Cervical cancer is the second most common cause of cancer deaths in women worldwide. Routine screening and treatment have substantially decreased the cervical cancer mortality rate in the United States. However, according to the World Health Organization, there are approximately 500,000 new cases and 250,000 deaths from cervical cancer every year worldwide. Although Pap tests and colposcopy have contributed tremendously to the decreased mortality of cervical cancer, they are subjective tests, prone to human error, and not always conclusive. Therefore, it is extremely important to develop more objective screening tools that can identify patients who are most at risk for developing cervical cancer (8
Human papillomavirus (HPV) infection is a major cause of virtually all invasive cervical cancers (2
). Of the 40 HPV types that infect the genital tract, only a subset of HPV subtypes are classified as “high-risk” HPV (HR-HPV) types that were found in cancers (33
). Most of these HPV infections are transient, are resolved by the body's immune system, and have no major clinical consequences. However, persistent HPV infections are found in 5 to 10% of infected women and represent a high risk factor for progression to cervical cancer (3
). Thus, it is important to identify the small percentage of women with HPV infections who are truly at risk for developing cervical cancer. Unfortunately, current screening tests cannot accurately predict the risk of dysplasia or cancer. Therefore, there is a significant need to develop a test that could better predict progression to these outcomes.
The current paradigm for cervical cancer screening is based on the Pap test, which is a cytologically based test using cells scraped from the cervix that are examined microscopically to detect dysplastic lesions (9
). There are approximately 4 million abnormal Pap tests each year in the United States. Under current practice guidelines, most of these patients are referred for colposcopy and cervical biopsy to identify the subset that has clinically significant high-grade precancers, such as c
eoplasia grade 2/3 (CIN2/3) (15a
). However, the Pap test is subjective, with significant interobserver variability, and is limited by low sensitivity. In addition, high false-positive rates, defined as a positive Pap result with no clinically significant disease by subsequent biopsy (i.e., histology results of less than CIN2/3), were observed in two-thirds of patients with abnormal Pap smears (6
). As a result, approximately 3 million colposcopic examinations performed each year, at a cost exceeding $2 billion dollars annually, may not be necessary.
In the last few years, HR-HPV DNA testing has been included in routine screening to increase the sensitivity and negative predictive value of the Pap test. While these tests can detect the presence of HPV DNA, they cannot differentiate a true precancerous state from self-limited HPV infection (1
), which represents the majority of infections. The low specificity of HPV DNA testing potentially results in overdiagnosis and inefficient disease management (26
). In addition to DNA tests, a number of host cellular proteins, including p16INK4a
, Ki67, and ProExC, have also been identified as biomarkers for cervical cancer diagnosis. However, they are considered surrogate markers and not specific to HPV.
HPVs are DNA viruses that code for several functional (E1 to E7) genes and two late structural (L1 and L2) genes. When high-risk HPV types integrate into the host genome, loss of negative-feedback control results in increased expression of viral E6 and E7 oncogenes, which in turn inactivate tumor suppressor genes that operate at key cell cycle checkpoints (10
). Since these two oncogenes are integral to the development of cervical cancer, their gene products could potentially serve as highly specific biomarkers to identify high-grade precancerous lesions that may progress to cervical cancer if left untreated. Indeed, evidence suggests that elevated levels of the E6 and E7 oncoproteins are better indicators of increased risk for cervical cancer than the presence of HPV DNA (6
). The recent FDA approval of the Aptima HPV E6/E7 RNA test is a significant milestone for the application of E6/E7 as specific biomarkers for cervical cancer screening. However, RNA is prone to degradation, and its detection requires expensive instrumentation and cumbersome procedures; tests based on the detection of E6/E7 mRNA may have limited clinical application in routine gynecological practice. Diagnostic tests based on the direct detection of the E6/E7 oncoproteins may have advantages over detection of HPV DNA or HPV E6/E7 mRNA. Until recently, most of the antibodies developed against the HPV E6 or E7 protein used either peptides or denatured proteins, as it has been difficult to purify recombinant nondenatured E6 and E7 proteins suitable for antibody production (31
). Antibodies produced using these denatured proteins do not have sufficient sensitivity for clinical use (16
). We have recently overcome the technical hurdles and have purified recombinant HPV E6 and E7 proteins in their native form to generate monoclonal antibodies that recognize HPV E6 and E7 from many high-risk HPV types.
In this study, we describe a simple whole-cell enzyme-linked immunoabsorbent assay (ELISA) using a pan-HPV anti-E6 monoclonal antibody to detect HPV E6 protein in previously collected and frozen cytology samples. Using cervical biopsy results as the gold standard, ELISA results are compared to HPV DNA test results. The ability to detect the E6 oncoprotein in clinical samples is a critical advance that will facilitate the development of diagnostic testing to distinguish benign HPV infections from precancers, thus preventing unnecessary colposcopies and biopsies.