Infection with high-risk types of human papillomavirus

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1 Clinical Performance of a Human Papillomavirus Messenger RNA Test (Aptima HPV Assay) on Residual Material From Archived 3-Year-Old PreservCyt Samples With Low-Grade Squamous Intraepithelial Lesion Marianne Waldstrøm, MD; Dorthe Ørnskov, PhD N Context. Human papillomavirus (HPV) testing is widely used in the triage of women with a borderline smear result but the efficiency of testing women with low-grade squamous intraepithelial lesion (LSIL) is less clear, mainly because of lack of specificity. New HPV tests are emerging, which detect E6/E7messenger RNA (mrna), and preliminary data suggest that they might have a higher specificity. However, mrna is less stable than DNA, thus posing a challenge to the preservation abilities of the cell-collecting medium. Objective. To evaluate the clinical performance of an HPV mrna assay on 3-year-old archived liquid-based samples, all with a diagnosis of LSIL. Design. The residual material from old archived PreservCyt samples from 442 women were tested with the Aptima HPV Assay, which detects E6/E7 mrna from 14 high-risk HPV types. The samples had been stored at room temperature without any further handling. Infection with high-risk types of human papillomavirus (HPV) is the leading cause of cervical cancer and its precursor lesion, cervical intraepithelial neoplasia (CIN). 1,2 This knowledge is widely being used in Europe and the United States in the management of cervical disease. It is recommended that an HPV DNA test be used to triage cytologic samples with borderline lesions to direct the further management of the patient, as well as for surveillance after treatment of CIN lesions. 3,4 Several studies have shown that, compared to repeated cytology, HPV DNA assays have a better sensitivity and equal specificity in finding high-grade CIN, and a negative HPV DNA test result has a very high negative predictive value. 5 Most cytologic samples with low-grade squamous intraepithelial lesion (LSIL) harbor HPV DNA, but many of these lesions regress spontaneously. Therefore, it is generally not considered effective to triage these samples Accepted for publication November 15, From the Department of Pathology and Microbiology, Vejle Hospital, Vejle, Denmark. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Marianne Waldstrøm, MD, Department of Pathology, Vejle Hospital, Kabbeltoft 25, 7100 Vejle, Denmark ( Marianne. waldstroem@slb.regionsyddanmark.dk). Results. Follow-up was available for 405 women, 67 of whom had histologic confirmed cervical intraepithelial neoplasia (CIN) 2+ and 31 with CIN 3+. The sensitivity and specificity for the mrna assay was 92.5% and 38.2%, respectively, for detecting CIN 2+, and 93.9% and 35.5%, respectively, for detecting CIN 3+. When evaluating separately the performance of the test for women younger than 30 years and for women 30 years or older, the sensitivity was found to be similar in the 2 groups, but the specificity was significantly lower for the younger women. Conclusion. Messenger RNA is well preserved in old archived PreservCyt samples. Triaging women with LSIL, using the Aptima HPV Assay, seems to be effective with a good sensitivity and a good specificity, especially for women 30 years or older. (Arch Pathol Lab Med. 2011;135: ) with a HPV DNA assay because of lack of specificity for high-grade lesions. 6 New methods claiming to have a higher specificity for detecting high-grade lesions are currently emerging and being evaluated. One of the most promising is mrnabased HPV assays. It is widely accepted that for HPV to cause cervical cancer, persistent infection and a cellular environment that allows high expression of the viral oncogenes E6 and E7 are necessary. 7 The E6 and E7 oncogenes are necessary for viral replication. They are overexpressed when regulation of the cell is disrupted during transformation of the cell to a malignant phenotype. Therefore, it seems reasonable to hypothesize that detecting the mrna of E6/E7 oncogenes may identify lesions that are more clinically relevant. Indeed several studies 8,9 have supported this hypothesis, with findings showing a better specificity for detecting high-grade lesions by mrna-based HPV assay than by HPV DNAbased assay. Still, there are few studies on the subject and more data are needed to prove the clinical applications of these tests. Previous studies 10 have documented that HPV DNA is well preserved in certain liquid-based media used to collect the exfoliated cells from the cervix. The widely used PreservCyt solution used with the ThinPrep system (Hologic UK Ltd, Crawley, West Sussex, United King Arch Pathol Lab Med Vol 135, August 2011 HPV Testing in Cervical Cytology Waldstrøm & Ørnskov

2 dom) has been approved by the US Food and Drug Administration (FDA) for adjunctive testing for HPV DNA with the Digene Hybrid Capture 2 (QIAGEN Inc, Gaithersburg, Maryland) and Cervista HPV HR (Hologic UK Ltd) systems for samples stored for periods of up to 6 weeks. Castle et al 11 found that HPV DNA, as detected by the Hybrid Capture 2 test, was unaffected by storage in those samples collected in the PreservCyt medium that had been stored at ambient temperature for up to 9 years. Messenger RNA, on the other hand, is generally less stable than DNA, which challenges the ability of the sample collection medium to preserve it. Some studies have indeed reported difficulties in preserving HPV mrna of sufficient quality in the media, for instance, in the SurePath preservative fluid solution (Tripath Inc, Burlington, North Carolina). 12 On the other hand, when Murphy et al 13 used an RNA extraction procedure containing proteinase on the residual material of 2- to 5- week-old SurePath samples, they obtained HPV mrna of sufficient quality and quantity to be used for applications such as real-time reverse transcription-polymerase chain reaction (PCR). 13 Cuschieri et al 14 have reported high preservation of E6/E7 HPV mrna for storage periods of at least 2 weeks, and Habis et al 15 have documented a high yield of HPV mrna in the PreservCyt medium. However, studies on the long-term effects of storage on the quality and quantity of HPV mrna in different cytologic collection media are still very sparse. The aim of this study was to evaluate the clinical performance of the E6/E7 mrna-based HPV test, the Aptima HPV Assay (Gen-Probe Inc, San Diego, California), on the residual material from 3-year-old archived liquid-based PreservCyt samples with the cytologic diagnosis of LSIL. The result of the HPV mrna test was compared with the long-term cytologic and histologic follow-up of the women. MATERIALS AND METHODS In this retrospective study, we retrieved 442 samples from the year 2006 from the files of the Department of Pathology, Vejle Hospital, Denmark, all with a cytologic diagnosis of LSIL. The residual material of the PreservCyt liquid-based specimens had been stored for more than 3 years in the basement of the hospital at room temperature without any further handling. Data from the clinical follow-up, with histologic and/or cytologic findings, were retrieved for each patient from the Danish National Register of Pathology, Patobank, which contains all histologic and cytologic diagnoses from all the pathology departments in Denmark. The data were collected in January 2010, such that the women had at least 3 years of followup. The follow-up had been in accordance with the regional guidelines in 2006, which recommended cytologic control after 3 months; if the control smear revealed atypical squamous cells of undetermined significance or a higher-grade lesion, the women were referred to colposcopy. If the control smear result was negative, 1 other cytologic control after 6 months was recommended before return to the normal screening program with recall every 3 years. The original cytologic diagnoses were made by using a modified World Health Organization (WHO) classification, with separate diagnosis for condyloma and mild dysplasia. For the purpose of this study, these entities were classified according to the Bethesda 2001 guidelines, and these 2 categories were therefore put together in the LSIL category. All the histopathologic diagnoses were made according to the WHO recommendation by using the CIN classification. The study was approved by the local Danish authority according to Danish law. The statistical analysis was calculated Table 1. Cytologic or Histologic Follow-up Diagnosis and Positivity Rate for Aptima HPV a E6/E7 Messenger RNA Test Follow-up Diagnosis No. of Women Positive for Aptima HPV, % Cytology only Normal LSIL ASCUS 5 80 Total Histology Normal CIN CIN CIN Adenocarcinoma in situ Adenocarcinoma Total Abbreviations: ASCUS, atypical squamous cells of undetermined significance; CIN, cervical squamous intraepithelial neoplasia; HPV, human papillomavirus; LSIL, low-grade squamous intraepithelial lesion. a Gen-Probe Inc, San Diego, California. with GraphPad Prism 5.0 (GraphPad Software Inc, San Diego, California). Detection of HPV mrna was performed by using the Aptima HPV Assay. This assay is a multiplex nucleic acid amplification test, which detects the E6/E7 mrna of 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The Aptima HPV Assay is based on 3 main steps: (1) target capture during which the mrna is captured by using target-specific oligomers linked to magnetic particles; (2) transcription-mediated amplification during which the captured mrna is amplified (using RNA polymerase and reverse transcriptase); and (3) detection of the mrna with dual kinetic assay, which is a variation of the hybridization protection assay. Using chemiluminescent-labeled probes, dual kinetic assay allows detection of both the target mrna and an internal control, which is introduced in step 1 to avoid false negatives. Briefly, 1 ml of a well-mixed PreservCyt liquid-based sample was added to a test tube containing lysis buffer, which destroys the cells and releases the mrna. The test tube was loaded onto the fully automated TIGRIS DTS instrument (Gen-Probe Inc), with the analysis performed according to the manufacturer s instructions. Positive and negative calibrators were included in each run to determine the validity of the run and the cutoff value for the internal control as well as the analyte. Five samples, representing histologic CIN 2+ lesions, were negative for HPV mrna. To pursue this observation, these samples were further analysed with the Linear array HPV genotyping assay (Roche Molecular Systems Inc, Alameda, California). Linear array is a DNA-based test, which is able to detect 37 HPV types, including the 14 high-risk types. The PCR targets a 450-bp region of the L1 gene, and detection is performed with type-specific oligonucleotide probes with colorimetric detection. Briefly, 1 ml of a well-mixed PreservCyt liquid-based sample was centrifuged and the pellet was solubilized in 200 ml of PBS. The DNA was purified by using the MagNA Pure LC instrument (Roche Diagnostics, GmbH, Mannheim, Germany) and subsequently subjected to the Linear array assay according to the manufacturer s instructions. RESULTS Histologic and/or cytologic follow-up was available for 405 of 442 women, and these women were included for further analyses. The age range of the women included in the study was 16 to 65 years, with a mean age of 32 and a median age of 30. Arch Pathol Lab Med Vol 135, August 2011 HPV Testing in Cervical Cytology Waldstrøm & Ørnskov 1053

3 Table 2. Sensitivity, Specificity, and Negative and Positive Predictive Values of the Aptima HPV a Messenger RNA Assay for Detecting Cervical Intraepithelial Neoplasia (CIN) Grade 2 or Higher and for Detecting CIN Grade 3 and Higher Disease Sensitivity, % (95% CI) Specificity, % (95% CI) NPV, % (95% CI) PPV, % (95% CI) CIN ( ) 38.2 ( ) 96.3 ( ) 22.9 ( ) CIN ( ) 35.5 ( ) 98.5 ( ) 11.4 ( ) Abbreviations: CI, confidence interval; NPV, negative predictive value; PPV, positive predictive value. a Gen-Probe Inc, San Diego, California. Two hundred and twelve women had a cytologic follow-up, with at least 1 Papanicolaou smear, and 193 women underwent biopsy (punch biopsies or cone biopsies). For each of the women, the most severe diagnosis was recorded and used in the further analysis. The results from the follow-up diagnoses are listed in Table 1. Sixty-seven women had lesions with a histologic diagnosis of CIN 2 or higher grade (CIN 2+) at follow-up and 33 women had histologically confirmed CIN 3 or a higher-grade lesion (CIN 3+). Two hundred and seventy-one women (67%) had a positive test result for Aptima HPV E6/E7 mrna and 134 (33%) tested negatively. The distribution of negative and positive test results according to age is shown in the Figure, and the percentage of positive test results according to the follow-up diagnosis is shown in Table 1. The percentage of positive samples increases with the severity of the cytologic or histologic diagnosis, from 57% among women with normal histology or cytology at follow-up, up to 91% for CIN 2+ lesions, and 94 % for CIN 3+ lesions. The 2 women with glandular lesions, included in the analysis, both tested positive. Using histologically confirmed CIN 2+ and CIN 3+ lesions as the gold standard, we calculated the sensitivity and specificity for the Aptima HPV Assay, as listed in Table 2. The positive predictive values (PPVs) and negative predictive values for CIN 2+ were 22.9% and 96.3%, respectively, and for CIN 3+, 11.4% and 98.5%, respectively. To investigate the effect of age on the clinical performance, sensitivity and specificity were calculated separately for the women younger than 30 years and for women 30 years or older (Table 3). The sensitivity for the 2 age groups was quite similar for CIN 2+ lesions (94.3%; 95% confidence interval [CI], versus 90.6 [95% CI, ]), but the specificity was lower for the women younger than 30 (27.3%; 95% CI, versus 47.3%; 95% CI, ). The same was true when using CIN 3+ lesions as the gold standard. Five samples with a follow-up diagnosis of CIN 2+, including 2 samples with histologically confirmed CIN 3, had a negative Aptima HPV test result. We retested these 5 samples with the LINEAR ARRAY HPV genotyping assay. The results are listed in Table 4. COMMENT In the present retrospective study, we have evaluated the clinical performance of a HPV test by detecting E6/E7 mrna in 14 oncogenic HPV types from 3-year-old archived PreservCyt samples, all with a cytologic diagnosis of LSIL. On the basis of these results, we conclude that the mrna in the PreservCyt samples was sufficiently well preserved and usable for nucleic acid analysis. The overall positivity rate of the Aptima HPV Assay was 67%, which is lower than the rate expected when using a HPV DNA test. In a meta-analysis, Arbyn et al 6 have reported a positivity rate of 76% (95% CI, 71% 81%) for HPV DNA in the LSIL group. The relative number of HPV-positive LSIL samples in our material decreases with women s age (Figure) and increases with the severity of the diagnosis (Table 1), as also reported in several other studies. 6,16 Fiftyseven percent of women with normal cytologic follow-up, as well as 57% of women with normal histologic followup, had a positive Aptima mrna test in their index LSIL sample, indicating that many mrna-positive, low-grade lesions regress and clear spontaneously. Three samples with histologically confirmed CIN 2 lesions and 2 samples with CIN 3 lesions tested negatively with the Aptima HPV Assay. All 5 samples had a positive result when retested for HPV DNA. One sample with a CIN 2 diagnosis and 1 sample diagnosed as CIN 3 showed only HPV types considered nononcogenic (HPV type 6 and HPV type 54), which are not a target for the Aptima HPV Assay. The second biopsy specimen with histologically confirmed CIN 3 and a negative mrna test result had a weak but positive reaction for HPV type 31. Indeed, CIN 3 is a precursor lesion for cervical cancer, but some CIN 3 lesions may regress. 17 However, little is known about factors involved in the progression or regression of a CIN 3 lesion. Some authors 18 have postulated that lesions that are HPV DNA positive but mrna negative are more likely to regress, but this still has to be proved. Studies using the Aptima HPV Assay are so far very few. However, the clinical performance of the Aptima Table 3. Sensitivity, Specificity, and Negative and Positive Predictive Values for Women Younger Than 30 and for Women 30 Years and Older for Detecting Cervical Intraepithelial Neoplasia (CIN) Grade 2 or Higher and Grade 3 and Higher Age Disease Sensitivity, % (95% CI) Specificity, % (95% CI) NPV, % (95% CI) PPV, % (95% CI),30 (n 5 189) CIN ( ) 27.3 ( ) 95.5 ( ) 22.8 ( ) CIN ( ) 24.6 ( ) 97.7 ( ) 9.0 ( ) $30 (n 5 216) CIN ( ) 47.3 ( ) 96.7 ( ) 23.0 ( ) CIN ( ) 45.2 ( ) 98.9 ( ) 14.3 ( ) Abbreviations: CI, confidence interval; NPV, negative predictive value; PPV, positive predictive value Arch Pathol Lab Med Vol 135, August 2011 HPV Testing in Cervical Cytology Waldstrøm & Ørnskov

4 Table 4. Linear Array a Test Results of Samples Negative by Aptima HPV b Messenger RNA Array and With a Histologic Diagnosis of Cervical Intraepithelial Neoplasia (CIN) Grade 2 or More Case No. Age Histologic Follow-up Diagnosis Biopsy Material HPV Types Found by Linear array a 1 41 CIN 2 Cone 16, 51, 52, 59, 61, 62, CIN 2 Cone CIN 2 Cone 42, CIN 3 Cone CIN 3 Cone 31 Abbreviation: HPV, human papillomavirus. a Roche Molecular Systems Inc, Alameda, California. b Gen-Probe Inc, San Diego, California. HPV test with our material seems comparable to the data reported by Szarewski et al. 8 These authors found a sensitivity of 97.4% and a specificity of 38.8% for detecting CIN 3+ lesions in a population of women referred for colposcopy. These figures are slightly higher but not significantly different from our data, which showed a sensitivity of 93.9% (95% CI, ) and specificity of 35.5% (95% CI, ). The same applies for detecting CIN 2+ lesions. In their study, the authors calculated PPV separately for detecting high-grade disease for the group of women having a referral smear with a diagnosis of mild dyskaryosis or a lesser abnormality. They found a PPV of 23.3% for CIN 2+ lesions and 12.9% for CIN 3 lesions, which is comparable to our results, with a PPV for CIN 2+ and CIN 3+ of 22.9% and 11.4%, respectively. In a study by Dockter et al, 19 the authors found a similar sensitivity for detecting high-grade disease with the Aptima HPV Assay, but a higher specificity and a higher PPV for both CIN 2+ and CIN 3+ lesions as endpoints. However, the design of their study was different from ours, as they evaluated the clinical performance in a referred population and biopsy specimens were available The number of positive and negative Aptima HPV (Gen-Probe Inc, San Diego, California) mrna test results by women s age. HPV, human papillomavirus; mrna, messenger RNA. in only 141 of 753 patients. If the colposcopy results were negative, no histologic or cytologic sample was taken and the women were considered to be disease negative. Data on the referral diagnosis of the women included are not available, and differences in the population studied might explain the differences in the PPV values between the study cited and ours. In the present study we did not directly compare our mrna HPV results with those of a HPV DNA test, but in a meta-analysis by Arbyn et al, 5 lower specificity has been reported for triage of women with LSIL, when using Hybrid Capture 2 or HPV DNA PCR. They found specificities (pooled estimates) of 28.6% (95% CI, ) for detecting CIN 2+ lesions and 21.6% (95% CI, ) for CIN 3+ lesions. In our material of old achieved LSIL samples, we found higher specificities for CIN 2+ and CIN 3+ lesions (38.2% and 35.5%, respectively) and high sensitivities as well (Table 2), suggesting a better clinical performance for the Aptima HPV Assay. As reported for DNA-based HPV tests, the specificity for detecting high-grade disease increases with age both for the group of women with borderline (atypical squamous cells of undetermined significance) lesions and those with LSIL and is reported to be quite low for women younger than 30 years. 5,6,16,20 This limits the effectiveness of triaging the younger-age group with a HPV DNA test and therefore, more specific tests are needed. Previous studies 21 have indicated a relatively higher specificity for women younger than 30 years when using an mrna-based test. Molden et al 21 evaluated the mrna expression in women younger than 30 years by using a mrna test detecting E6/E7 mrna of 5 oncogenic HPV types (PreTect HPV-Proofer, Norchip, Klokkarstua, Norway) and compared the results to a Gp5+/6+ consensus HPV DNA PCR test and a type-specific HPV DNA test. They found a significantly lower number of HPV-positive women with the HPV mrna test than with the HPV DNA tests, which might indicate that the mrna test is more specific. However, they did not evaluate the clinical performance by having a follow-up with histologic evaluation. In the predictor study, Szarewski et al 8 found that for all HPV tests included in their study, the clinical performance for detecting high-grade disease was better for women older than 30 years than for younger women. This also applied for the 2 mrna HPV tests evaluated (Aptima HPV Assay and the PreTect HPV-Proofer). These data correlate well with our results: for the group of women younger than 30 years, the tests retained sensitivity for detecting CIN 2+/CIN 3+ lesions but had a lower specificity than the tests performed on older women. Arch Pathol Lab Med Vol 135, August 2011 HPV Testing in Cervical Cytology Waldstrøm & Ørnskov 1055

5 CONCLUSION We found that mrna is well preserved in old, archived PreservCyt samples, stored at room temperature for more than 3 years without any further handling. Furthermore, using the Aptima HPV Assay to triage women with LSIL could prove to be effective, with a good sensitivity and with a higher specificity than the HPV DNA tests, especially among older women. Still, prospective studies are needed to get data with sufficient power to make a conclusion. We gratefully acknowledge laboratory technologist Susanne Hillbrandt for her skilled technical assistance. References 1. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. JPathol. 1999;189(1): Munoz N, Bosch FX, de SS, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003;348(6): Jordan J, Arbyn M, Martin-Hirsch P, et al. European guidelines for quality assurance in cervical cancer screening: recommendations for clinical management of abnormal cervical cytology, part 1. Cytopathology. 2008;19(6): Solomon D, Papillo JL, Davey DD. Statement on HPV DNA test utilization. Diagn Cytopathol. 2009;37(7): Arbyn M, Sasieni P, Meijer CJ, Clavel C, Koliopoulos G, Dillner J. Chapter 9 clinical applications of HPV testing: a summary of meta-analyses. Vaccine. 2006;24(suppl 3):S3/78 S3/ Arbyn M, Martin-Hirsch P, Buntinx F, Van RM, Paraskevaidis E, Dillner J. Triage of women with equivocal or low-grade cervical cytology results: a metaanalysis of the HPV test positivity rate. J Cell Mol Med. 2009;13(4): Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer. Clin Sci (Lond). 2006;110(5): Szarewski A, Ambroisine L, Cadman L, et al. Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears. Cancer Epidemiol Biomarkers Prev. 2008;17(11): Molden T, Kraus I, Karlsen F, Skomedal H, Nygard JF, Hagmar B. Comparison of human papillomavirus messenger RNA and DNA detection: a cross-sectional study of 4,136 women.30 years of age with a 2-year follow-up of high-grade squamous intraepithelial lesion. Cancer Epidemiol Biomarkers Prev. 2005;14(2): Boulet GA, Horvath CA, Berghmans S, et al. Cervical cytology biobanking: quality of DNA from archival cervical Pap-stained smears. J Clin Pathol. 2008; 61(5): Castle PE, Solomon D, Hildesheim A, et al. Stability of archived liquidbased cervical cytologic specimens. Cancer. 2003;99(2): Horvath CA, Boulet G, Sahebali S, et al. Effects of fixation on RNA integrity in a liquid-based cervical cytology setting. J Clin Pathol. 2008;61(1): Murphy PG, Henderson DT, Adams MD, et al. Isolation of RNA from cell lines and cervical cytology specimens stored in BD SurePath preservative fluid and downstream detection of housekeeping gene and HPV E6 expression using real time RT-PCR. J Virol Methods. 2009;156(1 2): Cuschieri KS, Beattie G, Hassan S, Robertson K, Cubie H. Assessment of human papillomavirus mrna detection over time in cervical specimens collected in liquid based cytology medium. J Virol Methods. 2005;124(1 2): Habis AH, Vernon SD, Lee DR, Verma M, Unger ER. Molecular quality of exfoliated cervical cells: implications for molecular epidemiology and biomarker discovery. Cancer Epidemiol Biomarkers Prev. 2004;13(3): Thrall MJ, Smith DA, Mody DR. Women $30 years of age with low grade squamous intraepithelial lesion (LSIL) have low positivity rates when cotested for high-risk human papillomavirus: should we reconsider HPV triage for LSIL in older women? Diagn Cytopathol. 2010;38(6): McCredie MR, Sharples KJ, Paul C, et al. Natural history of cervical neoplasia and risk of invasive cancer in women with cervical intraepithelial neoplasia 3: a retrospective cohort study. Lancet Oncol. 2008;9(5): Lie AK, Risberg B, Borge B, et al. DNA- versus RNA-based methods for human papillomavirus detection in cervical neoplasia. Gynecol Oncol. 2005; 97(3): Dockter J, Schroder A, Hill C, Guzenski L, Monsonego J, Giachetti C. Clinical performance of the APTIMA HPV Assay for the detection of high-risk HPV and high-grade cervical lesions. J Clin Virol. 2009;45(suppl 1):S55 S Ronco G, Cuzick J, Segnan N, et al. HPV triage for low grade (L-SIL) cytology is appropriate for women over 35 in mass cervical cancer screening using liquid based cytology. Eur J Cancer. 2007;43(3): Molden T, Kraus I, Karlsen F, Skomedal H, Hagmar B. Human papillomavirus E6/E7 mrna expression in women younger than 30 years of age. Gynecol Oncol. 2006;100(1): Arch Pathol Lab Med Vol 135, August 2011 HPV Testing in Cervical Cytology Waldstrøm & Ørnskov