Human Papillomavirus E6/E7 mRNA Testing, Predictive Marker, Cervical Cancer

HPV Detection Methods

Since HPV cannot be cultured, HPV tests currently in use rely on the detection of viral nucleic acids in the infected tissue.^[14] The most commonly used HPV tests are based on direct hybridization or DNA-based amplification techniques.

In situ hybridization (ISH) by chromogenic or fluorescence techniques is based on the complementary pairing of a labeled probe to HPV antigens or nucleic acids (DNA or mRNA) within either paraffin-embedded tissue biopsies or cervical smears. This is the only method to demonstrate the precise localization of viral genomes in individual cells. In addition, the physical state of the virus can be evaluated by the presence of punctuate signals for integrated virus and diffuse signals for episomal virus.^[15] However, diffuse staining that may arise from the episomal genome will mask the punctuate staining when both episomal and integrated genomes are present. The INFORM^® HPV assay (Ventana Medical Systems, Inc., AZ, USA) is based upon chromogenic ISH and includes a low-risk (6, 11, 42, 43 and 44) and a high-risk cocktail (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66). This assay has been validated technically but insufficiently validated clinically.^{[16,17,18,19]} ISH is too laborious and lacks sufficient clinical sensitivity to be used in screening.

Hybrid Capture^® 2 (HC2; Digene Corporation, MD, USA) is a signal-amplified hybridization microplate-based assay, and at present the only US FDA-approved HPV test. This is the most widely used and clinically validated assay on the market.^[20,21,22] HC2 is a semiquantitative measure of viral load relative to 1 pg/ml and uses RNA probes that react with 13 high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) or five low-risk HPV types (6, 11, 42, 43 and 44). As testing for low-risk HPV types has no clinical significance, testing is usually performed only with the high-risk probe set. The analytical sensitivity is lower than PCR but the clinical sensitivity is equal. Limitations of this test are lack of internal control for the amount of input of DNA and cross-reactivity with HPV types not included in the probe mix resulting in false-negative and false-positive results.^[23,24,25] Neither INFORM HPV nor HC2 will identify specific HPV types, hence other techniques have to be used for genotyping.

PCR is regarded as the most sensitive technique and allows testing on samples with less tissue or fewer cells, poorer DNA quality and fewer viral copies. In order to avoid contamination, separate rooms are necessary for sample preparation and PCR reactions. The reaction mix should include adequate internal controls to avoid false-positive and false-negative results. The PCR tests are based on consensus or type-specific assays. The most commonly used consensus PCR target is the highly conserved L1-region. L1-based PCR tests can give false-negative results in screening since integration of the HPV genome into the human chromosomes may result in loss of the L1 region. New technologies based on DNA microarray formate (DNA chips) and Luminex xMAP^® (Luminex Corp., TX, USA) are developed for more automated detection and genotyping.^[26,27,28] Commercial HPV assays based on L1 or E1 PCR for high-risk HPV DNA detection and genotyping are now available from different companies: among others Amplicor^® and Linear Array^™ (Roche Molecular Systems, CA, USA), INNO-LiPA^® (Innogenetics, Ghent, Belgium), PapilloCheck (Greiner Bio-One GmbH, Germany) and Multiplex HPV genotyping kit (Multimetrix GmbH, Heidelberg, Germany). Limited clinical validation exists only for the Amplicor test.^{[29,30,31,32]}

RNA-based Amplification Techniques

Detection of HPV E6/E7 mRNA and the presence of oncogene activity in cervical specimens can be performed by reverse-transcriptase (RT)-PCR or by nucleic acid sequence-based amplification (NASBA).^[33,34] So far, few clinical studies have been published; the most important studies are summarized in ( Table 2 ). The predictive values of HPV mRNA testing in screening have not been investigated in population-based, randomized trials.

A NASBA-based assay detecting E6/E7 transcripts from the five most common high-risk HPV types in cervical carcinoma (16, 18, 31, 33 and 45) is now available (same product, marketed under different brand names: PreTect^™ HPV-Proofer, NorChip AS, Klokkarstua, Norway and NucliSENS EasyQ^®, BioMerieux SA, France). In NASBA assays, single-stranded nucleic acids or RNA equivalents (e.g., viral genomic RNA, mRNA or rRNA) are amplified in a background of double-stranded DNA.^[35] Real-time multiplex NASBA with molecular beacons is a one-tube amplification and detection method, and the risk of carry-over contamination is therefore minimized (Figure 4). The advantage with PreTect HPV-Proofer and NucliSENS EasyQ is that both HPV detection and genotyping are performed in the same reaction. RT-PCR techniques are not commercially available, suffer from being rather laborious and are therefore not suitable for large-scale HPV testing. In contrast to the NASBA technology, RT-PCR may have problems with DNA contamination. Gen-Probe is currently developing the APTIMA^® HPV Assay, targeting E6/E7 mRNA from 14 carcinogenic HPV genotypes; a prototype of this assay has been evaluated in one cross-sectional study.^[36]

(Enlarge Image)

Schematic presentation of the NASBA technology including molecular beacons for real-time detection. (A) Two specific primers and three enzymes are required. The reaction is performed at 41°C and is started by the annealing of one primer to the target RNA sequence followed by building of a RNA/DNA hybrid by RT. This hybrid is recognized by Rnase H, which degrades the RNA strand. The other primer anneals to the newly synthesized DNA strand and RT creates a ds DNA molecule with the T7 RNA polymerase which anneals to this sequence and creates single-stranded anti-sense RNAs which in turn are targets for the primers. The reaction repeats itself and within 90 min, more than 1 million new copies are obtained. (B) Molecular beacons are DNA probes with modified ends. Upon binding to complementary RNA transcripts the probe undergoes conformational change and a fluorescence signal is emitted. ds: Double-stranded; RT: Reverse transcriptase. Reproduced with permission from Biomerieux.

E6 and E7 mRNA levels have been found to increase with lesion severity,^{[24,34,36,37]} therefore the detection of E6/E7 mRNA may be of higher prognostic value and may improve the specificity and positive predictive value compared with HPV DNA testing in screening. A Norwegian cross-sectional study has shown that 3% of women older than 30 years are positive with PreTect HPV-Proofer and for women with normal or low-grade cytology HPV detection by Gp5+/6+ consensus PCR is significantly more often positive than by PreTect HPV-Proofer.^[38] The prognostic value of this test has also been evaluated in a 2-year follow-up study of 77 women with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL) cytology.^[39] Women who tested positive with PreTect HPV-Proofer were 70-times more likely to be diagnosed with histologically confirmed cervical intraepithelial neoplasia (CIN) 2+ than women who tested negative. Furthermore, compared with PCR, PreTect HPV-Proofer showed equal clinical sensitivity for CIN 2+ as well as a higher clinical specificity, suggesting that the mRNA-based assay may offer an improvement for the triage of women with equivocal or low-grade cytology. In a study of women younger than 30 years,^[40] the positivity rate by PreTect HPV-Proofer was 14.5% compared with 32.5 and 20.8% by consensus PCR and type-specific PCR, repectively, for the five HPV types. The lower number of women testing positive with the mRNA test represents fewer women to be followed up; however, any conclusions with respect to clinical specificity in this study cannot be drawn due to the lack of histological data for these women. Still, a potential value of HPV mRNA testing among women younger than 30 years of age remains possible. HPV DNA testing is not recommended for this age group due to the generally high prevalence of transient HPV infections. In a Norwegian study of 190 cervical biopsies showing different degrees of CIN, E6 and E7 mRNA were detected in all but one of the PCR-positive cases of high-grade CIN lesions; the detection rate for HPV 16, 18, 31, 33 and 45 in CIN 3 biopsies was 92% both with PCR and with the NASBA assay.^[41]

Additional Norwegian hospital-based and cross-sectional studies have shown that PreTect HPV-Proofer is positive in 89% of squamous cell carcinoma of the cervix and in 77% of high-grade precursor lesions (CIN 2/3 or adenocarcinoma in situ).^[24,42] High-grade histology (CIN 2+) was detected in 83% of the women with normal cytology and positive PreTect HPV-Proofer.^[24] The predictive values of HPV testing were not calculated in this study; however, the clinical specificity of mRNA testing seems to be improved compared with HPV DNA testing. It is reported that a single positive mRNA result may be more predictive of a persistent infection than a DNA test.^[43] Although few samples were included in this study, this is an interesting finding that should be further investigated.

Gen-Probe is currently developing the APTIMA HPV Assay, targeting E6/E7 mRNA from 14 carcinogenic HPV genotypes. A prototype of this assay was evaluated recently in 536 women with histological outcomes.^[36] Detection of E6/E7 mRNA was strongly correlated with severity of the lesion; more than 90% of CIN 3 cases and all five carcinomas revealed E6/E7 mRNA.

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