening was performed with conventional Pap staining by trained cytologists.
All slides were evaluated according to the criteria of the Bethesda System
[15]. Precancerous lesions of the uterine cervix were classified as normal,
ASCUS, LSIL (mild dysplasia/CIN
grade I), and HSIL (moderate dysplasia/CIN grade
II, severe dysplasia/CIN grade III and carcinoma in situ/CIS).


## 2.3. Criteria for Management of Precancerous Lesions

To understand the biological behavior
of cervical precancerous lesions, the following criteria were followed.(i)
The cases initially diagnosed as LSIL (CIN I) and HSIL
(CIN II and III) were made to undergo a second Pap smear test after 30 days.(ii)
Cytological confirmed dysplasia
cases were enrolled for the followup with the consent of the patients.(iii)
Cases initially diagnosed as CIN
III were subjected to colposcopy directed biopsy on day 30 and were directed to
gynecologist for further management. A cytological control examination was done
after every 6 and 3 months for LSIL and HSIL subjects, respectively, and were
categorized as “returned to normal” (after the morphology normalized);
progressed (when presented with higher grade of dysplasia); and with no change
(when remained at the same grade of dysplasia). The samples were also tested
for the DNA ploidy pattern.(iv)
ASCUS and all aneuploid cases from control group were also followed for a
period of one year.


## 2.4. Flow-Cytometric DNA Analysis

To determine the cellular DNA content, the protocol of Nicoletti
et al. was followed [16]. In brief, cells were fixed with chilled ethanol for
30 minutes at 4°C temperature. After adding propidium
iodide (Sigma-Aldrich, Miss, USA), the mixture was stored in dark for 1 hour at
room temperature. Lymphocytes were used for calibration. The maximum permitted
coefficient of variation (CV) for calibration was 2%. A total of 15,000 cells
were counted in each sample. DNA histogram cell-cycle analysis was performed as
described by Rabinovitch [17], using ModFit LT for Mac V2.0
software
after excluding debris by using electronic gate. The events in G0-G1, S, and G2-M
phases of the cell cycle were counted. Cases were regarded as acceptable for
analysis if the CV of the G0-G1 peak was 7.0. Lesions were classified as
diploid, tetraploid, or aneuploid. If two distinct G0-G1 peaks were present
with a DNA index of ≥1.15 (each containing ≥10% of
total cell population), the histogram was considered as aneuploid (DNA
content). By convention, the first G0-G1 peak represented the diploid peak.
Diploid and tetraploid lesions were classified as nonaneuploid. Histogram
patterns were termed diploid if there was a single G0-G1 peak. A histogram was
classified as tetraploid only if a large peak observed in G2-M (DNA index of
1.80–2.2), containing ≥20% of
the total curve and was also associated with a corresponding 8 N peak.


## 2.5. Statistical Analysis

EPI-INFO software was used for
calculating the odd ratio andχ2 (chi-square) values. A P-value
of .05 or less was
considered statistically significant (95% CI).


## 3. Results

The cervical smears were divided into four groups (see Figure1). The first group qualified as normal and grouped 69 cases. The second group
(57 cases) qualified as ASCUS. The third and fourth groups comprised of LSIL
(79 cases) and HSIL (48 cases; 36 cases in moderate dysplasia and 12 cases in
severe dysplasia).Showing representative cytological images of cervical smears stained according
to Pap staining method (magnification: 40X): (a) showing cells with normal
morphology, (b) showing “atypical squamous cells of unknown
significance,” (c) showing cells of mild dysplasia (LSIL/CIN I), (d) showing
cells of moderate dysplasia (HSIL/CIN II), (e) showing cells of severe dysplasia
(HSIL/CIN III). LSIL: low-grade squamous intraepithelial lesion;
HSIL: high-grade squamous intraepithelial lesion; CIN: cervical intraepithelial neoplasia.
(a)(b)(c)(d)(e)A comparison of DNA content analysis
with behavior of LSIL or HSIL in
patients having cytologically diagnosed various grades of dysplasia along with
the control and ASCUS groups were made (Table1). 127 cases of different grades
of dysplasia were employed for analysis of DNA ploidy out of which 78 (62.99%)
cases were aneuploid (39 mild, 28 moderate, and 11 cases of severe dysplasia)
and remaining 49 (37.01%) cases (40 mild, 8 moderate, and 1 severe) were nonaneuploid,
that is, diploid (see Figure 2). In the control and ASCUS groups, 6 (8.70%) and
8 (14.04%) cases were found to be aneuploid, respectively (see Figure 2). The
association of aneuploidy with increasing grades of dysplasia was found to be
significant (P ≤ .001;
Table 1, see Figure 3).Table 1
DNA ploidy pattern in control, ASCUS, and various grades of dysplasia cases.
DNAControlASCUSORMildORModerateORSevereORploidy (n)n (%)n (%)(95% CI)dysplasian (%)(95% CI)dysplasian (%)(95% CI)dysplasian (%)(95% CI)Aneuploidy681.71NS3910.2*2836.75*11115.50*(92)(8.69%)(14.03%)(0.47–6.26)(49.36%)(3.71–29.7)(77.77%)(10.35–140.74)(91.66%)(11.75–5041.27)Diploidy63491.00401.0081.0011.00(161)(91.03%)(85.96%)(50.36%)(22.22%)(8.33%)Total (253)6957793612Odd ratios (OR) were computed considering control cases as the base line category for the ASCUS and dysplasia
groups.Not significantNSP ≥ .05, Statically significant *P ≤ .001, n: Number of cases.Showing representative histograms of flow-cytometric analysis of DNA
contents of cervical smears in different stages of dysplasia. In each sample,
25,000 events were counted at FL2-A following staining with propidium iodide 50μg/mL for 30 minutes in dark at 4°C.
Y-axis showing number of cells and X axis represents DNA ploidy. (a) Showing a
diploid peak of DNA. (b) Showing a near diploid aneuploidy. (c) Showing a hyperaneuploidy from severe dysplasia patient. DI shows DNA index.
(a)(b)(c)Figure 3
Histogram showing percent incidence of anuploidy in control, atypical squamous cells of unknown significance and
various grade of dysplasia.All these cases with aneuploid DNA
content and cytologically diagnosed ASCUS and various grades of dysplasia were
followed up for 1 year at aforementioned intervals, in order to study the
behavior of aneuploid DNA in progression/regression/persistence of disease.
Among 57 patients with ASCUS, 49 (85.96%) diploid cases had smears that returned
to normal cytology and remaining 8 (14.04%) aneuploid cases showed signs of
progression/regression. One patient presented with HSIL and three with LSIL
after one year period of followup. Remaining four aneuploid cases of ASCUS
group regressed to normal, as evident from diploid DNA. Out of six aneuploid
cases with normal cytological diagnosis, 4 cases were followed, where only one
case represented progression of disease from normal to mild dysplasia after a
year.A total of 79 patients who had diagnosis
of LSIL, 75 cases were followed up. Out of 39 aneuploid cases, 7 developed
moderate dysplasia (CIN II) and 30 cases presented with no change in cytology
(CIN I), after a period of six months from the date of registration. All cases
showed aneuploid DNA in their followup study. 35 cases who had LSIL with nonaneuploid
DNA returned to normal and one patient presented with no change in cytology, that
is, remained at CIN I. The sample for this particular subject showed aneuploid
DNA content of cell in followup study while 35 normal cases were diploid (Table2).Table 2
Progression/regression pattern of diploid and aneuploid cases of dysplasia during follow up.
GroupsTotal casesPloidy StatusProgressionRegressionChisquare(n)(n)(n)(n)valueMild75Aneuploid (39)37263.5**dysplasiaDiploid (36)135Moderate/severe33Aneuploid (28)2538.4*dysplasiaDiploid (5)14*P ≤ .01, **P ≤ .001.n: Number of cases.Out of 36 cases of moderate dysplasia, only 30 patients were available for followup
study and 6 patients underwent hysterectomy. 20 patients out of 25 who were
diagnosed with moderate dysplasia (HSIL/CIN II) and with aneuploid DNA
remained CIN II (71.79%) with an aneuploid DNA in their followup study after
three months of registration. In addition, 2 other patients were diagnosed
later with CIS, who were positive for aneuploid DNA in their followup. 1 patient
with diploid DNA remained at CIN II in cytological followup and other 4 diploid
showed a regressive pattern. Out of 11 only 3 aneuploid cases of severe
dysplasia (CIN III) were followed, rest underwent prescribed treatment. Of the 3
cases, which were aneuploid, 1 case progressed to CIS and 2 remained at CIN
III. The association of aneuploidy with progression of disease was found to be
significant (Table2). χ2 test value for LSIL
(P ≤ .001) and HSIL (P ≤ .001) was significant.


## 4. Discussion

There is evidence that cervical
carcinoma evolves on the basis of persistence of aneuploidy [10]. Studies have
shown that aneuploid conditions indicate either invasive carcinoma or
prospective neoplastic development in cervical dysplasia [18, 19] and play a
causal role in neoplastic development [20, 21].Genetic abnormalities, such as numerical chromosomal aberrations (aneuploidy and
tetraploidy), have been reported in women diagnosed with precancerous and
cancerous cervical lesions and are observed frequently in the presence of HPV
infections [10, 22, 23]. According to Melsheimer et al. [22], deregulated viral
oncogene expression appears to result first in chromosomal instability and
aneuploidization and is subsequently followed by integration of HPV genomes in
the affected cell clones. In CIN and
invasive cervical cancer, DNA ploidy assessment by flow cytometric technique has
been established as a prognostic factor that allows the estimation of the
relative progressive risk into more advanced lesions [8, 24–27]. Dysplastic
epithelium represents potentially precancerous conditions in which the risk of
progression to cancer is usually unknown. Bocking and Motherby [28] suggested
that the identification of DNA aneuploidy in dysplastic squamous epithelia
could increase the predictive value for malignant transformation to over 90%.
Similarly, the results of our study have also confirmed the predictive value of
aneuploidy with 92% aneuploid cases in all grades of dysplasia showing
progressive pattern of disease (Table 2).Evidences indicate aberrant DNA content
in cells as a cause rather than as a consequence of malignant transformation [29, 30]. Duesberg et al. pointed out that aneuploid cancers
are generally considered to be more dangerous and aggressive than the diploid
[31]. Boecking et al. carried out a prospective study to determine the
progression to malignancy of cervical smears with ASCUS [32]. Based on “the
high prognostic validity of DNA-aneuploidy” researchers concluded that the
“aneuploid lesions should immediately be controlled histologically or be removed”. In
accordance with the above findings, the results of the present study also
demonstrate the progressive pattern of disease among aneuploid cases from ASCUS
group. Bollmann et al. suggested that ASCUS cases, those with high-risk HPV and
with abnormally high DNA content, represent similar biologic features as HSIL
with elevated risk to develop cancer [33]. Thus, DNA content may reflect
genomic instability of the cells very early in the malignant process, at a
point where morphological changes in malignancies or premalignancies are not
yet evident [34]. Conceivably, lesions that are defined as nondysplastic, and
therefore considered to represent minimal or no risk for developing cancer
cervix, may contain readily detectable large-scale genomic alteration viz DNA
aneuploidy which could signal a malignant transformation [34, 35]. The
identification of a molecular biomarker of persistent HPV infection in women
who had been diagnosed as normal/ASCUS in Pap smear could be a valuable tool in
the proper management of this disease. We have also found 6 aneuploid cases
from control group and among them, after one-year duration, one case presented
the progression of disease.In this study, we have shown that
aneuploid DNA content could be used as a biomarker of progressive cervical
changes towards malignancy. Progressive
increase in aneuploidy increases the severity of the lesions (49.36% for mild
dysplasia, 77.77% for moderate dysplasia versus 91.66% for severe dysplasia).
Our followup results clearly demonstrate that more and more progressive
pattern of disease was noted in aneuploid cases and that DNA ploidy could be
used as a prognostic marker for cervical malignancies. Comparison of results obtained with Pap screening and DNA ploidy
measurement provide a positive correlation between aneuploid DNA and
progression of disease (Table2). Furthermore, the percentage of aneuploid cases
towards the progression of disease was higher in followup data that supported
the prognostic value of aneuploidy as a marker to identify the progressive
behavior of lesion. Occurrence of aneuploidy was exceptional in patients
who had samples with morphology that returned to normal, whereas, patients who
had cytological signs of disease progression were found with a significantly
greater frequency (P<.01, P<.001; Table 2). High-grade lesions (according
to Bethesda classification) can be detected using cytodiagnosis method with
generally good accuracy, but the diagnosis occasionally relies on subjective
parameters. Moreover, inadequate sampling may result in a mild morphologic
change despite early progressive behavior [36]. Thus, the measurement of DNA content by flow cytometry provides qualitative
information and presence of aberrant aneuploid cells in cytological samples,
which could serve as a useful parameter along with cytology with good
specificity and sensitivity [37, 38]. If patients at risk could be identified earlier
by analysis of parameter that reflects the true biological properties of
lesion, treatment regimes could be given selectively to improve the current
poor survival rate of cancer cervix cases [39]. Flow cytometric analysis of DNA
ploidy may provide a strategic diagnostic tool for early detection of carcinoma
cervix [8, 25]. Therefore,
we propose the concept of an HPV screening with reflex cytology in combination
with DNA flow cytometry to detect progressive lesions with the greatest
possible sensitivity and specificity.



---

*Source: 298495-2008-01-29.xml*