European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 501–506

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European Journal of Obstetrics & Gynecology and

Reproductive Biology
journal homepage: www.elsevier.com/locate/ejogrb

Progression of cervical low grade squamous intraepithelial lesions:

in search of prognostic biomarkers
Koen D. Quint a, Maurits N.C. de Koning a, Wim G.V. Quint a, Edyta C. Pirog b,*
a
DDL Diagnostic Laboratory, Rijswijk, the Netherlands
b
Department of Pathology, Weill Medical College of Cornell University, New York, USA

A R T I C L E I N F O A B S T R A C T

Article history: Objective: It has been reported that approximately 10% of low grade squamous intraepithelial lesions
Received 21 February 2013 (LSIL) progress to high grade squamous intraepithelial lesions (HSIL) within a 2-year follow up. The
Received in revised form 18 June 2013 factors related to lesion progression are currently unknown. The aim of the study was to identify
Accepted 6 July 2013
prognostic markers of the course of LSIL. This retrospective study was designed to correlate regression,
persistence and progression of biopsy-proven LSIL with patients’ age, HPV genotypes and
Keywords: immunohistochemical expression of the main cell cycle regulating proteins: p53, pRb, p16, and Ki-67.
LSIL
Study design: A total of 584 consecutive patients with biopsy proven LSIL and 2-year follow-up were
HPV
included in the age analysis. HPV genotyping was performed in 328 LSIL cases using the SPF10 PCR-
p16
Ki-67 LiPA25 (version 1), 238 LSIL cases were immunostained for Ki-67 and p16, and 101 cases were
p53 immunostained for pRb and p53.
pRb Results: The odds of LSIL persistence and progression were significantly higher in women 30–39, 40–49
and 50+ years old, as compared to women 20–29 years old (OR 1.89, 2.52 and 2.39, respectively). The
odds of persistence and progression were higher in women infected with HPV16, 18, 33 and 52 (OR 3.5,
3.1, 3.5 and 2.9, respectively). There were no significant differences in expression of immunomarkers
(p16, p53, pRB and Ki-67) between the lesions that regressed versus the lesions that persisted or
progressed.
Conclusions: Patients 30 years of age and older have a higher risk of LSIL progression or persistence as
compared to 20–29 year olds. In addition, HPV genotyping, but not the cell cycle markers, may aid in
prognosis of LSIL course.
ß 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Human papillomavirus (HPV) infection is now recognized as the
main pathogenetic factor of cervical carcinoma. Eighteen mucosal
HPV types have been detected in cancers of genital and
oropharyngeal mucosa and those types are identified as ‘‘high
oncogenic risk.’’ HPV16 alone accounts for 50–60% of cervical
cancers cases [1].
Invasive cervical carcinoma is preceded by a long period of
cervical intraepithelial neoplasia (CIN), also termed squamous
intraepithelial lesion (SIL). SIL is a heterogeneous group of lesions
graded as either low grade (LSIL) or high grade squamous
intraepithelial lesions (HSIL), depending on the severity of their
histological abnormalities. LSIL reflects a productive viral infection
* Corresponding author at: Department of Pathology, Weill Medical College of
Cornell University, 525 E 68th Street, ST-1041, New York, NY 10065, USA.
Tel.: +1 212 746 2722; fax: +1 212 746 8624.
E-mail address: ecpirog@med.cornell.edu (E.C. Pirog).
and it may follow various clinical courses. Most LSIL (60%) are
transient and regress spontaneously within 12–24 months, but
30% of LSIL persist, and approximately 10% of LSIL progress to pre-
malignant HSIL within 2-year follow-up [2–4]. Since only a small
fraction of patients with LSIL are expected to develop HSIL,
optimally such patients should be prospectively identified and
triaged for excisional treatment, while all other patients with LSIL
could be safely followed with repeat Pap tests and colposcopic
examinations until the resolution of the lesion. In some medical
centers patients with LSIL are offered a loop electrosurgical
excision procedure (LEEP) in order to prevent progression to HSIL.
Such an approach results in over-treatment of patients who have
only a transient HPV infection, contributing to unnecessary high
costs of medical care and occasional iatrogenic complications.
Understanding the factors involved in progression from benign,
productive HPV infection to premalignant HSIL and invasive
carcinoma could be utilized to identify patients who are truly at
risk for carcinoma. Such markers may help in developing efficient
individualized therapy and cost-effective triage protocols for
patients with diagnosis of LSIL. The aim of this study was to

0301-2115/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ejogrb.2013.07.012
502 K.D. Quint et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 501–506

identify prognostic markers of the course of LSIL in order to predict
which patients with LSIL may require excisional treatment.
This study examined the relationship between LSIL regression,
persistence and progression and patients’ age, HPV genotype and
immunohistochemical expression of four main cell cycle regulat-
ing proteins – p53, pRb, p16, and Ki-67.
2. Materials and methods
2.1. Case selection
The Institutional Review Board’s permission was obtained for
the study. The surgical pathology files of the Department of
Pathology at Weill Medical College of Cornell University were
searched from 1995 to 2005 to identify consecutive cervical punch
biopsies with the diagnosis of LSIL (‘‘index biopsy’’). Only LSIL
cases with available 2-year follow-up information (cervical
cytology or biopsy) were selected from the computer-database.
Regression of LSIL was defined as normal cervical cytology or
cervical biopsy at the end of 2-year follow up. Persistence was
defined as LSIL or atypical cells of undetermined significance
(ASCUS) on cervical cytology or biopsy at the end of 2-year follow
up. Progression was defined as diagnosis of HSIL any time within
the 2 years of follow-up on cervical cytology, punch biopsy or cone
biopsy. If LSIL and HSIL lesions were detected simultaneously in
the initial ‘‘index’’ cervical biopsy, such a case was also classified
as progression.
All slides were re-reviewed to confirm the diagnosis. Of the 584
LSIL cases with clinical follow up, 328 (57%) cases had enough
remaining lesional tissue available for both HPV detection and
immunohistochemical studies.
2.2. HPV DNA amplification and genotyping
2.3. Immunohistochemistry
Immunostaining was performed on 3-mm tissue sections from
randomly computer-selected cases; 238 LSIL cases were immu-
nostained for Ki-67 and p16, and 101 cases were stained for pRb
and p53.
The sections were subjected to heat-induced antigen retrieval
and incubated in an automated stainer with p16 antibody (Dako,
Glostrup, Denmark) at a dilution of 1:25; p53 antibody (Dako,
Glostrup, Denmark) at a dilution of 1:200; pRB antibody (Dako,
Glostrup, Denmark) at a dilution of 1:250; and Ki-67 antibody
(Zymed, San Francisco, CA) at a dilution of 1:50. For p16 the
staining was graded as either ‘‘no overexpression’’ (completely
negative or weak, focal nuclear or cytoplasmic blush), or ‘‘over-
expression’’ (moderate to strong intensity nuclear and cytoplasmic
staining with diffuse or patchy distribution). For p53 and pRB the
staining was graded in the basal and parabasal epithelial layer as
either ‘‘retained staining’’ (strong nuclear staining in >50% of basal
and parabasal cells) or ‘‘loss of staining’’ (no staining or only spotty
staining in the basal and parabasal cells). For Ki-67 the staining was
assessed semi-quantitatively in three epithelial strata – lower,
middle and upper 1/3 of the epithelial thickness. Presence of
strongly staining nuclei was expressed as a percentage of all nuclei,
and rounded up to the closest tenth percentage.
2.4. Statistical analyses
The two-sample t-test was used to compare age at diagnosis
between groups and the Pearson’s chi-square test or Fisher’s exact
was used, as appropriate, to assess relationships between
categorical variables. All p-values are two-sided with statistical
significance evaluated at the 0.05 alpha level. All analyses were
performed in SPSS Version 21.0 (SPSS Inc., Chicago, IL).

HPV testing was performed on all available 328 cases of LSIL.
Tissue digestion and DNA release were performed using
standard methods. Briefly, 250 mL of the proteinase K solution
was added to the 1.5 mL tubes containing tissue sections and
incubated for 18 h at 70 8C. Proteinase K was subsequently
inactivated at 95 8C for 10 min. 10 mL of the supernatant was
used for PCR reaction.
Broad-spectrum HPV DNA amplification and genotyping was
performed using HPV SPF10 PCR-DEIA-LiPA25, version 1 (Labo
Biomedical Products, Rijswijk, The Netherlands). The PCR-
products were analyzed on a 3% agarose gel. All HPV gel-positive
samples were further genotyped using HPV LiPA25, version 1 kit
(Labo Biomedical Products, Rijswijk, the Netherlands). This
system is a reverse hybridization method that can identify 15
high risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59,
66 and 68/73, and 10 low risk HPV types 6, 11, 34, 40, 42, 43, 44, 54,
70 and 74 [5].
3. Results
A total of 584 cervical biopsies with the diagnosis of LSIL and 2-
year follow-up were identified, including 336 (57.5%) LSIL that
regressed, 116 (19.8%) persistent LSIL and 132 (22.6%) LSIL that
progressed to HSIL within 2 years. Age analysis was performed on
all 584 cases, and out of these cases, 328 had available tissue
material for HPV testing and immunohistochemical analysis.
3.1. Age analysis
The mean age of patients at index LSIL diagnosis was 29.8 years
in the regression group and 33.2 years in the persistence/
progression group. The difference was statistically significant
(t-test, p = 0.001).
In the age group of 10–19 years, 61.4% of LSIL lesions regressed,
compared to 66.8%, 51.6%, 44.4% and 45.7% in the age groups of

Table 1
Regression, persistence and progression of LSIL in different age groups.

Age group n LSIL regression LSIL persistence LSIL progression OR of persistence OR of progression OR of persistence or p*
% (n) % (n) % (n) (95% CI) (95% CI) progression (95% CI)

10–19 44 61.4% (27) 15.9% (7) 22.7% (10) 1.19 (0.48–2.94) 1.33 (0.60–2.94) 1.27 (0.65–2.46) 0.483
**
20–29 247 66.8% (165) 14.6% (36) 18.6% (46) 1.00 1.00 1.00
30–39 186 51.6% (96) 23.1% (43) 25.3% (47) 2.05 (1.23–3.42) 1.76 (1.09–2.83) 1.89 (1.28–2.79) 0.001
40–49 72 44.4% (32) 30.6% (22) 25.0% (18) 3.15 (1.64–6.05) 2.02 (1.04–3.92) 2.52 (1.47–4.30) 0.0005
50+ 35 45.7% (16) 22.9% (8) 31.4% (11) 2.29 (0.91–5.76) 2.47 (1.07–5.68) 2.39 (1.17–4.89) 0.014
All age groups 584 57.5% (336) 19.8% (116) 22.6% (132) – – –

OR = odds ratio, CI = confidence intervals.

*
x2 test of regression versus persistence or progression.
**
Reference group.
 K.D. Quint et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 501–506 503

Table 2
HPV genotype distribution in consecutive 300 cases of LSIL.

HPV type Single infection (n = 205) Multiple infection (n = 95) Single + multiple (n = 300)

Frequency Percentage Frequency Percentage Frequency Percentage

LR-HPV
6 21 10.3 8 3.5 29 6.7
11 4 2.0 5 2.2 9 2.1
40 2 1.0 4 1.7 6 1.4
42 1 0.5 1 0.4 2 0.5
43 1 0.5 1 0.4 2 0.5
44 2 1.0 3 1.3 5 1.1
54 2 1.0 4 1.7 6 1.4
70 3 1.5 10 4.3 13 3.0
74 2 1.0 2 0.9 4 0.9

Total 38 18.5 38 16.5 76 17.5

HR-HPV type
16 15 7.3 9 3.9 24 5.5
18 8 3.9 7 3.0 15 3.4
31 18 8.8 17 7.4 35 8.0
33 3 1.5 5 2.2 8 1.8
35 6 2.9 7 3.0 13 3.0
39 7 3.4 9 3.9 16 3.7
45 2 1.0 7 3.0 9 2.1
51 22 10.7 32 13.9 54 12.4
52 19 9.3 29 12.6 48 11.0
53 11 5.4 17 7.4 28 6.4
56 15 7.3 11 4.8 26 6.0
58 14 6.8 9 3.9 23 5.3
59 4 2.0 7 3.0 11 2.5
66 15 7.3 16 7.0 31 7.1
68/73 8 3.9 10 4.3 18 4.1

Total 167 81.5 192 83.5 359 82.5

Overall total 205 100 230 100 435 100

20–29, 30–39, 40–49 and 50+ years, respectively (Table 1). The
overall difference of LSIL regression in different age groups was
statistically significant (x2 test, p = 0.001).
The odds ratio for LSIL persistence or progression in the
different age groups is shown in Table 1. The 20–29 years age group
was used as a reference, because it is the group of the highest
detection of LSIL. The group of 10–19 year olds did not show a
significantly increased risk of non-regression as compared to the
reference group (p = 0.483), but all the older age groups, 30–39,
40–49 and 50+, showed a significantly increased risk of progres-
sion or persistence as compared to 20–29 year olds.
3.2. HPV detection results
HPV testing was done in 328 LSIL cases with available tissue
material. HPV DNA was detected in 300 cases (91.5%) and the

Table 3
Odds ratio for LSIL persistence or progression in relation to HPV genotype.

Genotype No. cases LSIL persistence or progression % OR of persistence or progression (95% CI) p**
*
LR-positive 34 32.3 1
HR-positive 261 47.5 1.89 (0.88–4.04) 0.095
HPV16 24 62.5 3.48 (1.16–10.41) 0.023
HPV18 15 60.0 3.14 (0.89–11.03) 0.070
HPV31 35 48.6 1.97 (0.74–5.24) 0.170
HPV33 8 62.5 3.48 (0.70–17.28) 0.114#
HPV35 13 53.8 2.44 (0.66– 9.00) 0.175
HPV39 16 37.5 1.25 (0.36–4.34) 0.720
HPV45 9 55.6 2.61 (0.58–11.69) 0.200#
HPV51 54 44.4 1.67 (0.68–4.10) 0.259
HPV52 48 58.3 2.93 (1.16– 7.34) 0.020
HPV53 28 53.6 2.41 (0.85– 6.78) 0.092
HPV56 26 50.0 2.09 (0.73–5.98) 0.167
HPV58 23 47.8 1.92 (0.64–5.69) 0.239
HPV59 11 36.4 1.19 (0.28–4.95) 0.806#
HPV66 31 25.8 0.73 (0.24–2.13) 0.562
HPV68/73 18 33.3 1.05 (0.31–3.52) 1.000

OR = odds ratio, CI = confidence intervals.

*
Reference group.
**
x2 test of regression versus persistence or progression.
#
Fisher’s exact test of regression versus persistence or progression.
504 K.D. Quint et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 501–506

Table 4
Results of immunostaining for pRb, p53 and p16 in LSIL cases stratified according to the follow up.

Immuno-marker Staining result % (n) LSIL regression LSIL persistence LSIL progression OR of persistence or p*
% (n) % (n) % (n) progression (95%CI)

p53 Retained staining 100% (48) 41.6% (20) 27.0% (13) 31.2% (15) 1.00**
n = 101 Loss of staining 100% (53) 35.8% (19) 26.4% (14) 37.7% (20) 1.27 (0.57–2.85) 0.548

pRb Retained staining 100% (45) 42.2% (19) 33.3% (15) 24.4% (11) 1.00**
n = 101 Loss of staining 100% (56) 35.7% (20) 21.4% (12) 42.8% (24) 1.31 (0.58–2.94) 0.504

p16 No overexpression 100% (141) 48.2% (68) 28.3% (40) 23.4% (33) 1.00**
n = 238 Overexpression 100% (97) 47.4% (46) 12.3% (12) 40.2% (39) 1.03 (0.61–1.73) 1
* 2
x test of regression versus persistence or progression.
**
Reference group.

results of genotyping analysis are shown in Table 2. Of HPV-
positive cases, 68% (n = 205) were single-type infections and 32%
(n = 95) were positive for two or more viral types. Low risk HPV
types accounted for 17.4% of LSIL cases and among the low risk
types, HPV6 was the most common (38% of low risk genotypes).
High risk HPV types accounted for 82.5% of LSIL cases. The most
common high risk types were HPV51, 52 and 31. HPV16 and HPV18
accounted for 5.5% and 3.4% of all LSIL cases, respectively.
Presence of multiple type infection was not associated with
significantly increased risk of persistence or progression (OR = 1.17
95%CI = 0.71–1.90). Presence of any high risk HPV type conferred
an increased risk of persistence or progression as compared to any
low risk type (OR = 1.89, 95%CI = 0.88–4.04). Of 28 cases in which
HPV was not detected, 15 regressed, 9 persisted and 4 progressed.
Lack of detection of HPV type conferred an increased risk of
persistence or progression as compared to any low risk type
(OR = 1.81, 95%CI = 0.64–5.09).
Table 3 shows the odds ratio for LSIL persistence or progression
depending on HPV type detected in the case. The odds of
persistence or progression were higher in women infected with
HPV16, 18, 33 and 52 (OR 3.5, 3.1, 3.5 and 2.9, respectively), as
compared to any low risk type, and the difference was statistically
significant for HPV16 and HPV52 (p = 0.023 and p = 0.020,
respectively).
3.3. Expression of markers of the cell cycle
Table 4 shows results of immunostaining for pRb, p53 and p16
in LSIL stratified by follow up. Stain scoring definitions are
described in detail in Section 2; briefly, staining for p53 and pRb

Fig. 1. LSIL (right part of the picture, arrow), positive for HPV18, immunostained for pRb (a), Ki-67 (b), p16 (c) and p53 (d). (a) The normal squamous epithelium (on the left)
shows pRb staining in the basal/parabasal layer with extension to the midlayer. LSIL (right part of the picture, arrow) shows markedly diminished staining in the basal/
parabasal layer (scored as ‘‘loss of staining’’), presumably due to downregulation of pRb caused by HPV infection and reflecting a more active cell-cycle. (b) The normal
squamous epithelium (on the left) is positive for the proliferation marker Ki-67 only in the parabasal layer, highlighting the normal proliferation zone of the squamous
epithelium. LSIL (right part of the picture, arrow) shows Ki-67 positivity throughout the parabasal and entire midlayer reflecting abnormal expansion of cell proliferation. (c)
The normal squamous epithelium (on the left) does not stain for the cell cycle inhibitor p16. LSIL (right part of the picture, arrow) shows overexpression of p16 protein. (d) The
normal squamous epithelium (on the left) stains for p53 as a continuous band of positive nuclei in the basal/parabasal epithelial layer. LSIL (right part of the picture, arrow)
shows positive staining in the basal/parabasal layer and scattered positive cells in the upper layers of the epithelium (scored as ‘‘retained staining’’).
 K.D. Quint et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 501–506
Table 5
Mean percentage of Ki-67 positive nuclei in three epithelial compartments in LSIL cases stratified according to the follow up.
Epithelial compartment LSIL regression LSIL persistence
n = 114 n = 52
Mean percentage of Ki-67 positive nuclei
Lower 1/3 53.5% 49.0%
Middle 1/3 35.3% 35.1%
Upper 1/3 8.5% 10.1%
Full thickness 28.5% 27.5%
*
t test of regression versus persistence or progression.
was assessed in the basal and parabasal area and loss of normal
positivity in more than 50% of cells was qualified as ‘‘loss of
staining’’. p16 was assessed in the full thickness epithelium and
strong and diffuse staining was scored as ‘‘overexpression.’’ Ki-67
staining was scored as a percentage of positive nuclei to all nuclei.
The example of LSIL with immunostaining results is shown in
Fig. 1.
Loss of normal basal/parabasal expression of pRb and p53, and
overexpression of p16 were seen in 53%, 56% and 40% cases,
respectively. Loss of p53 and pRB expression and p16 over-
expression did not show a significant correlation with LSIL
persistence or progression (Table 4).
Table 5 shows the mean percentage of Ki-67 staining in three
epithelial compartments in LSIL stratified by follow up. There were
no significant differences in Ki-67 staining pattern between LSILs
that regressed and lesions that persisted or progressed.
4. Discussion
This retrospective study was designed to correlate regression,
persistence and progression of biopsy-proven LSIL with patients’
age, HPV genotypes and immunohistochemical expression of four
main cell cycle regulating proteins: p53, pRb, p16, and Ki-67. The
results of our study showed that HPV genotypes 16, 18, 33 and 52
were associated with increased risk of LSIL persistence or
progression. In addition, patients 30 years of age and older showed
a significantly increased risk of progression or persistence as
compared to 20–29 year olds. However, expression of none of four
markers of the cell cycle activity – p16, Ki-67, p53 and pRb –
showed significant correlation with LSIL course. These results
suggest that HPV genotyping, specifically for HPV 16 and 18, along
with consideration of the patient’s age may guide an individualized
follow up and treatment in patients with LSIL.
Association between high risk HPV genotypes and CIN
persistence/progression has been previously reported in studies
from Asia and South America. A recent prospective study from
Japan by Matsumoto et al. [6] examined the risk factors for CIN1
and CIN2 progression to CIN3. Their findings parallel the results of
our current study: specifically, women aged 30–54 had a higher
probability of CIN1 non-regression, as compared to women aged
18–29 years. Patients with multiple-type infection had higher risk
of lesion persistence but not of progression. LSIL associated with
HPV types 16, 33, 52 and 58 showed a significantly higher risk of
non-regression in 2-year follow up. The authors concluded that
type-specific HPV testing for women with LSIL may be useful in
identifying individuals at increased or decreased risk of disease
progression, and that genotype-specific HPV testing might be
applied for women younger than 30 years old, for whom the broad
spectrum high-risk HPV testing is not recommended.
In a longitudinal study of HPV infection and cervical neoplasia
in a cohort of patients from Brazil, cervical specimens were taken
from 2404 women for Pap cytology and HPV testing every 4–6
months over a period of 8 years [4]. The authors reported that LSIL
persisted longer and progressed more quickly in women with
505
LSIL progression LSIL persistence or progression p*
n = 72 n = 124
56.3% 53.4% 0.416
40.5% 38.2% 0.538
13.8% 12.2% 0.257
33.4% 30.7% 0.562
oncogenic HPV infections as compared to women with non-
oncogenic infections or without detectable HPV. Mean times for
LSIL progression were 73.3 and 83.5 months for low risk and high
risk HPV types, respectively. Half of LSILs regressed to normal or
ASCUS within 6 months. Mean times of regression from LSIL to
ASCUS or normal were longer for women with oncogenic HPV
types (13.8 months) than for women with non-oncogenic HPV
types (7.8 months). The authors concluded that testing for
oncogenic HPVs may help identify those lesions that are likely
to progress rapidly.
On the molecular level, CIN1 and some CIN2 lesions are thought
to represent productive viral infections which do not cause
significant disruption of the host cell cycle [7] and therefore such
lesions frequently undergo a spontaneous regression. Some of
these lesions, however, evolve into transforming infections with
progressive deregulation of the cell cycle by HPV, resulting in
increased cellular proliferation and decreased or arrested epithe-
lial maturation. Derangement of the cell cycle may become
irreversible and lead to a fully transformed malignant phenotype
[7]. With this concept of molecular progression from CIN1 to
cancer, we thought that it is important to examine the expression
of the main regulatory proteins of the mitotic cycle, namely p53,
pRb, p16, and Ki-67 [7–11] in correlation with LSIL follow up.
While our study failed to identify significant correlation between
expression of the above markers and the course of LSIL, a few prior
studies with smaller number of cases showed promising value for
those markers. A study of Negri et al. [12] consisted of 31 CIN1 cases
that regressed and 32 cases that progressed to CIN3. Among the CIN1
cases with p16 overexpression 62% progressed, compared to 30%
progression in CIN1 without p16 overexpression. In another study of
Negri et al. [13], p16 overexpression correlated with non-regression
of CIN1, but in that study 57% cases of CIN1 that regressed showed
lack of HPV L1 immunoexpression, and as no histopathologic re-
review of diagnoses was conducted, accuracy of CIN1 diagnosis was
questionable. In a study of a population-based cohort in Costa Rica,
Wang et al. [14] reported that 9% (18/199) of LSIL had strong, diffuse
p16 positivity. Among these cases, 44% of patients had LSIL
progression. In the p16-negative group only 15% of patients
progressed in 5–7 years follow up. Opposite results were reported
by Nam et al. [15], who found that decreased level of p16 expression
in CIN1–CIN3 was associated with increased risk of CIN recurrence.
Ki-67 was examined by Kruse et al. [16], who collected 25 CIN1 and
65 CIN2 biopsy-proven cases and reported that cases which
progressed had significantly increased Ki-67 positivity in all three
layers of the epithelium as well as decreased expression of pRb in the
lower half of the epithelial thickness. In that study, however, there
were only 2 cases of CIN1 that showed progression to a higher grade
and therefore this heterogeneous group of cases could not be
compared with our study.
There are quite a number of studies that have reported on viral,
behavioral and host factors influencing HPV clearance and persis-
tence [reviewed in 17], but similar studies of risk factors influencing
regression/progression of SIL are scarce. While the natural history of
HPV infections is important from the epidemiologic point of view, it
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