Utilization of Biomarkers to Improve Cervical Cancer Screening

By Charlotte Brown, PhD, FACMG and Douglas P. Malinowski, PhD

Cervical cancer is the third most common malignancy of the female genital system and the second most common cancer in women worldwide, with an estimated 470,000 new cases diagnosed annually. The American Cancer Society estimates that about 11,070 American women will be diagnosed with cervical cancer in 2008 and that 3,870 will die from the disease.

Cervical dysplasia is a term used to describe the appearance of abnormal cells on the surface of the cervix, the lowest part of the uterus. These changes in cervical tissue are classified as low-grade or high-grade dysplasia. While dysplasia itself does not cause health problems, it is considered to be a precancerous condition. Left untreated, dysplasia can progress to an early form of cancer known as cervical carcinoma in situ, and eventually to invasive cervical cancer. Approximately 330,000 new cases of high-grade dysplasia (Cervical Intraepithelial Neoplasia 2/3 or CIN2/3) and 1.4 million new cases of low-grade dysplasia (CIN1) are diagnosed in the United States every year. Human Papillomaviruses (HPV) are known to be a major causative agent in the development of cervical cancer and its precursor lesions.

Cervical screening has proven to be a model for successful cancer prevention and is largely responsible for the 70% decrease in cervical cancer mortality in the United States over the last 50 years. Cervical cytology screening, also known as the Papanicolaou (Pap) test, is based on examining cells from the cervix and is considered an integral part of women's preventive healthcare. In the conventional Pap smear procedure, scrapings of both the endocervical and ectocervical cells from the cervix are collected, deposited on a microscope slide, stained with the Pap stain and examined microscopically for the presence of abnormal cells. In spite of its success, however, the Pap test is subject to marked inter- and intraobserver variability as well as having a relative low sensitivity for detection of cervical dysplasia on a single sample.

Since the introduction of the Pap smear, investigators have attempted by various means to enhance its sensitivity, including the introduction of liquid-based methods to address issues of specimen collection and preparation and the use of computer-assisted screening systems to address screening errors. Even with these improvements to the cervical screening process, the Pap test is still far from perfect with both false positive and false negative results obtained. The success of current screening programs results from the frequent screening of women and the laborious workup of inconclusive or mildly abnormal test results. In fact, most of the money spent in cervical cancer prevention today is used to identify patients truly in need of medical intervention.

The most common abnormal Pap result, referred to as Atypical Squamous Cells of Undetermined Significance (ASC-US), signifies that there were mild cellular changes detected in some of the examined cervical cells. The majority of these abnormal cells, however, are not caused by cervical cancer, but more likely by inflammation or a vaginal infection. A mildly abnormal result, referred to as a Low-grade Squamous Intraepithelial Lesion ( LSIL) indicates that there are early changes in the size and shape of the cervical cells, most often due to the presence of HPV infection.

While the majority of women with ASC-US or LSIL results on Pap testing are not found to have significant cervical disease, this group of women are at increased risk, with one large study finding severe dysplasia (CIN3) in 8-9% of the ASC-US cases and in about 15% of LSIL cases [1,2]. Therefore, additional testing is currently recommended for all women with an ASCUS or LSIL Pap result in order to identify which cases are truly associated with significant disease. Three management strategies are currently performed: repeat cytology, direct colposcopy, and HPV DNA testing [3].

For the repeat cytology option, a woman will undergo additional Pap tests at 6 and 12 months after the original abnormal test. If both repeat tests result in a normal cytology result, the patient can return to routine screening. If an abnormal result is obtained with either of the repeat Pap tests, a direct colposcopic examination is then recommended. Colposcopy is a procedure done during a pelvic exam with the aide of a colposcope, a microscope-like device. By applying acetic acid on the cervix and examining it with a colposcope, a physician can look for abnormal areas of the cervix. A biopsy of an abnormal area may also be taken during the procedure, as a microscopic examination of a biopsy is the only way to definitively diagnose severe dysplasia or cancer. Poor sensitivity of colposcopic biopsy, however, often requires numerous patient visits for colposcopy and biopsy to obtain tissue confirmation of an abnormal Pap diagnosis.

With clinical performance issues affecting both Pap testing and colposcopy, it is evident that additional biomarkers are needed that allow the identification of women truly at risk to develop cervical cancer at a time point that still allows for successful curative intervention. Biomarkers are surrogate markers used to detect the presence or severity of disease. In the case of cervical dysplasia and cancer, analysis of a biopsy taken from the cervix is the true diagnostic test. Biomarker detection from cells obtained from the Pap test, however, could provide a better indication of individuals requiring additional work-up prior to performing the invasive colposcopic/biopsy procedure. The use of biomarkers could improve the sensitivity of the Pap test by being able to highlight abnormal cells on a slide, bringing rare abnormal cells to the attention of screeners. In addition, biomarkers could decrease the number of false positive results and colposcopic procedures, by triaging cells with possible abnormalities to low and high-risk classifications.

Prior to discussing individual biomarkers utilized for cervical cancer screening, it is important to note how the performance of biomarkers are evaluated. Biomarker test performance is characterized by measures of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). A lack of sensitivity will result in cases that require treatment being missed. A specificity of less than 100% indicates that patients with no disease will have a positive test, possibly resulting in unnecessary follow-up visits and procedures. The PPV specifies the proportion of individuals tested positive that truly do have disease, while the NPV indicates how often a negative test result is obtained when no disease is present.

Currently, the most frequently used biomarker in cervical cancer screening is the detection of HPV DNA. For women over 30 years of age, testing for the presence of DNA from high-risk HPV types has been accepted as an adjunct to ASC-US cytology in primary screening. Women testing negative for HPV are considered at very low risk of developing cervical cancer and will be returned to routine screening. Women testing positive for HPV, however, are considered at higher risk of harboring cervical dysplasia or cancer and are generally referred for colposcopic examination. This HPV DNA triage testing is not recommended for younger women and women with LSIL cytology due to the high prevalence of HPV DNA in these groups. It is important to note that the majority of HPV infections induce low grade precursor lesions that are cleared spontaneously within one to two years of exposure in more than 90% of cases, with less than 10% eventually progressing to high grade lesions or invasive cancer. Thus, while HPV DNA testing has a very high sensitivity for the detection of high grade cervical disease, it has a very low specificity and PPV, unnecessarily raising patient anxiety levels and referrals to invasive colposcopic procedures [4]. The HPV DNA story, therefore, clearly demonstrates that there is a need for additional biomarkers to discriminate lesions with a high risk of progression from those that will spontaneously regress.

When the immune system is unable to clear an HPV infection, or the cervical cells are coping with a continual re-infection, an increased risk of cervical neoplasia exists. During persistent infection with HPV, the continued expression of two HPV oncoproteins, referred to as E6 and E7, alter the integrity of the cell cycle and disrupt the controlled processes of DNA repair, replication , and subsequent cell division. As the effects of E6 and E7 are critical for neoplastic transformation of HPV infected cervical cells, the detection of E6/E7 mRNA of high risk HPV genotypes may be a useful biomarker for revealing underlying cell abnormalities at greatest risk for developing cervical neoplasia and carcinoma. In fact, preliminary studies have provided evidence to support the increased specificity of HPV E6/E7 mRNA-based tests for the detection of severe cervical dysplasia, suggesting that HPV mRNA testing may represent a valuable biomarker in prevention of cervical cancer as an adjunct to cytology [5].

The improved understanding of the molecular pathways of HPV-induced cervical carcinogenesis has led to the discovery of additional clinically useful biomarkers, many of which are proteins involved in the regulation of the cell cycle. The detection of abnormally increased levels of these proteins may reflect the deregulation of the cell cycle in cervical neoplasia. Protein biomarkers, including p16 INK4a, Cyclin E, the MCM proteins, and TOP2A, have been used to detect the cells on a Pap test most likely to represent true cervical dysplasia or cancer.

The protein p16 INK4a is a cell-cycle regulator, with its expression tightly controlled in normal cells. The presence of the HPV E6 and E7 oncoproteins, however, results in increased levels of p16 INK4a, the detection of which can serve as a surrogate biomarker for persistent infection with high-risk HPV. It is widely accepted that p16 INK4a is a sensitive and specific marker of dysplastic cells of the cervix and is a useful biomarker in cervical cancer lesion diagnosis and cervical screening [7]. The cyclins, a family of regulatory proteins also involved in the coordination of the cell cycle, have been analyzed in cervical cancer and precancer. Cyclins A, B, and E have been found to be overexpressed in premalignant cervical lesions, with a strong association between the expression of Cyclin E in liquid based cytology specimens and HPV induced cellular abnormalities being reported [5,8].

BD ProEx™ C is a protein-based biomarker reagent containing antibodies to minichromosome maintenance protein 2 ( MCM 2) and topoisomerase II- a ( TOP2A) , proteins that have been shown to accumulate in HPV-transformed cells. [5,8] Increased expression of the MCM and TOP2A proteins are present in cervical dysplasia and cancer. Studies performed using BD ProEx™ C on Pap samples have demonstrated increased sensitivity for the detection of biopsy proven high grade disease compared with cytology alone and improved positive predictive value for the detection of high grade disease compared with high risk HPV DNA testing (9,10). In addition, a recent study demonstrated that the use of BD ProEx™ C in women found to have abnormal cytology results upon routine screening predicted a 73% reduction in colposcopic procedures compared with the number of procedures predicted based on Pap results alone.

The use of biomarkers such as p16 INK4a, TOP2A, and the MCM proteins have been reported to facilitate the detection of abnormal cells within a Pap cytology sample based upon simple immunocytochemistry assay formats. The published reports on the use of MCM2 and TOP2A in Pap cytology samples have highlighted the use of these biomarkers both to detect dysplastic cells (i.e., a highlighter function) as well as to increase the detection of CIN2+ within abnormal cases (i.e., an interpreter function) [9,10].

A number of potential biomarkers for cervical screening have been analyzed that appear to improve the detection of women at greatest risk for developing cervical cancer. The recent introduction of prophylactic HPV vaccines will eventually reduce the incidence of cervical cancers and its malignant precursors, therefore increasing the importance of biomarkers in future cervical cancer screening programs to identify for treatment only those women truly at high risk for developing cervical cancer. As such, it is anticipated that the use of these biomarkers and immunocytochemistry assays can be applied both as a reflex test from an atypical Pap specimen but also as a primary screen to improve the overall accuracy of the Pap test.

References:

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