Approved assays for detecting HPV DNA—
design, indications, and validation

CAP Today

January 2012
Cytopathology And More

Chengquan Zhao, MD
Huaitao Yang, MD, PhD

Cervical cancer is the second most common women’s cancer worldwide, less common only than breast cancer. Human papillomavirus is the known dominant cause. HPV classification is based on nucleotide sequence homology. The L1 open reading frame (ORF) is the most conserved gene and has been used to identify new HPV types. A new type is defined when its L1 ORF DNA sequence differs by more than 10 percent from the closest known papillomavirus type. About 150 HPV types have now been identified, of which about 40 are commonly transmitted through sexual contact and infect the anogenital region. HPV types are often referred to as low-risk (wart-causing) or high-risk (cancer-causing), based on whether they put a person at risk for cancer. High-risk HPV may cause abnormal Pap test results and can lead to precancerous lesions, such as high-grade cervical intraepithelial neoplasia (CIN), vaginal intraepithelial neoplasia (VAIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), and anal intraepithelial neoplasia (AIN), as well as invasive cancers of the cervix, vulva, vagina, anus, or penis. Carcinogenic types of HPV cause growths that are usually flat and nearly invisible. Low-risk types of HPV, on the other hand, often cause no symptoms, may sometimes cause conditions such as genital warts, but virtually never cause cervical cancer or true precancerous lesions.

Unlike other DNA viruses, conventional cell cultures cannot detect HPV. Due to limited sensitivity and specificity, classical direct virological diagnostic techniques, such as electron microscopy and immunohistochemistry, are not used routinely to detect HPV. Serological detection of anti-HPV antibodies has not been used routinely for clinical diagnosis because of its limited analytical accuracy. Consequently, all commercially available HPV tests are designed for the detection of HPV nucleic acids in clinical specimens. Although many in-house HPV nucleic acid detection methods have been used successfully in research laboratories worldwide for more than two decades, most of them are not approved by the FDA for clinical use. The currently available commercial assays for the multiplex detection of alpha-HPVs are classified into the following groups: 1) FDA-approved high-risk–HPV DNA-based screening assays, 2) FDA-approved high-risk–HPV DNA-based screening assays with concurrent or reflex HPV 16 and HPV 18 genotyping, 3) FDA-approved HPV DNA-based genotyping assays, 4) FDA-approved high-risk–HPV E6/E7 mRNA-based screening assays, and 5) in situ hybridization (none FDA-approved at this time).

Currently, there are five FDA-approved assays that can be used to detect high-risk HPV. These include the Hybrid Capture 2 HPV DNA test and the Cervista HPV HR test, which are high-risk–HPV DNA-based screening assays. The Cervista HPV 16/18 test, which is a high-risk–HPV DNA-based genotyping assay, is approved for reflex testing of patients with positive high-risk–HPV Cervista test results. The recently FDA-approved Cobas 4800 HPV test is a high-risk–HPV DNA-based PCR screening assay with capability for concurrent genotyping for HPV 16 and HPV 18 by real-time PCR. Most recently, the Aptima HPV assay for the detection of E6 and E7 mRNA received FDA approval for detection of high-risk HPV.

In this review, we will briefly discuss assay design, clinical indications, high-risk HPV types detected by each assay, analytical and clinical validation, and potential problems and resolutions.

Hybrid Capture 2 HPV DNA test

Digene Corp. of Gaithersburg, Md., developed in 1997 the Hybrid Capture 2 (HC2) HPV DNA test, now marketed by Qiagen. This assay received FDA approval in 1999 for reflex testing of patients with atypical squamous cells of undetermined significance, or ASC-US, cytology results. In March 2003, the FDA approved its use in conjunction with routine Pap testing of women over age 30. The HC2 High-Risk HPV DNA test is the most frequently used diagnostic HPV test worldwide. It has been recommended that new HPV assays should possess clinical characteristics similar to those of HC2 in the process of so-called clinical validation of each new high-risk–HPV test.

The HC2 HPV DNA test is an in vitro nucleic acid hybridization assay with signal amplification using microplate chemiluminescence for the qualitative detection of 13 high-risk types of HPV DNA (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). The HC2 High-Risk HPV DNA test cannot determine the specific HPV genotype present. Cervical specimens containing the target DNA hybridize with a specific high-risk–HPV RNA probe cocktail. The resultant hybrids of RNA probes and targeted high-risk HPV DNAs are captured onto a microplate well coated with specific antibodies against RNA-DNA hybrids. After alkaline phosphatase conjugated antibodies bind with immobilized hybrids, the signals are detected with a chemiluminescent substrate. Multiple conjugated antibodies bind to each captured hybrid resulting in substantial signal amplification. The signal of emission light is measured as relative light units (RLUs) on a luminometer. The intensity of the chemiluminescence denotes the presence or absence of target high-risk HPV DNAs in a clinical cervical specimen. An RLU measurement equal to or greater than the cutoff (CO) value indicates the presence of high-risk–HPV DNA sequences. An RLU measurement less than the cutoff value indicates either absence of targeted high-risk–HPV DNA sequences or high-risk–HPV DNA levels below the detection limit of the HC2 assay. Specimens with RLU/CO ratios ≥ 1.0 are considered positive. Specimens with RLU/CO ratios < 1.0 are considered negative for the 13 HPV types tested. For higher throughput, the HC2 testing Rapid Capture System (Qiagen) has been available for the past few years. Using this system, one technologist can process up to 352 patient specimens in an eight-hour shift, with 3.5 hours of hands-free operation.

The HC2 High-Risk HPV DNA test is designed for use with specimens collected and transported using the HC2 DNA Collection Device or HC Cervical Sampler (cervical broom) with samples deposited in either Digene Specimen Transport Medium or a Cytyc PreservCyt vial. Specimens can be stored at room temperature for up to two weeks and shipped to the testing laboratory. After two weeks, specimens can be stored for an additional week at 2–8°C. If the assay cannot be performed within three weeks of collection, specimens can be placed at -20°C for up to three months prior to testing. Collected specimens placed in Cytyc PreservCyt solution used in preparing Cytyc ThinPrep Pap test slides can be used in the HC2 High-Risk HPV DNA test. There must be at least 4 mL of PreservCyt solution remaining for the HC2 High-Risk HPV DNA test. PreservCyt solution specimens can be stored for up to three months at temperatures of 2–30°C, after collection and before processing for the HC2 High-Risk HPV DNA test. PreservCyt solution specimens cannot be frozen.

The main problems of the current version of HC2 are analytical inaccuracy due to the cross-reactivity of its probe cocktail with untargeted HPV types (11, 53, 54, 55, 66, MM4, MM7, MM8, or MM9.39) and lack of an internal control to evaluate specimen adequacy or the presence of potentially interfering substances. Recent studies have shown that HC2 has an additional five percent false-positive rate when no HPV DNA is present in the clinical specimen, based on the use of different usually highly sensitive and broad-range PCR tests.

Cervista HPV HR test

The FDA approved the Cervista HPV HR test from Hologic of Bedford, Mass., in March 2009. The Cervista test can be used for the following two indications: 1) to screen patients with ASC-US cervical cytology results to determine the need for referral to colposcopy, and 2) with cervical cytology in women age 30 and older to adjunctively screen for the presence or absence of high-risk HPV types. Cervista is FDA approved for use with cervical specimens collected in ThinPrep PreservCyt solution.

The Cervista HPV assay includes two tests: Cervista HPV HR test and Cervista HPV 16/18 test. The HPV HR test cannot determine the specific HPV genotype present. It is an in vitro diagnostic test for the qualitative detection of high-risk HPV DNA from 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) in cervical specimens.

Cervista HPV HR test uses the Invader chemistry. It is a signal amplification method for detecting specific nucleic acid sequences. This method uses two types of isothermal reactions: a primary reaction that occurs on the targeted DNA sequence and a secondary reaction that produces a fluorescent signal. In the primary reaction, there are two types of sequence specific oligonucleotides: a probe oligonucleotide and an Invader oligonucleotide. They bind to the DNA target sequence. An invasive structure forms when these oligonucleotides overlap by at least one base pair on the target sequence. Cleavase enzyme cleaves the 5’ portion (flap) of the probe at the position of the overlap. The probes are present in large molar excess. Cycle runs rapidly on and off the target sequence. They produce many cleaved 5’ flaps per target sequence. These flaps bind to a universal hairpin fluorescence resonance energy transfer (FRET) oligonucleotide that creates another invasive structure recognized by Cleavase enzyme as a substrate. After enzymes cleave the FRET oligonucleotides between the fluorophore and quencher molecule, fluorescence signal is produced as the cleaved flaps cycle on and off (Cervista HPV HR, revision C, 2009).

The reagents for this Cervista HPV HR test are provided as three oligonucleotide mixtures, which detect the 14 types of high-risk HPV. Internal and external controls, including negative and positive controls, are used as quality control in this test. Controls must be run on each assay. Sample results are valid when the positive and negative controls yield the correct results. Oligonucleotides that bind to the human histone 2 gene (H2be, HIST2H2BE) are also present and serve as an internal control. The format of the HPV HR test makes it possible to detect HPV DNA sequences and HIST2H2BE simultaneously in a single well by using two sets of different 5’-flap sequences on the probes and two different FRET oligonucleotides, each with a spectrally distinct fluorophore (FAM and Red). A positive result indicates that at least one of the 14 high-risk HPV types is present in the DNA sample. This positive result is represented by a FAM fluorescent signal, which is above an empirically derived cutoff value. For each reaction, a negative result is represented by a FAM fluorescent signal, which is below an empirically derived cutoff value. A final positive or negative or indeterminate result for any particular sample is generated based on signal-to- noise value. This signal-to-noise value is referred to as FOZ (Fold-Over-Zero).

Cervical specimens should be collected in PreservCyt solution of the ThinPrep Pap Test preservation system, using a broom-type device or endocervical brush/spatula. For Cervista HPV HR testing, cervical specimens preserved in PreservCyt can be stored at 20–30°C for up to 18 weeks before performing the test. PreservCyt solution specimens cannot be frozen.

The limitation of the Cervista HPV HR test is its potential cross-reactivity with other HPV types, such as types 67 and 70, which yield positive results with the Cervista HPV HR test.

Cervista HPV 16/18 test

The Hologic Cervista HPV 16/18 test is an in vitro diagnostic test for the qualitative detection of DNA from HPV types 16 and 18 in cervical specimens. The Cervista HPV 16/18 test uses the same Invader chemistry as the HPV HR test. In the primary reaction, probe oligonucleotides provided for this test bind to the DNA target sequence of high-risk HPV 16 and high-risk HPV 18.

The HPV 16/18 test is approved for two indications: 1) adjunctively with the Cervista HPV HR test in combination with cervical cytology in women age 30 and older to assess the presence or absence of high-risk HPV types 16 and 18, and 2) adjunctively with the HPV HR test in patients with ASC-US cytology results to assess the presence or absence of high-risk HPV types 16 and 18 (Cervista HPV 16/18, revision B, 2009). The specimen collection and storage requirements are the same as those for HPV HR.

The limitation of the HPV 16/18 test is its cross-reactivity and false negativity. It exhibits cross-reactivity to high levels of HPV high-risk type 31. Very low levels of infection or sampling error may cause a false-negative result.

Cobas 4800 HPV test

The Cobas 4800 HPV test from Roche Molecular Diagnostics of Pleasanton, Calif., was launched on the European market in November 2009. In April 2011, the FDA approved the Cobas 4800 HPV test in cervical specimens. It uses fluorescence signal to detect nucleic acids amplified by using real-time PCR methodology. The 4800 HPV test is performed using the Cobas 4800 system, which consists of two separate instruments: the Cobas z 480 and Cobas x 480 analyzers. The 4800 HPV test is FDA approved for use with cervical specimens collected in ThinPrep PreservCyt solution.

The Cobas 4800 system has software that integrates sample preparation, amplification and detection, and result management. The software has two different testing options for each sample screening: the pooled testing for all 14 targeted high-risk HPV types together, and pooled testing plus separate individual genotyping for HPV 16 and HPV 18. The Cobas HPV test uses amplification of target DNA by PCR and subsequent nucleic acid hybridization for the detection of 14 high-risk HPV types in a single analysis. The test specifically identifies HPV types 16 and 18 while concurrently detecting the 12 remaining high-risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) at clinically relevant infection levels (Cobas HPV test, draft package insert, 2011).

The Cobas HPV test has four indications: 1) to screen patients 21 years and older with ASC-US, 2) in patients 21 years and older with ASC-US cervical cytology results to assess the presence or absence of high-risk HPV genotypes 16 and 18, 3) with cervical cytology in women age 30 and older to adjunctively screen to assess the presence or absence of high-risk HPV types, and 4) in women 30 and older to assess the presence or absence of high-risk HPV genotypes 16 and 18. The assay is FDA-approved for use with cervical specimens collected in Cobas PCR Cell Collection Media (Roche) or ThinPrep PreservCyt solution.

The Cobas HPV test has quality controls (internal control, β-globin, plus positive and negative controls included in every run to validate the results), has high throughput (Cobas 4800 system is designed to process up to 280 samples in one day), is automated (loading and unloading the microwell plate is the only manual intervention), and is LIS compatible (Cobas 4800 system can be connected to a laboratory information system—operator’s manual software version 1.0, 2011).

There is limited literature on the analytical and clinical validation of the Cobas 4800 HPV test. The test’s clinical sensitivity and specificity were compared with those of HC2. It has sufficient intralaboratory and interlaboratory reproducibility. The data demonstrated that the test fulfills all requirements of international guidelines to consider this assay clinically validated for screening purposes.

Aptima HPV assay

The Aptima HPV assay from Gen-Probe of San Diego is the latest FDA-approved HPV test; it was approved on Oct. 31, 2011. The FDA approved the assay for use with Gen-Probe’s Tigris, the only fully automated testing system for molecular diagnostics. The Aptima HPV assay is a transcription-mediated amplification-based assay, which allows the detection of E6/E7 mRNA transcripts of 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68). This assay does not discriminate between the 14 high-risk HPV types.

The Aptima assay includes three main steps: target capture by oligomers, target amplification by transcription-mediated amplification (TMA), and amplicon detection by the Hybridization Protection Assay (HPA). After lysis of cells in collected specimens, the target mRNA is released and captured by capture oligomers that are linked to magnetic microparticles. The capture oligomers contain complementary sequences to specific regions of the high-risk–HPV mRNA target molecules. During the hybridization step, the captured oligomers bind to specific regions of the high-risk–HPV mRNA target molecule and form the capture oligomer:target complex. After the target capture is complete, the high-risk–HPV mRNA is amplified by using TMA. The TMA reaction uses two enzymes: MMLV reverse transcriptase and T7 RNA polymerase. The MMLV reverse transcriptase is used to generate a DNA copy of the target high-risk–HPV mRNA sequence containing a promoter sequence for T7 RNA polymerase. Subsequently, T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template. Detection of the amplicon is achieved by Hybridization Protection Assay. The HPA uses chemiluminescent-labeled single-stranded nucleic acid probes that are complementary to the amplicon. Light emitted from the labeled RNA:DNA hybrids is measured as photon signals and reported as relative light units. The internal control is used for quality control. The assay software automatically determines assay test results. Determined by the signal-to-cutoff (S/CO), a test result may be negative, positive, or invalid (Gen-Probe 502182EN revision A, 2011).

Cervical specimens are collected in ThinPrep Pap test vials containing PreservCyt solution or the Aptima Cervical Specimen Collection and Transport Kit. If a specimen is collected in PreservCyt, before transfer, PreservCyt liquid Pap specimens should be stored at 2–8°C, with no more than 30 days at temperatures up to 30°C. If a specimen collected in PreservCyt liquid has been transferred to an Aptima Specimen Transfer Tube, it may be stored at 2–30°C for up to 60 days. If longer storage is needed, the PreservCyt liquid Pap specimen may be stored at -20°C or colder for up to 24 months. If the specimens are collected and stored in the Aptima collection and transport kit, the specimens can be stored at 2–30°C for up to 60 days. If long-term storage is needed, the specimens can be stored at -20°C or colder for up to 24 months (Gen-Probe 502182EN revision A, 2011).

The advantage of the Aptima assay is that it does not show cross-reactivity with any tested high-risk HPV types or with normal flora and opportunistic organisms that may be found in cervical samples. The Aptima test shows high sensitivity and specificity. At the CIN3+ end point, the assay was equally sensitive (95 percent) as HC2 but Aptima was more specific than HC2 (Gen-Probe 502182EN revision A, 2011).

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Dr. Zhao is an associate professor of pathology and associate director of cytopathology, and Dr. Yang is a cytopathology fellow, Department of Pathology, Magee-Womens Hospital, University of Pittsburgh Medical Center. Dr. Zhao is a member of the CAP Cytopathology Committee.