Quantification of intracellular HPV E6/E7 mRNA expression increases the specificity and positive predictive value of cervical cancer screening compared to HPV DNA

Abstract

Objectives

Current methods for HPV screening rely on the detection of L1 DNA from high risk genotypes (HRHPV). These assays have very high negative predictive values (~ 99%), however, the specificity and positive predictive value of HPV DNA tests for pre-cancerous and cancerous lesions (CIN 2+) is less than 50%. The purpose of this study was to compare HPV DNA with intracellular HPV E6, E7 mRNA quantification in an effort to improve the performance of cervical cancer screening.

Methods

Liquid-based cervical cytology specimens collected in either PreservCyt or SurePath were processed for routing cytology, HPV HRDNA detection by Hybrid Capture 2 and HPV E6, E7 mRNA quantification in cells using the same sample. We analyzed a total of 2049 samples including 73 with CIN 2, CIN 3, or squamous cell carcinoma by biopsy and 1694 samples from women with normal cytology.

Results

The positive predictive value of HPV E6, E7 mRNA quantification in cells for CIN2+ was 78% which was greater than HPV DNA alone (43%). The specificity of HPV E6, E7 mRNA quantification was 96% based on normal cytology compared to 82% for HC2 while the specificity of HPV E6, E7 mRNA quantification based on CIN 2− histology was 85% compared to 35% for HC2.

Conclusions

With similar sensitivity and greater specificity/positive predictive value, HPV E6, E7 mRNA quantification in cells is an improvement over HPV DNA for cervical cancer screening.

Introduction

Effective cervical cancer screening programs like those implemented in the United States have reduced the incidence and the mortality of cervical cancer [1]. The Papanicolaou smear (Pap) has been used for over fifty years to screen for cervical cancer and over the last ten years, HPV DNA testing as a reflex test for ASCUS (atypical squamous cells of unknown significance) has also been employed [2], [3], [4], [5]. Based on current recommendations, HPV DNA testing is used as an adjunctive test for all cervical cytology samples (co-testing) in women over 30 years old [6], [7]. The Pap smear is limited by low sensitivity for high grade lesions (CIN 2, CIN 3, or squamous cell carcinoma) while the sensitivity of HPV DNA testing for high grade lesions is greater than 90% [6], [7], [8], [9]. HPV DNA testing alone, however, has lower specificity for high grade lesions than does Pap testing [6], [9]. Because of the poor specificity of HPV DNA testing, the positive predictive value of HPV DNA for high grade lesions by biopsy is 15%–25% which can lead to unnecessary colposcopy and biopsy examinations in women with abnormal Pap smears who are positive for high risk HPV DNA [10], [11], [12], [13], [14].

Since the overexpression of the HPV-encoded oncogenes E6 and E7 plays a significant albeit not solitary role in cervical carcinogenesis [15], [16], quantification of these oncogenes could improve the specificity and positive predictive value of cervical cancer screening with the goal of detecting CIN 2+ lesions with high specificity [17], [18], [19].

The combination of the Pap smear and HPV DNA has proven to be a useful, though a costly and labor intensive combination to effectively screen for cervical cancer in areas where highly skilled cytotechnologists, cytopathologists, and virologists can perform this combination of tests. The majority of the world, where greater than 85% of cervical cancer occurs, lacks the resources to implement such a rigorous screening program at the recommended intervals [14]. Therefore in the present study, we compared a new technology that quantifies the HPV-encoded genes (E6, E7 mRNA) that cause cervical cancer in individual cells [17], [18], [19]. Upregulation of E6, E7 mRNA in individual cells is the molecular trigger that begins the cycle of cellular transformation, p53 and Rb degradation, and cell cycle dysregulation that ultimately leads to cervical cancer [15], [16]. Other molecular tests such as the Aptima HPV Assay and the HPV Proofer assay detect the presence of E6, E7 mRNA from high risk HPV types but do not quantify the overexpression of E6, E7 mRNA on a cell-by-cell basis, the hallmark of cellular transformation [10]. Here, we demonstrate that quantification of E6, E7 mRNA at the cellular level and quantification of the percentage of cells expressing elevated levels of HPV E6, E7 mRNA have equivalent sensitivity and far superior specificity/PPV than the FDA-approved hybrid capture 2 assay for CIN2+ lesions detected by histology. Further, we demonstrate that quantification of cells expressing high levels of E6, E7 mRNA correlates with the severity of the cervical lesion on biopsy.