Abstract
Background: In recent years, efforts have been made to identify molecular markers as potential screening tools in the early detection of cervical cancer precursors. Patients and Methods: One-hundred-eighty-two women admitted to the Colposcopy Unit of Tor Vergata University Hospital were enrolled in this study. The inclusion criteria were: i) Pap test with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL); ii) normal cytology but human papillomavirus (HPV) DNA test positive for at least one of the most frequently detected five high-risk HPV types (16, 18, 31, 33 and 45). HPV DNA was detected with the HPV Sign kit and the type was assigned by pyrosequencing using the PyroMark ID System. E6/E7 transcripts of the high-risk HPV types 16, 18, 31, 33 and 45 were detected by the NucliSense EasyQ HPV kit. Results: Overall, 90 (49.5%) patients were HPV-DNA negative, whereas 92 (50.5%) were HPV-DNA positive. Single infections were detected in 55 women: HPV 16 ranked first (56.4%), followed by HPV 18 (21.8%), HPV 31 (9%), HPV 33 (7.3%), and HPV 45 (5.5%). Co-infections were detected in 37/92 (40.2%) positive cases; HPV 16 was detected most frequently (27/37), followed by HPV 18 and 31. All patients underwent HPV RNA testing: 47/182 (25.8%) tested positively while 135/182 (74.2%) were negative. HPV 16 E6/E7 transcripts was the most frequently detected. Conclusion: Detection of HPV E6/E7 oncogenic transcripts may be used as a molecular biomarker in women with ASCUS or LSIL to help identify women at risk of disease progression.
Cervical cancer is the second most common type of cancer in women worldwide. The majority of cases occur in the developing world, where, in some countries, cervical cancer represents the primary cause of cancer-related death (1). Human papillomavirus (HPV) is the recognized etiological agent of cervical cancer, being detected in almost all cervical cancer cases (2). To date, 120 human HPVs have been identified (3) and based on epidemiological studies, they are classified as low-risk types and high-risk types. Low-risk types are associated commonly with benign lesions such as warts and condylomata, while high-risk types are associated with high-grade lesions and cervical cancer (4).
HPV prevalence estimates are highest in women younger than 34 years, with prevalence decreasing in the 35-44 year group. A second peak is generally observed in the older age groups (45-54 years, and more than 54 years) in all regions, with the exception of Asia where rates continued to decrease (5). About 90% of the women are cleared of HPV infection within two years (6) and fewer than 10% develop persistent infections that can progresses towards high-grade lesions and invasive cancer. Persistent infection is sustained by oncogenic HPV which upon integration into the human genome cause a deregulation of the cell cycle control that can lead to cell transformation and invasive cervical cancer. Deregulation is caused by the inactivation of the cellular proteins p53 and pRB by the viral oncoproteins E6 and E7, respectively, which are over expressed after integration of the HPV genome into the human chromosome (7, 8).
As HPV infection is quite common among sexually active women, and the vast majority of low-grade squamous intraepithelial lesions (LSIL) are HPV positive, HPV testing is recommended for atypical squamous cells of undetermined significance (ASCUS) cytology and women aged 30 years or more. In these cases, HPV testing reduced the referral to colposcopy and was cost-effective (9, 10). In addition, it has been suggested that risk stratification by single typing of HPV might improve HPV-based triage both for ASCUS and LSIL (11).
Although HPV-DNA testing improved the management of women infected with HPV (12, 13), its positive predictive value is low (14-16) due to the high prevalence of HPV infection. Moreover, HPV-DNA testing cannot distinguish between transient and potentially persistent infections that favor cell transformation. Therefore testing for E6 and E7 oncogenic transcripts could be more specific and have a higher predictive value (17-19).
In this study, we evaluated the diagnostic value of the E6/E7 RNA testing for five high-risk HPV types (HPV 16, 18, 31, 33 and 45) in women with ASCUS, LSIL, and in those with normal cytology but with a HPV-DNA test positive for at least one of the five high-risk types. In particular, the results of RNA testing were correlated to those of HPV-DNA testing and cytology.
Patients and Methods
Study group. Of the women admitted to the Colposcopy Unit of the Department of Gynecology and Obstetrics of the Tor Vergata University Hospital, Rome, Italy, 182 were enrolled in this study between December 2009 and May 2011. The inclusion criteria were as follows: i) cytology of ASCUS or LSIL; ii) normal cytology but HPV-DNA test positive for at least one of the most frequent five high-risk HPV types (16, 18, 31, 33 and 45). The age of the women ranged from 17 to 65 years, with a median of 25 years.
Informed consent was obtained from all participants in accordance to our Institutional Review Board.
Cytology screening. Ecto- and endocervical cells were collected using an Ayre's spatula and cytobrush, respectively. The cells were streaked out on two distinct areas of a glass slide and spray fixed. Smears were classified according to the 2001 Bethesda system (20).
HPV-DNA testing and typing. Using the collection method described above, the cervical cells were rinsed in the PreservCyt solution (Hologic, Marlborough, MA, USA) and stored according to the manufacturer's instructions until analysis. Two aliquots of 5 ml each were transferred into collection tubes and after centrifugation at 839× g for 3 min the supernatant was discarded and the cellular pellet stored at −80°C until analysis.
Total DNA/RNA was extracted using an Easy-Mag automatic extractor (bioMerieux S.A., Marcy l'Etoile, France). HPV-DNA was detected with HPV Sign kit (Diatech, Jesi, Italy) following the manufacturer's instructions. HPV type was assigned by pyrosequencing using the PyroMark ID System (Qiagen, Hilden, Germany). The assay targets a hypervariable region of 30 nucleotides within the L1 gene. The obtained product is aligned to an HPV library and the viral type assigned by the software IdentiFire SW (Qiagen, Hilden, Germany).
Detection of the E6/E7 transcripts of HPV 16, 18, 31, 33 and 45. The oncogenic E6/E7 transcripts of the five high-risk HPV types 16, 18, 31, 33 and 45 were detected by the NucliSense EasyQ HPV kit (bioMerieux S.A.) that uses nucleic acid sequence-based amplification technology (NASBA) to amplify the viral target. Six different molecular beacons are used to identify and amplify the corresponding five HPV types and the U1A gene. Two different fluorophores, 6-carboxyfluorescein (6-FAM) for HPV 16, 31 e 33, and 6-carboxy-X-rhodamine (6-ROX) for U1A, HPV 18 and 45, allow simultaneous duplex amplification. Dedicated software reveals the presence or absence of the viral target.
Statistical analyses. Correlation analyses were carried out in order to determine assay relationships. Chi-square test was used to confirm the statistical significance. All p-values <0.05 were considered statistically significant. Agreement between tests was calculated by k statistics.
Results
Cytological and virological findings. Of the 182 women enrolled in the study, 62 (34.1%) had a normal cytology, 59 (32.4%) were diagnosed with ASCUS and 61 (33.5%) with L-SIL. Overall, 90 (49.5%) patients were HPV-DNA negative, whereas 92 (50.5%) were HPV-DNA positive. Single infections were detected in 55 women: HPV 16 ranked first (56.4%), followed by HPV 18 (21.8%), HPV 31 (9%), HPV 33 (7.3%), and HPV 45 (5.5%) (Table I). Co-infections were detected in 37/92 (40.2%) positive patients. HPV 16 was detected most frequently (27/37), followed by HPV 18 and 31.
All patients underwent HPV-RNA testing: 47/182 (25.8%) tested positively while 135/182 (74.2%) were negative. HPV 16 RNA transcript was the most frequently detected (Table II).
Cytological findings versus HPV DNA and RNA testings. Out of the 62 women with normal cytology, 32 (51.6%) tested HPV-DNA positive, and 15 of them (24.2%) also expressed the E6/E7 oncogenic viral transcripts. It is worth noting that among the women with a negative Pap test, 23 had no history of past HPV infection, whereas the remaining 39 had anamnesis positive for abnormal Pap test, HPV-DNA typing, cervical lesions at colposcopy, or conisation.
Of the women with no history of past HPV infection, 11/23 (47.8%) were HPV-DNA positive and 6/11 (54.5%) expressed HPV-RNA transcripts. Those with a history of HPV infection, 21/39 (53.8%) were HPV-DNA positive and 9/21 (42.8%) expressed the E6/E7 oncogenic transcripts.
Out of the 59 women with ASCUS, 23/59 (39%) were HPV-DNA positive and 10/59 (16.9%) expressed the E6/E7 transcripts. Finally, of the 61 women diagnosed with LSIL, 37/61 (60.7%) were HPV-DNA positive and in 22/61 (36.1%) E6/E7 transcripts were detected (Table III).
The statistical correlation between HPV-DNA and Pap tests findings was of borderline significance (p=0.0585), as was that between presence of E6/E7 transcripts and Pap test finding (p=0.0536).
Distribution of HPV types in the study population. In a total of 182 cases, 90 tested negatively and 92 positively.
HPV mRNA test results. In a total of 182 cases, 135 tested negatively for HPV mRNA, and 47 positively.
HPV-DNA test versus HPV-mRNA test. Out of the 182 cases examined with both molecular tests, 90 women had a negative HPV-DNA test and only two expressed the E6/E7 transcripts. Among the 92 women with a positive HPV-DNA test, 47 (51.1%) also expressed the E6/E7 transcripts (Table IV). Interestingly, seven cases expressed E6/E7 transcripts belonging to an HPV type not identified by the HPV-DNA test (Table V). The correlation co-efficient between the two assays was K=0.46 and p<0.0001.
Correlation of HPV DNA and mRNA test results with cytological data.
Discussion
In this study, we evaluated the diagnostic value of HPV E6/E7 transcripts by NucliSens EasyQ HPV assay versus the HPV-DNA test and the cytology in women with low-grade lesions and normal cytology but with a positive DNA test for at least one of the five genotypes also detected by the NucliSens EasyQ HPV assay. The high number (92/182) of women with a positive HPV-DNA test confirms that HPV infection is widespread among sexually active women, even though the majority of these infections will be cleared by the immune system (21). Interestingly, of the 92 women with a positive HPV-DNA test, about half of them (47/92) also had a positive HPV-RNA testing, despite the selection of women with low-grade lesions or normal cytology. It has been reported that the probability of viral integration increases with the progression of the lesion (15, 18, 22). In our study, women with normal cytology had a higher percentage (24.2%) of viral integration than that of women with ASCUS (16.9%), while in LSIL lesions, the percentage was the highest (36.1%). These data differ from that reported by Sǿrbye et al. (19), where of the 2099 women with minor cervical lesions, 406 (19.3%) had a positive RNA test, and from the results reported by Trope et al (23), who detected HPV-RNA transcripts in only 3.3% of the women with normal cytology using the same RNA assay. In contrast, Waldstrom et al (24) found RNA transcripts in 31.7% of the ASCUS lesions examined using the Aptima HPV assay (Gen-Probe, CA, USA) which detects the E6/E7 transcripts of 14 oncogenic HPV types. The histological examination revealed the presence of CIN2+ or CIN3+ lesions. Higher grade lesions are more likely to contain integrated HPV genome. Factors such as the presence of an underlying high-grade lesion, or the molecular biology technique used may affect the rate of detection of E6/E7 transcripts (24).
Agreement between HPV DNA and mRNA test results.
The genotype profile detected by the HPV-DNA and –RNA assays was substantially concordant: K=0.46, p<0.0001 as already reported (23). However, in seven cases, the two tests detected different genotypes (Table V). This discrepancy was due to the detection by the HPV-DNA assay of HPV genotypes not detected by the NucliSens EasyQ HPV assay due to the lack, in this latter assay, of specific primers/probes; in one case the HPV 31 detected at the DNA level was not found at the RNA level, probably because of the presence of the episomal form only (Table V).
A limitation of the study is the small number of women examined and the lack of follow-up data. However, the aim of the study at this stage was to identify women at risk of disease progression based on the detection of the E6/E7 oncogenic transcripts. The preliminary results of this study suggest the utility of the introduction of the RNA test in the management of women with a cytological diagnosis of ASCUS or LSIL. In addition, the detection of E6/E7 oncogenic transcripts in cases with normal Pap smear may anticipate the identification of women at risk of progression and suggest a closer follow-up is required.
HPV DNA and HPV mRNA test results of the seven discordant cases.
In conclusion, this study supports the notion (16, 17, 19, 22, 23, 24) that women with minor cervical lesions will benefit from the introduction of HPV-RNA testing in a routine setting. Further studies on a larger sample size and follow-up data are warranted.
Acknowledgements
This work was supported by grants from the Italian National Institute of Health, the Italian Ministry of Instruction University and Research (MIUR), “Progetto FILAS” and AVIRALIA Foundation.
- Received February 20, 2012.
- Revision received March 12, 2012.
- Accepted March 13, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.