Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 605615

Contents lists available at ScienceDirect

Best Practice & Research Clinical

Obstetrics and Gynaecology
journal homepage: www.elsevier.com/locate/bpobgyn

Histology of cervical intraepithelial neoplasia and the role of

biomarkers
Cara M. Martin, BSc, MSc, PhD, Lecturer in Molecular Pathology and Tumour
Biology a, b, *, John J. OLeary, MD, D.Phil, MSc, FRCPath, FFPathRCPI, Professor
of Pathology & Consultant Pathologist a, b, c
a

Department of Histopathology, Trinity College, Dublin, Ireland

Department of Pathology, The Coombe Women and Infants University Hospital, Dublin 8, Ireland
c
St Jamess Hospital, Dublin 8, Ireland
b

Keywords:
cervical intraepithelial neoplasia
human papillomavirus
E6/E7 oncogenes
cyclin-dependent kinase inhibitor p16
Ki-67 antigen

Accurate histological grading of cervical intraepithelial neoplasia

(CIN) lesions is important for clinical management of patients,
because CIN1 and CIN2 and 3 lesions are treated differently. In
general, there tends to be poor inter and intra-observer reproducibility of CIN grade evaluation among pathologists. In particular, the differential diagnosis between immature squamous
metaplasia and CIN1 and 2, or between low-grade (CIN1) and
high-grade (CIN2 and 3) lesions, tend to be difcult. These difculties mean that patients tend to be over-treated for CIN lesions,
which will naturally regress. Collectively, this highlights the need
for alternative approaches and specic biomarkers to aid objective
CIN lesion grading, and to identify true high-grade cervical disease.
In this review we focus on the aetiology, pathobiology, the natural
history of CIN, current issues with diagnosis and classication of
CIN and the diagnostic and prognostic utility of specic biomarkers
in identifying true cancerous precursor lesions.
2011 Published by Elsevier Ltd.

Introduction
Cervical cancer is the third most common cancer in women worldwide, with an estimated 529,000
new cases diagnosed in 2008.1 It is usually preceded by a long phase of pre-invasive disease. This pre* Corresponding author. Department of Histopathology, Trinity College, Dublin, Ireland. Tel.: 353 1 4085430; Fax: 353 1
4085499.
E-mail address: cara.martin@tcd.ie (C.M. Martin).
1521-6934/$ see front matter 2011 Published by Elsevier Ltd.
doi:10.1016/j.bpobgyn.2011.04.005

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invasive phase is characterised microscopically as a range of events progressing from cellular atypia to
various grades of dysplasia, including cervical intraepithelial neoplasia (CIN) before progression to
invasive carcinoma. This precursor phase is generally asymptomatic, and can occur over a long period
of 1020 years.2 The introduction of cervical screening programmes have greatly reduced the incidence
of cervical cancer; however, CIN rates remain signicantly high. In this review, we will focus on the
aetiology, pathobiology, the natural history of CIN, diagnosis and classication of CIN, and the utility of
biomarkers in identifying true cancerous precursor lesions.
Aetiology of cervical intraepithelial neoplasia
Human papillomavirus (HPV) is the single most important causative agent in the pathogenesis of
cervical cancer and pre-cancer.3 About 1520 types are associated with cervical cancer, of which
HPV16, 18, 31, and 45 accounts for 80% of cervical cancers.46 HPV 16 followed by HPV 18 are the most
frequently detected HPV types in squamous cell carcinomas of the cervix, whereas HPV 18 is more
strongly associated with adenocarcinoma of the cervix. Evidence of HPV infection can also be detected
in 6080% of high-grade CINs4 and in 75% of adenocarcinoma in situ cases.7,8
HPV infection is more common in younger women, reaching a peak of about 20% among women aged
between 20 and 24 years, with a subsequent decline among women aged over 30 years. Infection with
the HPV virus is a frequent phenomenon, with 80% of women showing evidence of infection at some
stage in their lives.9 Most of these infections are transient, with a median duration of 614 months;
however, in a small proportion, HPV becomes integrated into the host genome causing a persistent
infection as detected by the presence of HPV E6/E7 messenger RNA (mRNA).10,11 Women with integrated
HPV virus are signicantly more likely to develop severe dysplasia and malignancy than those who clear
the infection,11 and screening programmes are increasingly incorporating HPV testing in an effort to
improve accuracy. The role of HPV as biomarker for predicting CIN will be discussed later.
Other risks factors for developing CIN and cervical cancer include sexual behaviour, age, smoking
history, diet, parity and contraceptive use.12
Natural history of cervical intraepithelial neoplasia
HPV is the major causative agent in the development of CIN. Despite womens frequent exposure to
HPV, the development of cervical cancer is relatively rare. Most low-grade cervical abnormalities, such
as CIN1, are associated with benign viral replication, and will spontaneously regress without requiring
treatment. Studies in women have shown CIN1 regression rates of up to 7080%; however, in
adolescents and young women under 25 years, more than 90% show regression.1315 In contrast, highgrade abnormalities, specically CIN3, has a much greater potential to progress to invasive cancer, with
reported progression rates of between 0.24% within 12 months.16 A proportion of high-grade CIN,
however, will also regress or persist, and this is probably related to the increasing evidence that not all
CIN3 and, in particular, CIN2 lesions are true pre-cancerous lesions.17,18 Although HPV-induced precancerous lesions may in some instances rapidly lead to cancer, the average total time from infection
with a carcinogenic HPV type to development of invasive cervical carcinoma is 2530 years.19
CIN2 tends to be a controversial diagnosis that is the least reproducible of all cervical diagnoses. The
biological behaviour of CIN2 is not well understood. Many clinicians treat CIN2 as a true precancerous
lesion and routinely treat these lesions, whereas others argue that CIN2 lesions do not exist.20
Notwithstanding this, CIN2 regression rates are reported at between 1523%, with up to 55% of
cases regressing within 46 years.17
Of course, the risk of progression and regression of precancerous lesions is greatly inuenced by the
persistence of specic high-risk HPV types. CIN2 lesions that are HPV 16 positive seem less likely to
regress than CIN2 lesions that are HPV16 negative.13,17
Natural history of human papillomavirus infections
The HPV lifecycle is closely linked to stratied epithelium differentiation.21 HPV virions infect the
basal epithelium through micro-abrasions in the epidermis. The exact mechanism of invasion is still

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not fully understood, but several receptors, including heparan sulphate proteoglycans and alpha-6
integrin have been associated with this process.5 Upon migration to the basal cell nucleus, the viral
genomes are established as episomes, and the early promoter activated, resulting in low levels of viral
synthesis. During normal epithelium differentiation, the daughter cells migrate from the basal layer
upwards and undergo terminal differentiation. They ultimately reach the epithelial surface where they
form a cornied layer of dead cells, which are eventually sloughed off. In HPV-infected differentiating
cells, the late promoter is activated, leading to the vegetative state of the HPV lifecycle.22 During this
phase, high levels of viral DNA are replicated and packaged into capsids and released from the cell. The
virus relies on the host cell replication machinery to maintain viral synthesis in the epithelium. This is
where the HPV oncoproteins E6 and E7 come into play, maintaining the cell cycle and preventing
terminal differentiation. As HPV-infected daughter cells move up through the epithelium, the virally
infected basal cell layer is maintained with a low level of viral DNA synthesis. This typically occurs in
low-grade cervical disease. High-grade CIN lesions, such as CIN3, are typically associated with HPV
DNA that has integrated into the host genome. Viral integration often occurs in the E1 and E2 regions
downstream of the late genes. This can result in disruption and loss of these late genes, with subsequent loss of control of oncogene expression by the E2 viral gene.23 To maintain the HPV infection,
high-risk HPV types produce E6 and E7 oncogenes, which interfere with critical cellcycle checkpoint
pathways and proteins, namely p53 and retinoblastoma.
Diagnosis of cervical intraepithelial neoplasia
No specic clinical features or symptoms indicate the presence of CIN. Initial diagnosis is usually
made by cytological analysis of a Pap smear specimen. Alternatively on direct visualisation, CIN lesions
can turn white upon application of 35% acetic acid or they may be iodine negative on application of
Lugols iodine solution. Final diagnosis, however, is only conrmed by histopathological examination of
biopsy or excised tissue specimens. An accurate histopathological diagnosis is a key factor in the
decision to treat or not treat an individual patient.
Cytological assessment of CIN is based on nuclear and cytoplasmic changes and can often be quite
challenging. Nuclear enlargement, hyperchromasia, irregular chromatin distribution and clumping are
the most common features of CIN in a cytological preparation (Fig. 1A and B). The primary basis for
assessing the specic grade of CIN is the ratio of nucleus to cytoplasm in a given cell. Higher ratios are
associated with higher grades of CIN.
Histological diagnosis and grading of cervical intraepithelial neoplasia
Traditionally, cervical intraepithelial abnormalities are graded as CIN1, CIN2 and CIN3, depending
on the degree of differentiation. A diagnosis of CIN relies on histological features, including differentiation, maturation and stratication of cells and nuclear abnormalities. In addition, the proportion
of the thickness of the epithelium and differentiated cells is used for grading CIN.More severe grades of
CIN are likely to have a thicker epithelium composed of undifferentiated cells with a narrow layer of

Fig. 1. Pap staining in ThinPrep smears from (A) moderate dyskaryosis and high-grade squamous intraepithelial lesion; (B) severe
dyskaryosis high-grade squamous intraepithelial lesion and cervical intraepithelial neoplasia 3; and (C) immunocytochemical
staining for p16INK4A in mildly dyskaryotic cells.

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mature undifferentiated cells on the surface. Nuclear abnormalities, such as enlarged nuclei, hyperchromasia and mitotic cell features are also assessed. HPV infections are also common in CIN, and are
characterised by the presence of koilocytosis.
In CIN1, the undifferentiated cells are conned to the lower layer of the epithelium. There tends to
be minimal nuclear abnormalities and few mitotic features (Fig. 2A and B). In CIN2, the dysplastic
cellular changes are restricted to the lower half of the epithelium. There also tend to be more marked
nuclear changes and more mitotic features (Fig. 2C). In CIN3, differentiation and stratication may be
totally absent or only present in supercial quarter of the epithelium. Nuclear abnormalities can be
seen throughout the thickness of the epithelium. Mitotic features are present throughout, with general
loss of polarity.
Accurate grading of CIN lesions is important for clinical management of patients, because CIN1 and
CIN2 and 3 lesions are treated differently. Histological diagnosis of CIN is complicated by a variety of
cellular changes associated with inammation, pregnancy, atrophy, or both. These changes may mimic
pre-cancerous cervical lesions, thereby making traditional cervical histology approaches subjective
and prone to variability. This is reected in poor inter-observer agreement between pathologists.2427
In particular, the differential diagnosis between immature squamous metaplasia and CIN1 and 2, or
between low-grade (CIN1) and high-grade (CIN2 and 3) lesions, tend to be difcult.24,26,27 To overcome
these problems, difcult lesions are usually adjudicated by more than one pathologist and a consensus
reached. Although CIN1 is generally over-interpreted in cervical pathology practice, clinical trials, such
as the New Technologies in Cervical Cancer24 and the ASCUS Low grade trial study,28 have shown that
most CIN1 cases downgraded at review were in fact HPV positive. The issues relating to accurate
diagnosis of CIN can result in over- or under-treatment of the lesion. Moreover, in early CIN lesions, the
progression rates to high-grade lesions are low (CIN1 and 2 to CIN3 is 922% and to invasive cancer is
15%). In spite of these low-risk rates, frequent cytological and colposcopical follow up (in CIN1) with
large biopsies are often carried out in women with these lesions. This effectively means that many
women unlikely to progress are being over-treated and followed up unnecessarily, causing a huge
burden on healthcare systems. Collectively, this emphasises the need for specic biomarkers to aid
objective CIN lesion grading, and to identify true high-grade cervical disease.
Utility of biomarkers for diagnosis of cervical intraepithelial neoplasia
The limitations that exist with current diagnostic strategies have accelerated the use of alternative,
more objective methods as adjuncts to histology, to resolve difcult to diagnose and uncertain cases.
With the enormous advances in gene proling and biomarker discovery technologies in recent years,
several molecular biomarkers have been described for CIN and cervical cancer, many of which are
involved in HPV-induced molecular alterations. A number of these markers have already been tested
and validated to identify dysplastic cells in cervical smear specimens, and therefore have potential to
enhance and improve current cervical screening performance and, in some instances, have therapeutic
potential.29 Of these, the most extensively used are Ki-67 and p16. Other biomarkers, including topoisomerase IIa (TOP2A), Survivin, MYBL2 and some of the minichromosome maintenance (MCM)
markers, show promising results. A selection of these will be discussed in detail below.

Fig. 2. Haematoxylin and eosin staining in CIN: (A) and (B) represent a CIN1 case (C) represents a CIN2 and 3 lesion in the cervix.
CIN, cervical intraepithelial neoplasia.

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Human papillomavirus, DNA and messenger RNA testing

In recent years, HPV testing has been implemented in triage cervical screening to help more
accurately diagnose CIN lesions and substantiate persistent HPV infections. Although HPV testing is
predominantly carried out on cervical cytology specimens, there is clear merit to providing this
information on HPV status to the histopathologist to assist in reviewing the case and assessing grade of
CIN. HPV DNA-based tests focus on detecting viral genomes from high-risk HPV types, whereas RNAbased tests detect expression of HPV oncogenes E6 and E7. As described above, over-expression of
these two oncogenes is necessary in the malignant transformation of HPV-infected cells. It is argued
that detection of these oncogenes allows better distinction between transient HPV infections and those
active infections that are likely to progress to a cancerous lesion.
Until relatively recently, HPV testing has focused on HPV DNA testing. Although it seems to be more
sensitive than cytology, it lacks specicity.3033 In contrast, detection of HPV mRNA generally seems to
be more specic (0.420.85, depending on which mRNA test was used), with a higher positive
predictive value (0.400.92 depending on which mRNA test was used), but less sensitive than DNA
testing for detection of high-grade disease.34 In general, E6/E7 mRNA testing seems to have clinical
utility for diagnosing CIN; however, the number of studies remain small and are variable in study
design and approach.34 Given the high prevalence of HPV DNA in the population, it is likely that HPV
mRNA may be more appropriate for triage of women with atypical squamous cells of undetermined
signicance and low-grade squamous intraepithelial lesions. Similarly, given the reported high specicity of mRNA testing, a combinatorial approach of HPV mRNA testing in triage, with cytology and
histology, may reduce the incidence of unnecessary treatment in women. Large-scale HPV mRNA
testing trials are obviously needed on well-dened populations of women to completely and accurately
assess the diagnostic and prognostic utility of HPV mRNA testing for CIN.
Ki-67
Ki-67 is a cellular marker for proliferation that is expressed at all stages during the cell cycle except
G0. Although Ki-67 has been used as a marker of cellular proliferation and as an aid for grading CIN, it is
not thought to be involved specically in the cervical carcinogenic process.3539 Ki-67 immunoquantitative analysis of CIN1 and CIN2 biopsies has been shown to have a strong independent
predictive value for grade and disease progression.3537 There is also clear evidence for the prognostic
value of Ki-67 staining in CIN, with reports suggesting that Ki-67 staining is superior to standard
histopathological grading to predict CIN progression.38 Kruse et al.38 present an attractive Ki-67
progression risk model that assesses the stratication index (Si90) and the percentage of Ki-67 positive cells in the middle-third layer of the epithelium to classify women into low risk or high risk
progression categories. Women at low risk are classied as such, where there is a combined Si90 less
than 0.57 and per cent of Ki-67 positive cells in the middle third of the less than 30%. All other scores
are considered high risk, with reported high reproducibility among pathologists.38 Several other
reports have described the utility of Ki67 in a combined approach with several additional markers,
including p16INK4a, MCM2 and TOPO2A. Ki67 staining in high-grade CIN (CIN2/3) is predominantly
nuclear, although the reported staining patterns across all CIN lesions are not necessarily characteristic
and discordance has been observed.39
p16INK4a (CDKN2A)
One of the most widely investigated biomarkers in cervical pre-cancer and cancer is p16INK4a.
p16INK4a is a cyclin-dependent kinase inhibitor (CDK4/6) involved in cellcycle regulation, through the
retinoblastoma gene complex. Cellular levels of p16INK4a protein are generally low, but it has been
found to be upregulated in HPV-infected cervical cells due to inactivation of the Rb complex by HPV E7
oncoprotein. E7 disrupts the protein of retinoblastoma from its binding to E2F transcription factor, and
thereby promotes cellcycle progression, a molecular switch that is usually activated by CDK4/6. HPVinfected cells producing E7 oncoprotein strongly express p16 to counteract the irregular cellcycle
activation; however, as E2F is no longer released through CDK4/6 action, p16 expression has no effect

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on cellcycle activation and accumulates in the cell over time. As a result, p16INK4a can be used as
a surrogate marker of active high-risk HPV infection. Over-expression of p16INK4a has been shown in
CIN and cervical cancer,4043 which increases with increasing grade of disease41,44 More recently,
p16INK4a has been described as a marker of progression, as its over-expression is strongly associated
with histologically conrmed CIN2 lesions.45
Studies on smear preparations and biopsy material have shown signicant over-expression of
p16INK4a in CIN and cGIN46,47 (Fig. 3A and 1C). In addition, a strong correlation between p16INK4a protein
expression in cervical dysplasias, and the presence of high-risk HPV infection has been shown.48 In this
review, we will focus of p16INK4a expression in histology specimens. A meta-analysis on p16INK4a
immunostaining on cytological and histological cervical specimens estimated that 2% of normal
biopsies and 38% of CIN1 showed diffuse staining for p16INK4a compared with 68% of CIN2 and 82% of
CIN3.47
The staining pattern of p16INK4a in cervical biopsy and large loop excision of the transformation
zone specimens is generally nuclear, although cytoplasmic staining has also been shown (Fig. 2A). No
clear guidelines, however, have been produced for histopathological assessment of nuclear compared
with cytoplasmic staining or indeed intensity of staining. The semi-quantitative scoring system
described by Klaes et al.42 is the most widely used approach. This scoring system denes less than 1%
positive cells as negative staining, less than 5% positive cells as sporadic staining, 520% positive
staining dened as focal staining, and less than 25% positive staining is considered diffuse staining. On
the whole, it is considered that a diffuse, positive, parabasal staining pattern is suggestive of a transforming hrHPV infection and accompanied high-grade CIN lesion, whereas p16INK4a immunoreactivity restricted to the lower part of the epithelium (one-third), focal scattered staining or absence of
staining is indicative of a CIN1 diagnosis.44,49,5052 In support of this, Negri et al.46 assessed the role of
p16 in predicting CIN1 lesions that were likely to progress to CIN3 in a 4-year follow up. The investigators concluded that, although p16 may be expressed in low-grade squamous lesions (CIN1) that
undergo spontaneous regression, cases with diffuse staining (> 25% cells stained) had a signicantly
higher tendency to progress to a high-grade lesion than p16-negative cases.
Clear evidence shows that a combined interpretation of haematoxylin and eosin and p16INK4a
staining is a good approach to improve diagnostic accuracy. This combined approach has been shown
to reduce the number of high-grade CIN diagnoses, and seems to offer more reassurance to pathologists in grading some of the more difcult low-grade lesions.50,53,54 Similarly, a reduced incidence of
missed high-grade CIN cases has been reported when a combined H&E and p16INK4a staining approach
is used.53,54
Despite this, there remains considerable reluctance among histopathologists to incorporate
p16INK4a staining into the routine gynae-pathology repertoire of tests. This reluctance may, in part,
stem from the confusion that surrounds the literature (i.e. correlation of p16INK4a positivity and integration, and grading of p16INK4a positivity). Different studies use different antibodies and have different
interpretation of staining intensity. In order to implement p16INK4a staining into routine gynaepathology, standardised protocols for interpreting p16INK4a immunoreactivity, with standardised
validated antibodies and scoring schemes, are required. In addition, interpretation of p16INK4a staining

Fig. 3. Biomarkers in CIN: (A) p16INK4A immunohistochemical staining in CIN2; (B) MCM3 immunohistochemical staining in cGIN;
(C) MCM3 immunohistochemical staining in maturing metaplasia. CIN, cervical intraepithelial neoplasia. MCM, minichromosome
maintenance.

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can be hampered by staining of non-dysplastic cells. Endometrial, metaplastic and endocervical cells,as
well as tubo-endometrioid metaplasia, have all been observed to stain positive for p16INK4a.31,55 Trained
cytologists and histopathologists should be adept at distinguishing between benign cells and abnormal
cells to interpret p16INK4a immunostaining accurately.
Topoisomerase IIa and minichromosome maintenance proteins
Topoisomerase IIa (TOP2A) is a nuclear enzyme involved in DNA replication. Its expression is related
to the cell cycle, with lowest levels of expression occurring in G0 and G1 phases of the cell cycle. Gene
expression proling studies have shown increased expression of TOP2A in cervical cancer cell lines and
invasive cervical cancer.56,57 Immunohistochemical analysis has also shown increased TOP2A protein
expression levels in CIN and cervical cancer compared with normal cervical epithelium.58,59
Minichromosome maintenance (MCM2) is a member of the DNA licensing protein family, which is
involved with licensing DNA for replication, and thus is a marker of proliferation. This process of DNA
licensing requires the regulated assembly of pre-replicative complexes consisting of MCM proteins, cell
division cycle protein 6 (cdc6) and cdt1, onto the origins of replication scattered along each chromosome.60 Cdc6 and cdt1 act as MCM-loading proteins. Once loaded onto the origin of replication, the MCM
complex forms a hexameric ring, which acts as a rotary motor that pumps DNA along its helicase axis. As
DNA replication proceeds, MCM proteins are phosphorylated, the MCM complex becomes dissociated
from the chromatin and is prevented from re-binding DNA until late mitosis, by the inactivation of its
loading factors, cdc6 and cdt1. Cdc6 inactivation is achieved by phosphorylation, whereas cdt1 is
inactivated via binding to its inhibitor, geminin.61,62 Overexpression of MCM 2,4,5 and 6, cdc6 and cdt1
have all been identied in gene expression proling experiments56,57 in keeping with the premise that
cellcycle dysregulation is a key factor in the development of cervical cancer. Both immunohistochemical analysis and polymerase chain reaction studies have shown over-expression of cdc6, MCM3
and MCM5 in cervical cancer6365 (Fig. 3B and C). Again, several studies have shown increased expression
of MCM2 and some of the other MCM family members in cervical pre-cancer and cancer.
ProEx C is an immunocytochemical assay composed of two monoclonal antibodies directed
against TOP2A and MCM2 protein. Studies using ProExC have described the characteristic staining
patterns seen in CIN,39,66 which seem to increase with increasing grades of disease.67 The staining
pattern in high-grade CIN shows strong nuclear staining in more than 50% of dysplastic cells, whereas
in low-grade CIN, varying degrees of scattered positive cells are observed.39 In a combined approach
with p16 immunostaining, ProExC demonstrates higher specicity but lower sensitivity than p16
staining for detection of CIN3,67 suggesting that a combined approach using these two biomarkers
can be used together to distinguish CIN 2/3 from its mimics in cervical biopsy specimens.67 The utility
of ProExC for diagnosis of low-grade CIN is more contentious.
Survivin
Survivin is a recently discovered member of the family of inhibitor of apoptosis proteins, and is thought
to have a role in cell death and cell division. Over-expression of survivin has been shown in a wide range of
cancers,68 including cervical cancer.56 Its expression is associated with resistance to treatment, an
increased risk of recurrence, and a poor survival rate.68 Expression of survivin protein and mRNA in
cervical pre-cancer increases with increasing grades of dysplasia, and is associated with high-risk HPV
infection.69,70 Degradation of the p53 tumour-suppressor protein, through the interaction with HPV E6
oncoproteins, is suggested as the mechanism of over-expression of survivin in cervical dysplasia.71
MYBL2
MYBL2 (B-MYB) is a member of the MYB proto-oncogene family that encode DNA-binding proteins.
These proteins are involved in cell proliferation and control of cellular differentiation.72 Its transcription
levels have been shown to be tightly regulated during the cell cycle by an E2F-dependent mechanism,
being induced to a high level only in late G1 and S phase.73 This suggests that MYBL2 is involved in
activating genes involved in G1/S phase progression. It has also been shown to play a role in preventing

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apoptosis74 and, in addition, its expression levels are affected by HPV 16.75 Furthermore, our group76
have shown over-expression of MYBL2 cervical pre-cancer CIN, cGIN and invasive cancer. The immunohistochemical pattern of expression of MYBL2 in all cases of CIN, cGIN and invasive carcinoma of the
cervix is nuclear, with a staining score of 2. MYBL2 is not identied in most normal cervical epithelial
cells. In cases of CIN, MYBL2 staining is nuclear and strong in most cells, with the exception of the most
terminally differentiated cells. These staining patterns are in keeping with previously published tissue
microarray data showing absent MYBL2 expression in normal cervix, with positive staining in CIN and
cervical cancer, suggesting a potential role for this marker in histological diagnosis of CIN.57,76
Conclusion
An accurate diagnosis of CIN is important for clinical management because CIN1 and CIN2/3 lesions
are treated differently. Histological diagnosis of CIN can be complicated by the variety of cellular
changes that can be associated with inammation, pregnancy and hormonal therapies, which can
mimic pre-cancerous lesions. This makes histological diagnosis prone to subjectivity and variability,
which is clearly reected in the poor intra- and inter-observer agreement between pathologists.
Incorporation of biomarkers into diagnostic strategies has the potential to improve current cervical
screening performance and management of disease. In addition to HPV DNA and mRNA testing,
a number of biomarkers, including ki-67 and p16INK4A, are extensively used to assist with diagnosis of
difcult-to-diagnose cases. Other markers, such as some of the minichromosome maintenance
proteins, and TOPO2A, MYBL2 and survivin have also shown clear utility. For cervical cancer diagnosis,
we believe a combined approach of histological diagnosis, and biomarkers such as HPV DNA/mRNA,
p16ink4A/ki67, and TOPO2A/MCM2, has the potential to stratify women into different diagnostic
categories associated with different levels of risk.

Practice points
 Persistent infection with one of the oncogenic HPV subtypes is a necessary cause for cervical
neoplasia.
 CIN can be categorised into three grades, CIN1, 2 and 3, depending on the proportion of the
thickness of the epithelium showing abnormalities.
 Most low-grade CIN lesions regress within a relatively short period and do not progress to
high-grade lesions.
 High-grade lesions are signicantly more likely to progress to invasive cancer.
 The combined interpretation of haematoxylin and eosin and p16INK4a staining can signicantly improve the accuracy of interpreting and grading cervical lesions on biopsy samples.

Research agenda
 A large-scale clinical trial is required to demonstrate the utility of HPV mRNA and p16ink4a
expression for diagnosis and prognosis of CIN.
 A randomised HPV and biomarker intervention trial is required to fully evaluate the utility of
this combined approach in managing CIN.
 Independent validation of new biomarkers, such as MYBL2 and surviving, in diagnosing lowand high-grade CIN is required.
 The utility of combined HPV and biomarker testing for difcult to diagnose and rare lesions
such as adenocarcinoma and villoglandular adenocarcinoma is required.
 HPV genotyping of low- and high-grade lesions in women vaccinated against HPV is required.

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Acknowledgements
The authors wish to acknowledge members of the Cervical Cancer Research Group at the Coombe
Women and Infants University Hospital and Trinity College, Dublin and CERVIVA; The Irish Cervical
Screening Research Consortium. The group are funded by the Health Research Board, Cancer Research
Ireland, The Royal City Of Dublin Trust, The Meath Foundation, Friends of the Coombe, Science
Foundation Ireland and The European Union.
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