Cervical cancer is the third most frequent malignancy in women worldwide with almost half a million cases diagnosed each year.¹ In Ireland, the annual incidence in 2007 was 286 cases, resulting in 83 deaths.²

Cervical cancer is initiated by progression through a well-defined pre-malignant phase known as cervical intraepithelial neoplasia (CIN). This begins as a slow progressive disruption of normal epithelium surrounding the transformation zone. It is staged from CIN1-CIN3 based on the degree of neoplasia, if left untreated lesions can develop into cervical cancer. 

In Ireland, the National Cervical Screening Programme, CervicalCheck, was introduced in late 2008 and provides free smear tests for detecting pre-invasive neoplasia to women aged 25-60. Since its launch the programme has proved very successful with an average of 1,000 women availing of a free smear test per day during the first two years of operation. The latest statistics available from the programme indicate that in excess of 280,000 women were screened during the second year, the majority of whom (85%) had a negative smear test; of the remaining 15%, low-grade abnormalities accounted for 12.5% and high-grade abnormalities 1.7%. In total, 16,811 women attended a colposcopy clinic for the first time, with 7,546 receiving treatment.²

HPV infection

Human papillomavirus (HPV) is the single most important aetiological agent in the pathogenesis of cervical cancer and pre-cancer. There are 15-20 types associated with cervical cancer, of which HPV 16, 18, 31, and 45 account for 80% of cervical cancers.³ Up to 80% of sexually active women will acquire a HPV infection at some stage in their lifetime. 

HPV infection is more common in younger women, reaching a peak of approximately 20% among women between 20 and 24 years of age, with a subsequent decline among women over 30 years of age.⁴ In Ireland, HPV prevalence is estimated at 19% in women aged 25-60 years and as high as 30% in women under 30 years of age.⁵

Secondary to HPV infection, cigarette smoking is one of the main risk factors associated with progression of lesions and development of high-grade CIN.⁶ The high prevalence of smoking among young women will have a profound effect on future incidence of cervical cancer. 

Cytology screening based on the Pap smear is the method used for cervical screening, however, the Pap test is a subjective test with many limitations, specifically in relation to diagnosis of low-grade abnormalities LSIL (low-grade squamous intraepithelial lesions ) and ASCUS (atypical squamous cells of undetermined significance).⁷ Issues including low sensitivity, equivocal results and inability to distinguish progressive disease have provoked the use of HPV tests in screening programs. It is now widely accepted that HPV testing has a role in cervical cancer prevention in both the pre and post HPV vaccination era. 

The roles include: 

• As a primary screening tool in women over 30 years
• For triage of women with low grade abnormalities; and 
• In the follow-up of women treated for cervical intraepithelial neoplasia (CIN).⁸

CervicalCheck, like other screening programmes internationally, is due to introduce HPV testing into the Irish screening programme. Since autumn 2011, HPV testing has been used in conjunction with cytology in the follow-up of women treated for CIN. HPV DNA testing is currently used in cervical screening in the United States⁹ and has recently been approved for triage of LSIL/ASCUS in the UK.¹⁰

Issues in management

A significant proportion of cervical abnormalities will regress spontaneously and do not require treatment, however, currently there is no test to distinguish between those that will progress to malignancy and those women that will regress. There remains considerable controversy over the appropriate management of low-grade abnormalities with standard management consisting of cytology screening and/or colposcopic referral. The proportion of women presenting with persistent low-grade abnormalities (LSIL/ASCUS) that go on to develop a high-grade lesion is in the region of 28%. There is a clear need to develop an improved screening algorithm to identify which of these low-grade lesions will progress to a high-grade lesion. 

HPV DNA testing, although useful, is still likely to result in over-treatment and unnecessary follow-up of women with low-grade abnormalities, given the high prevalence of transient HPV infection among sexually active women. 

The Irish Cervical Screening Research Consortium (CERVIVA) has demonstrated that HPV DNA testing, while it seems to be more sensitive than cytology, is lacking in specificity. In a cross-sectional study of women referred to colposcopy with low-grade abnormalities, the sensitivity of HPV DNA testing for detection of high-grade disease was 90% (95% CI 0.8168-0.9569) and specificity 55% (95% CI 0.4849-0.6327). 

There is similar evidence from the ALTS Study (US) and the TOMBOLA study (UK), that HPV DNA testing in particular in low-grade cytology is of limited value.^11,12 Early evidence suggests that detection of HPV E6/E7 mRNA is more indicative of a clinically significant infection than a HPV DNA testing approach.^13,14

Incorporation of HPV mRNA and other biomarkers into cervical screening programs has the potential to stratify patients based on their real risk of developing a high-grade lesion or cancer.

Approaches to HPV testing

There are three basic approaches to HPV testing:

• HPV DNA testing
• HPV genotyping
• HPV mRNA testing. 

HPV DNA testing and genotyping methods are all based on detection of the L1 region of the HPV genome and will not distinguish between transient and integrated oncogene expressing HPV infection.

Persistent infection can lead to integration into the host genome, these transforming infections are considerably more likely to develop high-grade CIN and initiate malignant transformation than women who clear the infection. 

HPV mRNA tests detect the viral transforming genes HPV E6/E7. The E6 oncogene interacts with cellular factors, primarily p53, resulting in deregulation of the cell cycle and DNA repair mechanism. E7 targets pRB disrupting its association with transcription factor E2F, resulting in transactivation of genes involved in DNA synthesis and progression through S phase including p16INK4a, TOPO2A, MCMs.³ Evidence to date indicates that HPV mRNA detection has the potential to offer a more specific test for identifying women at risk of developing high-grade disease.^14-16 The data suggests that HPV mRNA testing has an overall specificity of between 76-86% for detection of CIN2+. However, many of these studies focus on women in a colposcopy setting. There are few large scale studies that concentrate on HPV mRNA testing as a triage for LSIL/ASCUS. 

CERVIVA has so far examined the HPV status of 960 women between the ages of 18 and 65 years (median age 30 years), referred to colposcopy for investigation of persistent LSIL/ASCUS. The HPV DNA and mRNA prevalence at first visit to colposcopy is shown in Figure 1. In contrast to HPV DNA, the prevalence of HPV mRNA in this population was 38% compared to 61% for HPV DNA. CERVIVA also established that detection of HPV mRNA appears to be more specific, 79% (95% CI 0.7277-0.8495) than HPV DNA, 55% (95% CI 0.4849-0.6327) for detection of high-grade disease. However, mRNA testing alone is not optimal, since sensitivity (71%) is lower than DNA testing and cytology. 

 (click to enlarge)

If HPV triage is to be incorporated into cervical screening programmes, there is clear need for additional markers to achieve a screening test with improved specificity and at least equivalent sensitivity to current methods. 

Biomarkers

Several host cell biomarkers have been evaluated for their potential to improve diagnostic specificity of cervical screening. These markers include p16INK4a, which plays a major role in cell cycle regulation, Ki-67 a proliferation marker, topoisomerase 11-α (TOPO2A) a regulator of DNA topology and minichromosome maintenance protein 2 (MCM2), a DNA licensing factor. 

It has recently been proposed that combined detection of these novel biomarkers has utility as a surrogate marker of transforming HPV infections.^17,18

This has led to the development of commercial assays, such as ProExC (Becton Dickinson [BD]), which simultaneously detects TOPO2A/MCM2, and CINtec PLUS (MTM Laboratories), which detects over expression of p16INK4a/Ki-67 proteins. 

CERVIVA is currently evaluating a selection of novel biomarkers, including p16INK4a in conjunction with HPV DNA and mRNA, for their utility in stratifying women presenting with low-grade abnormalities. The findings to date demonstrate dual overexpression of p16INK4a and Ki-67 in women who go on to develop high-grade abnormalities (see Figure 2 [D]). 

 (click to enlarge)

From these findings CERVIVA propose that combining HPV mRNA and p16INK4a/Ki-67 detection will be more specific and have a higher PPV with equivalent sensitivity to HPV DNA and cytology. 

Smoking and cervical pre-cancer

Persistent HPV infection is considered the strongest epidemiology risk factor for cervical cancer. However, while it is necessary it is not a sufficient cause given the high prevalence of transient HPV infections in younger women. 

Work performed under CERVIVA examined the relationship between cigarette smoking and HPV infection for the first time using analysis of cotinine concentrations. 

Data from this study found 61% of LSIL/ASCUS population were positive for HR HPV DNA. Only a small percentage of these will go on to develop cervical cancer or its immediate precursor CIN3, thus co factors are believed to be involved in the risk of transition from cervical HPV infection to cervical cancer. 

The involvement of smoking and development of cervical cancer was first suggested by Winkelstein in 1977.¹⁹ In 2003, an International Agency for Research on Cancer (IACR) multicentre study by Plummer et al reported that smoking increases risk of cervical cancer in HPV positive women. The IACR went on to list cervical cancer as causally related to smoking.²⁰ Evidence is based on a number of factors including the presence of nicotine derivates in cervical mucus,²¹ increased presence of DNA adducts in the cervical epithelium of smokers,²² local immunosuppression in the cervix.²³

CERVIVA found that heavy smokers (representing approximately > 10 cigarettes per day) had a significantly higher risk of developing high-grade disease. 

This supports evidence of a synergy between cigarette smoking and HPV infection in development of high-grade cervical disease,²⁴ highlighting the importance of awareness of patient history. Counselling of HPV positive women to abstain from smoking may be warranted.

Conclusion 

There is a high prevalence of HR-HPV infections in women presenting to colposcopy with ASCUS/LSIL smears. Using a combined mRNA/ p16INK4a/Ki-67 approach in the management of women referred to colposcopy with abnormal smears could help to stratify women at high risk of developing high-grade cervical disease from those at minimum or no risk. 

This will allow clinicians to direct resources into monitoring women at high risk while those women with no risk or very low risk can be monitored by more conservative, less costly methods. 

This approach will help in reducing costs to healthcare providers in addition to reducing the psychological impact on women associated with receiving persistent abnormal smear results and attending follow-up investigations and treatments. 

Acknowledgments 

This work is carried out under the umbrella of CERVIVA the Irish Cervical Screening Research Consortium funded by the HRB and is supported by the Irish Cancer Society. Christine White is an Irish Cancer Society funded PhD scholar.

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