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 difficult. These difficulties mean that patients tend to be over-treated for CIN lesions, which will naturally regress. Collectively, this highlights the need for alternative approaches and specific 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 classification of CIN and the diagnostic and prognostic utility of specific biomarkers in identifying true cancerous precursor lesions.
Introduction
Cervical cancer is the third most common cancer in women worldwide, with an estimated 529,000 new cases diagnosed in 2008. It is usually preceded by a long phase of pre-invasive disease. This pre-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 10–20 years. The introduction of cervical screening programmes have greatly reduced the incidence of cervical cancer; however, CIN rates remain significantly high. In this review, we will focus on the aetiology, pathobiology, the natural history of CIN, diagnosis and classification 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. About 15–20 types are associated with cervical cancer, of which HPV16, 18, 31, and 45 accounts for 80% of cervical cancers. 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 60–80% of high-grade CINs and in 75% of adenocarcinoma in situ cases.
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. Most of these infections are transient, with a median duration of 6–14 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). Women with integrated HPV virus are significantly more likely to develop severe dysplasia and malignancy than those who clear the infection, 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.
Aetiology of cervical intraepithelial neoplasia
Human papillomavirus (HPV) is the single most important causative agent in the pathogenesis of cervical cancer and pre-cancer. About 15–20 types are associated with cervical cancer, of which HPV16, 18, 31, and 45 accounts for 80% of cervical cancers. 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 60–80% of high-grade CINs and in 75% of adenocarcinoma in situ cases.
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. Most of these infections are transient, with a median duration of 6–14 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). Women with integrated HPV virus are significantly more likely to develop severe dysplasia and malignancy than those who clear the infection, 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.
Natural history of cervical intraepithelial neoplasia
HPV is the major causative agent in the development of CIN. Despite women’s 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 70–80%; however, in adolescents and young women under 25 years, more than 90% show regression. In contrast, high-grade abnormalities, specifically CIN3, has a much greater potential to progress to invasive cancer, with reported progression rates of between 0.2–4% within 12 months. 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. Although HPV-induced pre-cancerous 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 25–30 years.
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. Notwithstanding this, CIN2 regression rates are reported at between 15–23%, with up to 55% of cases regressing within 4–6 years.
Of course, the risk of progression and regression of precancerous lesions is greatly influenced by the persistence of specific high-risk HPV types. CIN2 lesions that are HPV 16 positive seem less likely to regress than CIN2 lesions that are HPV16 negative.
Natural history of human papillomavirus infections
The HPV lifecycle is closely linked to stratified epithelium differentiation. HPV virions infect the basal epithelium through micro-abrasions in the epidermis. The exact mechanism of invasion is still not fully understood, but several receptors, including heparan sulphate proteoglycans and alpha-6 integrin have been associated with this process. 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 cornified 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. 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. To maintain the HPV infection, high-risk HPV types produce E6 and E7 oncogenes, which interfere with critical cell–cycle checkpoint pathways and proteins, namely p53 and retinoblastoma.
Diagnosis of cervical intraepithelial neoplasia
No specific 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 3–5% acetic acid or they may be iodine negative on application of Lugol’s iodine solution. Final diagnosis, however, is only confirmed 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. 1 A and B ). The primary basis for assessing the specific 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 stratification 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 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 confined to the lower layer of the epithelium. There tends to be minimal nuclear abnormalities and few mitotic features ( Fig. 2 A 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. 2 C). In CIN3, differentiation and stratification may be totally absent or only present in superficial 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 inflammation, 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 reflected in poor inter-observer agreement between 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 difficult. To overcome these problems, difficult 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 Cancer and the ASCUS Low grade trial study, 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 9–22% and to invasive cancer is 1–5%). 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 specific 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 difficult to diagnose and uncertain cases. With the enormous advances in gene profiling 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. 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.
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 RNA-based 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 specificity. In contrast, detection of HPV mRNA generally seems to be more specific (0.42–0.85, depending on which mRNA test was used), with a higher positive predictive value (0.40–0.92 depending on which mRNA test was used), but less sensitive than DNA testing for detection of high-grade disease. 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. 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 significance and low-grade squamous intraepithelial lesions. Similarly, given the reported high specificity 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-defined 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 specifically in the cervical carcinogenic process. Ki-67 immunoquantitative analysis of CIN1 and CIN2 biopsies has been shown to have a strong independent predictive value for grade and disease progression. 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. Kruse et al. present an attractive Ki-67 progression risk model that assesses the stratification 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 classified 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. Several other reports have described the utility of Ki67 in a combined approach with several additional markers, including p16 INK4a , 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.
p16 INK4a (CDKN2A)
One of the most widely investigated biomarkers in cervical pre-cancer and cancer is p16 INK4a . p16 INK4a is a cyclin-dependent kinase inhibitor (CDK4/6) involved in cell–cycle regulation, through the retinoblastoma gene complex. Cellular levels of p16 INK4a 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 cell–cycle progression, a molecular switch that is usually activated by CDK4/6. HPV-infected cells producing E7 oncoprotein strongly express p16 to counteract the irregular cell–cycle activation; however, as E2F is no longer released through CDK4/6 action, p16 expression has no effect on cell–cycle activation and accumulates in the cell over time. As a result, p16 INK4a can be used as a surrogate marker of active high-risk HPV infection. Over-expression of p16 INK4a has been shown in CIN and cervical cancer, which increases with increasing grade of disease More recently, p16 INK4a has been described as a marker of progression, as its over-expression is strongly associated with histologically confirmed CIN2 + lesions.
Studies on smear preparations and biopsy material have shown significant over-expression of p16 INK4a in CIN and cGIN (Fig. 3 A and 1 C ). In addition, a strong correlation between p16 INK4a protein expression in cervical dysplasias, and the presence of high-risk HPV infection has been shown. In this review, we will focus of p16 INK4a expression in histology specimens. A meta-analysis on p16 INK4a 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.
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