New technologies for cervical cancer screening seek to provide an accurate, efficient and cost-effective way of identifying women at risk for cervical cancer. Current screening uses human papilloma virus DNA testing combined with cytology, and requires multiple visits at a great cost to the patient and society. New methods for screening include HPV diagnostics (detection of either the presence of human papilloma virus or integration of the virus into the host cell), proliferation, and detection of epigenetic changes, either in the host or virus. These methods show promise in changing the way that current cervical cancer screening is undertaken in low-and high-resource settings.
Epidemiology of cervical cancer
We have known how to screen for squamous cell carcinoma of the cervix (SCCC) since the 1940s; however, it is still the second most common cancer diagnosed among women worldwide. Virtually all SCCC are caused by persistent infection with human papillomavirus (HPV), most commonly HPV types 16 and 18. In the last half century in high-resource settings, such as the USA, screening strategies that identify cervical high-grade squamous intraepithelial lesions (HSIL) have reduced the incidence and mortality of SCCC by over 50%. Current technologies, however, are relatively inefficient at identifying individuals at risk for disease, and require longitudinal testing over a woman’s lifetime. This type of screening is not feasible in low-resource settings. Accordingly, on a global scale, SCCC is the third most common cause of cancer-related death in women, resulting in 309,800 deaths worldwide in the year 2007.
Squamous cell carcinoma of the cervix is preventable because effective screening strategies that identify the precursor lesion may allow the disease to be cured. The two major histologic types of cervical cancer include SCCC and adenocarcinoma. Squamous cell carcinoma of the cervix is the most common type, representing 70% of cases. Adenocarcinoma, which is more commonly associated with HPV type 18, comprises about 25% of cases. In the USA, the incidence of adenocarcinoma seems to be rising. Adenosquamous carcinoma is the least common and comprises about 3–5% of cases.
In high-resource settings, cervical cancer is the seventh most common female cancer. In the USA, the annual incidence of SCCC is 12,200 women, and the annual mortality is 4210 women. Because of differences in access to medical care, cervical cancer is disproportionately diagnosed in minorities and among women of low socioeconomic status. According to the American Cancer Society, the incidence of disease in African–American women is 10.8 cases of cervical cancer per 100,000 women. The incidence of disease in Hispanic women is 12.7 cases of cervical cancer per 100,000 women. In contrast, the incidence of disease in white women is 8.2 cases per 100,000 women. Globally, cervical cancer is much more common in low-resource settings compared with high-resource settings. Eighty per cent of the 555,100 new cases worldwide per year are diagnosed in low-resource settings. Because disease is not diagnosed until it is late-stage, and because treatment also requires infrastructure and resources, more than 85% of the 309,800 deaths from SCCC in the year 2007 occurred in low-resource settings.
Aetiology of cervical cancer
Persistent mucosal infection with an oncogenic (high risk) HPV genotype, including types 16, 18, 33, 45, 31, 58, 52 and 35, is the most significant cause of cervical cancer. Human papilloma virus types 16 and 18 are the genotypes most commonly associated with disease, and are identified in 70% of SCCC cases. Human papilloma virus infection is transmitted by direct contact, and is common among sexually active men and women. The estimated prevalence of infection ranges from 50–80%. Risk factors for developing cervical disease include age of sexual debut, number of sexual partners, prolonged use of oral contraceptive pills, high parity, cigarette smoking, co-infection with human immunodeficiency virus or other sexually transmitted infections, and chronic immunosuppression.
Although HPV infection causes cervical cancer, most HPV infections do not lead to cervical cancer. Human papilloma virus infection is easily and silently transmitted, as it does not cause symptoms. About 90% of HPV infections resolve within several months of initial infection. Persistent viral infection is the single biggest risk factor for the development of high-grade dysplasia and progression to cervical cancer.
Transient HPV infections correlate with low-grade squamous intraepithelial lesion (LSIL) cytology or cervical intraepithelial neoplasia 1 (CIN1) histology. Persistent oncogenic HPV infections correlate with HSIL cytology or CIN2 and 3 histology. Persistent infections are associated with integration of the viral genome into the host genome and subsequent transformation. After viral integration, two viral gene products, E6 and E7, are expressed, both of which are necessary but not sufficient for disease initiation and persistence. These oncoproteins bind to, and disrupt, the function of tumour suppressor genes p53 and the retinoblastoma protein, respectively. Disruption of these genes causes blocked apoptosis and cell–cycle arrest, leading to dysplasia. The expression of viral oncoproteins in dysplastic epithelial cells, and the indolent biology of intraepithelial HPV lesions together present many opportunities to prevent the development of SCCC by carrying out routine screening.
Aetiology of cervical cancer
Persistent mucosal infection with an oncogenic (high risk) HPV genotype, including types 16, 18, 33, 45, 31, 58, 52 and 35, is the most significant cause of cervical cancer. Human papilloma virus types 16 and 18 are the genotypes most commonly associated with disease, and are identified in 70% of SCCC cases. Human papilloma virus infection is transmitted by direct contact, and is common among sexually active men and women. The estimated prevalence of infection ranges from 50–80%. Risk factors for developing cervical disease include age of sexual debut, number of sexual partners, prolonged use of oral contraceptive pills, high parity, cigarette smoking, co-infection with human immunodeficiency virus or other sexually transmitted infections, and chronic immunosuppression.
Although HPV infection causes cervical cancer, most HPV infections do not lead to cervical cancer. Human papilloma virus infection is easily and silently transmitted, as it does not cause symptoms. About 90% of HPV infections resolve within several months of initial infection. Persistent viral infection is the single biggest risk factor for the development of high-grade dysplasia and progression to cervical cancer.
Transient HPV infections correlate with low-grade squamous intraepithelial lesion (LSIL) cytology or cervical intraepithelial neoplasia 1 (CIN1) histology. Persistent oncogenic HPV infections correlate with HSIL cytology or CIN2 and 3 histology. Persistent infections are associated with integration of the viral genome into the host genome and subsequent transformation. After viral integration, two viral gene products, E6 and E7, are expressed, both of which are necessary but not sufficient for disease initiation and persistence. These oncoproteins bind to, and disrupt, the function of tumour suppressor genes p53 and the retinoblastoma protein, respectively. Disruption of these genes causes blocked apoptosis and cell–cycle arrest, leading to dysplasia. The expression of viral oncoproteins in dysplastic epithelial cells, and the indolent biology of intraepithelial HPV lesions together present many opportunities to prevent the development of SCCC by carrying out routine screening.
Current cervical cancer screening methods
The goal of cervical cancer screening is to identify women at risk for developing the disease: that is, those with the immediate precursor lesion, high-grade squamous intraepithelial lesions. Current screening for cervical cancer is highly dependent on the type of resources available in the population being screened. In high-resource settings, routine screening includes pap smears over the course of a lifetime to evaluate for cervical dysplasia. Evaluation may or may not include screening for high-risk HPV, depending on the age of the woman. If abnormal cytology is detected, then the woman may either have more frequent pap smears, or may be referred to colposcopy for further evaluation. This type of screening allows for close evaluation of the cervix and early excision of high-grade dysplasia in appropriate cases. The American Congress of Obstetricians and Gynecologists currently recommends that cervical cytology screening begins at age 21 years, and is repeated thereafter every 2 years for women aged 21–29 years, and every 3 years for women aged 30 years or older who have had three prior normal pap smears. More frequent screening is recommended for women who are immunosuppressed, women infected with human immunodeficiency virus, women exposed to diethylstilbestrol in utero , and women previously treated for CIN 2, CIN 3 or cancer. Screening may be discontinued in women aged 65–70 years with three prior consecutively normal pap smears, and no abnormal pap smears over a period of 10 years.
In addition to repetitive cytology screening, many providers in high-resource settings implement concurrent testing for oncogenic HPV DNA in women with either an atypical squamous cells of undetermined significance (ASCUS) pap smear or among women who are over 30 years. Three types of tests to detect oncogenic HPV DNA have been approved by the Food and Drug Administration (FDA). The Hybrid Capture 2 test, approved by the FDA in 2003, detects 13 oncogenic HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) using full genome probes complementary to HPV DNA, specific antibodies, signal amplification, and chemiluminescent detection. The Cervista ® HPV HR test, approved by the FDA in 2009, detects 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) using a signal amplification method for detecting specific nucleic acid sequences. This method uses a primary reaction that occurs on the targeted DNA sequence and a secondary reaction that produces a fluorescent signal. These two tests have two limitations. First, neither test differentiates between single HPV genotype infections and multiple concurrent HPV genotype infections. Second, neither test quantitates viral load. The third, and newest HPV DNA test, Cervista ® HPV 16/18, was approved by the FDA in 2009, and detects only HPV 16 and 18, the genotypes most commonly associated with cancer, using a similar method to the Cervista ® HPV HR assay. Among women with HSIL cytology, HPV 16 is detected in 45.4%, and HPV 18 in 6.9%.
Detection of oncogenic HPV with HPV DNA screening tests is an effective strategy in the triage of cytology interpreted as ASCUS. Substantial research suggests that, in women over 30 years, HPV testing may be a cost-effective and accurate means of primary screening. Cuzick et al. retrospectively examined HPV testing and cytology samples in 60,000 European and US women between the ages of 30 and 60 years. Human papilloma virus DNA testing was more sensitive in detecting cervical intraepithelial neoplasia grade 2 or 3 (CIN2+) than cytology (96.1% v 53.0%), but less specific (90.7% v 96.3%). The sensitivity of HPV testing was similar among different areas of Europe and the USA, whereas the sensitivity of cytology in these areas varied. Another study evaluating HPV testing and pap smear cytology in 10,154 Canadian women aged 30–69 years identified sensitivities and specificities similar to those shown in the study by Cuzick et al. In the Canadian cohort, the sensitivity of HPV DNA testing for identifying CIN2+ was 94.6% (95% CI 84.2 to 100) and the specificity was 94.1% (95% CI 93.4 to 94.8). In contrast, the sensitivity of Pap smear was significantly lower (55.4% 95% CI 33.6 to 77.2; P = 0.01). The specificity of Pap smears, however, was similar to HPV testing (96.8% 95% CI 96.3 to 97.3; P < 0.001). The sensitivity of both tests used concurrently was 100% with a specificity of 92.5% ( Table 1 ). Because these screening methods are complementary, many high-resource settings have implemented algorithms that incorporate both. The use of cytology and HPV detection has reduced the incidence of cervical cancer in the USA from 14.8 per 100,000 in 1975 to 6.8 per 100,000 in 2008.
| Test | Sensitivity (%) | Specificity (%) |
|---|---|---|
| Pap smear | 53–55.4 | 96.3–96.8 |
| High-risk human papilloma virus DNA testing | 94.6–96.1 | 90.7–94.1 |
| Pap smear plus high-risk human papilloma virus testing | 100 | 92.5 |
Despite the effectiveness of using cytology and HPV DNA testing to detect disease, it is expensive and cumbersome. Many women undergo repetitive Pap smears and colposcopy for evaluating low-grade dysplastic lesions that are likely to resolve over time. Repetitive clinic visits and testing places a psychological burden on the woman, but also places economic strain upon the society providing the screening. In the USA alone, it has been estimated that 6 billion dollars per year are spent on evaluating low-grade lesions.
Screening for cervical cancer is restricted by financial resources and the social infrastructure of the society being screened, relying on methods that are low-cost and require few visits to the clinic. Accordingly, alternative methods of screening that may be implemented quickly and cheaply, such as visual inspection alone or visual inspection with a magnifying device, are currently used in low-resource settings.
Visual inspection involves evaluating the cervix with the naked eye, using either dilute acetic acid solution (VIA) or Lugol’s iodine solution to identify cervical lesions. Visual inspection using acetic acid wash has a sensitivity of 79% (95% CI 73 to 85%) and a specificity of 85% (95% CI 81 to 89%) for the detection of CIN2+ lesions. The use of Lugol’s iodine solution can increase sensitivity marginally, by 10%, and does not change the specificity. The use of a magnifying device to aid in evaluating the cervix has similar sensitivity and specificity to VIA alone. The sensitivity and specificity of visual detection are dependent on the skill of the provider and vary widely.
Although these methods are imperfect, they may decrease rates of cervical cancer in low-resource settings. Using computer models, Goldie et al. analysed screening strategies among women between 35 and 39 years in India, Kenya, Peru, South Africa, and Thailand. They estimated that one-time screening of women at 35 years, using either visual inspection of the cervix or high-risk HPV testing, could reduce the lifetime risk of cancer by 25–36%, at a cost of less than $500 per year of life saved. Using this model, two screenings at age 35 and 40 years resulted in a relative reduction in lifetime risk by about 40%. Visual inspection, in combination with testing for oncogenic HPV, may be used in screen-and-treat programmes, which incorporate immediate excision of cervical lesions.
In a large prospective study in rural India, Sankaranarayanan et al. evaluated the effectiveness of three different screening tools: one-time, high-risk HPV screening, visual inspection, and cytologic testing in 131,746 women aged between 30 and 59 years. In this cohort, a single round of HPV testing led to a significant reduction in the incidence of stage II or higher cervical cancer (1 per 1000 in the HPV testing group compared with 2.5 per 1000 in the control group). A reduction in cervical cancer mortality was also seen in the HPV testing group. In contrast, neither cytology nor VIA resulted in a significant reduction in either the incidence of advanced cancer or mortality compared with controls. This study shows the potential effectiveness of one-time screening in unscreened populations with a high incidence of disease, but also emphasises the importance of using a reproducible, objective test, such as detection of oncogenic HPV genotypes, compared with subjective examinations that are critically dependent on the skill of the provider.
New cervical cancer screening methods
An ideal screening method would allow for the efficient and inexpensive screening of all women regardless of their social situation. Methods meeting these criteria would allow for effective screening to take place in low-resource settings and decrease the overall fiscal burden that current cervical cancer screening methods place on high-resource healthcare systems. Several new approaches are currently being developed. These screening methods may be classified into three broad areas: HPV diagnostics (detection of either the presence of HPV or of viral integration into the host genome), biomarkers of cellular proliferation, and detection of epigenetic changes, either in the host or virus. Several of these methods show promise in improving cervical cancer screening in low- and high-resource settings.
Screening methods using human papilloma virus diagnostics
Current recommendations of the American Society for Colposcopy and Cervical Pathology (ASCCP) state that women aged 30 years and older who have normal cytology but are high-risk HPV DNA positive may benefit from genotyping assays for the presence of HPV 16 and 18. Women in whom HPV 16 and 18 is detected should be referred for colposcopy. If other high-risk types are found, but no HPV 16 and 18 is detected, the woman should be followed with repeat cytology and testing for high-risk HPV DNA in 12 months. The American Society for Colposcopy and Cervical Pathology guidelines state that it is also acceptable to observe women with negative cytology who are high-risk HPV DNA positive with repeat cytology and high-risk HPV DNA screening in 1 year. In general, testing for HPV DNA is not a useful screening strategy in either women younger than 30 years of age or those with abnormal cytology. HPV infections in women less than 30 years of age are transient and likely to regress over time. Human papilloma virus testing in women with abnormal cytology is redundant because it will show the presence of oncogenic HPV.
In women aged 30 years or older, identification of oncogenic HPV DNA is currently being implemented in high-resource settings to function as a primary screening test, simultaneously with a Pap smear. The presence of HPV DNA in cervical samples of women aged 30 years or older is likely to reflect persistent infection, in contrast to cytology that may reflect transient abnormalities. Human papilloma virus DNA testing provides a quantitative means of HPV detection, compared with evaluating cellular changes in cervical cytology, which is more subjective. Human papilloma virus DNA testing is also carried out as a reflex test on any ASCUS pap smear. By directing the management of ASCUS cytology and triage of women aged 30 years or older, HPV testing has saved women and the healthcare system a significant amount of time and resources. Despite the overall success of this strategy in identifying CIN2+, the system remains cumbersome, requiring multiple visits. Cost–benefit analyses in high-resource settings suggest that high-risk HPV DNA testing alone may replace cytology as the primary means of cervical cancer screening in women aged 30 years or older.
Screening for oncogenic HPV DNA is useful in high-resource settings; however, the costs and time involved in running the currently available tests restrict their use in low-resource settings. A rapid, low-cost oncogenic HPV DNA screening test that could be used in low-resource settings has the potential to greatly decrease the worldwide incidence of cervical cancer. One assay currently under development is the careHPV™ assay (QIAGEN, Gaithersburg, MD, USA), which uses a signal-amplification assay that detects 14 different high-risk HPV DNA types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), requires only 25 × 50 cm of work space, does not require electricity or running water, and takes about 2.5 h to carry out. This assay time of 2.5 h, compared with the approximate 6 h required for HC2 high-risk HPV testing, allows for evaluation and treatment the same day if needed.
The careHPV™ assay has been evaluated by Qiao et al. in China in a prospective cohort of 2388 women aged between 30 and 54 years who had not previously been screened for cervical cancer. In this study, women self-collected a careHPV™ vaginal sample and then underwent provider-directed careHPV™ testing, HC2 testing, visual inspection by a midwife, and digital colposcopy by a physician with guided cervical biopsies as indicated. Using CIN2+ as the reference standard, the sensitivities and specificities of the careHPV™ test were 90.0% (95% CI 83.0 to 97.0) and 84.2% (95% CI 82.7 to 85.7), respectively, on provider-collected cervical specimens, and 81.4% (95% CI 72.3 to 90.5) and 82.4% (95% CI 80.8 to 83.9), respectively, on patient-collected vaginal specimens. These methods were both superior to visual inspection, which had a sensitivity of 41.4% (95% CI 29.9 to 53.0) and a specificity of 94.5% (95% CI 93.6 to 95.4). No significant difference was found in the incidence of CIN2+ between provider- and patient-collected samples. This approach provides logistical and economic advantages, although no plans are afoot to make it available in high-resource settings.
Another strategy using HPV diagnostics for screening involves identification of specific oncogenic HPV genotypes. Currently available assays detect a pool of 13–14 oncogenic HPV DNA types, but do not specify how many HPV genotypes or which genotypes are present. Given the transient nature of many HPV infections, many women may have detectable HPV DNA, but may be at low risk for disease. Currently, Cervista ® is the only FDA-approved HPV genotyping test that identifies only HPV 16 and 18. Many additional HPV genotyping assays are not currently FDA-approved, but are available for use outside the USA ( Table 2 ).
