Early Detection and Diagnosis
Predicting CIN2+ when detecting HPV mRNA and DNA by PreTect HPV-proofer and consensus PCR: A 2-year follow-up of women with ASCUS or LSIL pap smear
It has been suggested that human papillomavirus (HPV) testing improves follow-up of atypical cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) in cervical cancer screening programs. To evaluate the prognostic value of including HPV testing as an adjunct to cytology, we carried out a 2-year follow-up study of 77 women with ASCUS or LSIL Papanicolaou (Pap) smear in the Norwegian Cervical Cancer Screening Program (NCCSP) for detection of histological cervical intraepithelial neoplasia (CIN) 2+. The study includes a comparison between viral mRNA and DNA detection. PreTect HPV-Proofer was used for HPV E6/E7 mRNA detection from the 5 high-risk types 16, 18, 31, 33 and 45, and Gp5+/6+ consensus PCR was used for HPV DNA detection. Twice as many women were positive for HPV DNA (54.6%) than for HPV mRNA (23.4%). PreTect HPV-Proofer and consensus PCR had a sensitivity of 85.7% (95% confidence interval [CI] = 42.1–99.6) for detecting CIN2+ during follow-up. The specificity was significantly higher for PreTect HPV-Proofer, 84.9% (95% CI = 73.9–92.5), than for consensus PCR, 50.0% (95% CI = 37.4–62.6). PreTect HPV-Proofer positive women were 69.8 times (95% CI = 4.3–1137.3) more likely to be diagnosed with CIN2+ within 2 years than PreTect HPV-Proofer negative women. Consensus PCR-positive women were 5.7 times (95% CI = 0.6–52.0) more likely to be diagnosed with CIN2+ within 2 years than PCR-negative women. With equal sensitivity and higher specificity than consensus PCR, the PreTect HPV-Proofer might offer an improvement for the triage of women with ASCUS or LSIL Pap smear. © 2004 Wiley-Liss, Inc.
Cytological cervical cancer screening programs have been successful in reducing the incidence of cervical cancer, even though a single conventional Papanicolaou (Pap) smear is only moderately accurate and does not achieve concurrent high sensitivity and specificity.1, 2
The management of women with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) is problematic because only a small proportion will progress to cervical intraepithelial neoplasia (CIN) 3 and invasive cervical carcinoma (ICC). Histologically verified CIN has been found in 10–60% of women with an ASCUS diagnosis, with CIN2/3 present in more than 5%.3, 4, 5, 6, 7, 8, 9, 10, 11 Pap smear follow-up of women with an ASCUS smear fails to identify all women at higher risk of CIN2+, suggesting that cervical cancer screening programs might benefit from implementing new diagnostic tests in the triage of women with equivocal Pap smears.12
Infection with high-risk (HR) types of HPV is necessary for the development of ICC13, 14, 15, 16 and the expression of the E6/E7 oncogenes is necessary for conversion to and maintenance of malignancy in cervical tissue.17, 18, 19 Therefore, detection of the E6/E7 mRNA of HR-HPV types might serve as a better risk evaluation factor than mere DNA detection for the development of high-grade squamous intraepithelial lesion (HSIL) and ICC.20 The combination of cytology and HPV testing seems to save additional life at a reasonable cost compared to Pap testing alone.21, 22 Detection of E6/E7 mRNA can be achieved by using the commercial PreTect HPV-Proofer assay (NorChip AS, Klokkarstua, Norway), that utilizes nucleic acid sequence based amplification (NASBA).
The aim of our study was to assess whether a positive HPV mRNA or DNA test at the time of an ASCUS or LSIL Pap-smear identifies women diagnosed with a histological CIN2+ after 2 years of follow-up.
Material and methods
The study subjects comprise a subgroup from 4,136 women older than 30 years of age that visited a selection of gynecologists in Oslo, Norway, and have been tested in 2001 for the presence of HPV DNA by Gp5+/6+ consensus PCR and E6/E7 transcripts by real-time multiplex NASBA (PreTect HPV-Proofer, NorChip AS) in addition to cytology.35 PreTect HPV-Proofer detects mRNA from HPV types 16, 18, 31, 33 and 45, whereas Gp5+/6+ consensus PCR detects HPV DNA from the L1 region in >20 HPV types. We included all women with an index Pap smear diagnosis of ASCUS or LSIL (n = 77). The index Pap smear refers to the smear taken together with the HPV testing. Information on Pap smears in the 10-year period before the inclusion in our study was obtained from CRN registers. Former abnormal smears mean any smear that is not normal or unsatisfactory and has been taken before the index smear in 2001.
Seventy-seven women were followed up for 24 months in the registers of the Cancer Registry of Norway (CRN) with subsequent Pap smears and biopsies. During the follow-up period, only histological CIN2+ lesions were reported to the CRN. The women not diagnosed with CIN2+ after follow-up, have not a record of CIN2+ histology and at least one normal Pap smear during follow-up. The women diagnosed with CIN2+ have been treated and a new histological diagnosis has been established on the cone. We used the first histological diagnosis. Four women (3 = ASCUS, 1 = LSIL) did not have any record of Pap smear or biopsy and were excluded from the study. HPV DNA and mRNA have not been retested during follow-up.
The Norwegian Cervical Cancer Screening Program (NCCSP) recommends women with an ASCUS or LSIL Pap smear to have a new smear after 6 months. If ASCUS/LSIL persist, a biopsy is recommended. A detailed description of the NCCSP is published elsewhere.23 To reflect normal practice more closely, we used the original cytological diagnosis (ASCUS or LSIL) and histology diagnosis from the laboratories rather than the review diagnosis by an expert pathology quality control group. The HPV testing and cytology was conducted in 2001. In the NCCSP, a hybrid classification with both dysplasia (World Health Organization [WHO] classification) and CIN is used in cytology. We used the Bethesda nomenclature, before revision in 2001, and the cytological diagnoses HPV condyloma and CIN1 are defined as LSIL. For histopathological evaluation, the WHO nomenclature and criteria are used. The terms ASCUS and LSIL refer to cytological diagnoses and the term CIN refers to histological diagnoses.
Detection of HPV mRNA and DNA
HPV DNA from more than 20 HPV types was identified by Gp5+/6+ consensus PCR whereas the E6/E7 transcripts from the 5 carcinogenic HPV types 16, 18, 31, 33 and 45 were identified by PreTect HPV-Proofer (NorChip AS) at the time of index Pap-smear. A detailed description of the protocols for the HPV testing is found in the article by Molden et al.35 PCR results were analyzed by micro scale gel electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA) and not by in situ hybridization of the amplified products. The follow-up of the ASCUS/LSIL women was not influenced by the result of the HPV tests.
Odds ratio (OR), sensitivity, specificity and predictive values for detecting histological CIN2+ within 2-year follow-up were calculated for each of the HPV tests. The McNemar's test was used for comparison of the HPV tests in the cytological ASCUS and LSIL groups. The two-sample t-test was used for comparison of mean follow-up time and the mean number of Pap smears.
The mean age of the women included in our study was 46.4 and 47.8 years for the index ASCUS and LSIL groups, respectively (p = 0.63). The mean number of former Pap smears was 5.7 and 7.1 for the ASCUS and LSIL group, respectively (p = 0.08), whereas the history of former abnormal Pap smears showed a mean of 1.1 and 1.8, respectively (p = 0.19).
The detection of HPV mRNA and DNA in the index ASCUS or LSIL group by PreTect HPV-Proofer and PCR are shown in Table I. The overall prevalence of E6/E7 mRNA from the HR-HPV types 16, 18, 31, 33 and 45 (23.4%), was significantly lower than HPV DNA by consensus PCR (54.6%).
Table I. Detection of HPV in Index Cytological ASCUS or LSIL by Pretect HPV-Proofer and PCR
|PreTect HPV-Proofer||12 (21.1%)||6 (30.0%)||18 (23.4%)|
|Gp5+/6+ consensus PCR||27 (47.4%)1||15 (75.0%)1||42 (54.6%)1|
Seven women were diagnosed with CIN2+ within 2 years of follow-up. Two of these women had an ASCUS index Pap smear and 5 women had an LSIL index Pap smear. All 7 women with histological CIN2+ were treated by conization and histology on the subsequent cone was carried out. A total of 4 of 7 of these CIN2+ samples were diagnosed as CIN3 by the first histology. They were all confirmed to be CIN3 in the cone. Three cases were CIN2 in the biopsy preceding conization. One case was confirmed to be CIN2 in the cone, one was diagnosed as adenocarcinoma in situ (ACIS) and one was diagnosed with reactive changes. The index Pap smear in the last case was HPV DNA- and mRNA-negative. Four women (5.2%) had no documented follow-up by cytology or histology.
The detection of HPV mRNA and DNA by PreTect HPV-Proofer and PCR, respectively, in ASCUS and LSIL women with diagnosed CIN2+ during follow-up are shown in Table II. PreTect HPV-Proofer and consensus PCR identified the same 6/7 CIN2+ women. Among the 66 women not diagnosed with CIN2+, PreTect HPV-Proofer detected 10 mRNA-positive women in contrast to 33 women positive for HPV DNA by consensus PCR.
Table II. Detection of HPV mRNA and DNA in Baseline ASCUS and LSIL Women with 2-Year Follow-Up for Detection of CIN2+
|ASCUS (n = 54)||PreTect HPV-Proofer|| || |
| ||Negative||43 (82.7%)||0 (0.0%)|
| ||Positive||9 (17.3%)||2 (100.0%)|
| ||Gp5+/6+ consensus PCR|| || |
| ||Negative||29 (55.8%)||0 (0.0%)|
| ||Positive||23 (44.2%)||2 (100%)|
| ||Total||52 (100%)||2 (100%)|
|LSIL (n = 19)||PreTect HPV-Proofer|| || |
| ||Negative||13 (92.9%)||1 (20.0%)|
| ||Positive||1 (7.1%)||4 (80.0%)|
| ||Gp5+/6+ consensus PCR|| || |
| ||Negative||4 (28.6%)||1 (20.0%)|
| ||Positive||10 (71.4%)||4 (80.0%)|
| ||Total||14 (100%)||5 (100%)|
The specificity for detection of HPV in CIN2+ was 84.9% for PreTect HPV-Proofer, compared to 50.0% for consensus PCR, which was significantly different at the 5% level (Table III). Positive predictive value and negative predictive value was 37.5% and 98.3%, respectively for PreTect HPV-Proofer, compared to 15.4% and 97.1% for consensus PCR.
Table III. Sensitivity, Specificity, Predictive Values and OR for Pretect HPV-Proofer and Consensus PCR for Prediction of CIN2+ in 2 Year Follow-Up of ASCUS and LSIL Women
A PreTect HPV-Proofer positive ASCUS/LSIL Pap was 69.8 (95% confidence interval [CI] = 4.3, 1137.3) times more likely to be diagnosed as histological CIN2+ within 2 years than an ASCUS/LSIL PreTect HPV-Proofer negative (Table III). For consensus PCR, a positive HPV test result was 5.7 (95% CI = 0.6, 52.0) times more likely to be diagnosed with CIN2+ within 2 years.
Nine of 52 women with index ASCUS cytology found positive by HPV by PreTect HPV-Proofer, were not diagnosed as CIN 2+ during follow-up. For consensus PCR, the corresponding number was 23/52. For the index LSIL women, there was a significant difference between PreTect HPV-Proofer, which identified 1 of 14 women, and consensus PCR, which identified 10 of 14 women.
No differences in mean follow-up time has been found between women with CIN2+ compared to those without CIN2+ (Table IV).
Table IV. Time Between Index Cytology and Final Diagnosis and Average Number of Pap Smears Among Women Who Developed CIN2+ and Those Who did not
Our prospective study has investigated the detection of HPV in index cytological ASCUS or LSIL Pap smears with a 2-year follow-up for diagnosis of histological CIN2+. We found that a woman having an ASCUS/LSIL Pap smear and a positive PreTect HPV-Proofer result was 69.8 times more likely to be diagnosed with CIN2+ than a woman negative by the PreTect HPV-Proofer assay. For consensus PCR, the OR was 5.7. The significantly higher specificity of PreTect HPV-Proofer as compared to consensus PCR in detecting CIN2+ during follow-up, and the similar sensitivity and negative predictive values, suggest that detection of mRNA from the 5 high-risk HPV types 16, 18, 31, 33 and 45, is more suited for predicting CIN2+ than consensus PCR. The consequence of these findings is that the number of women referred to closer follow-up will remain low without missing the women most likely to progress to CIN2+ by using PreTect HPV-Proofer as an adjunct to ASCUS/LSIL Pap smear, as compared to consensus PCR. This aspect is essential in a screening setting to identify the women with concurrent or higher risk for later development of CIN2+. It is also desirable to keep the number of women that need close follow-up at a minimum because infections with the genital types of HPV are very common.24 Despite their oncogenic potential, most HPV infections typically resolve within 1 year.25, 26, 27 Cuschieri et al.28 carried out a 2-year follow-up of cytological normal women with repeat HPV genotyping by both PCR and PreTect HPV-Proofer. They reported that detection of HPV E6/E7 transcripts was less sensitive but more specific than detection of HPV DNA for the detection of disease at follow-up. Women who were positive for HPV DNA and mRNA transcripts at baseline were significantly more likely to harbor persistent infection compared to those in whom DNA only was detected at baseline.
In the index cytological LSIL group not having a CIN2+ during follow-up, the PreTect HPV-Proofer assay identified 1/14 women in contrast to consensus PCR, which identified 10 of 14 women. For the ASCUS group, this difference was less evident. Both methods identified the same 6 of 7 histological CIN2+. The American ASCUS-LSIL Triage Study (ALTS) concluded that the very high percentage of women with an LSIL diagnosis from Pap smears were positive for HPV DNA by Hybrid Capture 2 (HC2) testing (Digene, Gaithersburg, MD), there is limited potential for this assay to direct decisions about the clinical management of women with LSIL.29 For the women with an ASCUS Pap smear, HPV DNA testing can help to identify those who have underlying HSIL.29, 30, 31 It entails, however, a very high and costly use of colposcopy as complement. In women 25–69 years of age undergoing cytological screening in Norway, the proportion of ASCUS is 2.3%.23 About 70% of these women have a normal cytology in the subsequent Pap smear within the 2-year follow-up of the index smear and 6.9% have a histological CIN2+ diagnose.12 In this setting, the detection of E6/E7 transcripts from the high-risk HPV types may be more suitable for HPV triage of cytological ASCUS and LSIL than consensus HPV DNA detection, as suggested by our results.
One histological CIN2 sample was found HPV DNA- and mRNA-negative. This sample was diagnosed with reactive epithelial changes in the subsequent surgical cone. The difference in diagnoses may be due to removal of the lesion by the first biopsy, a wrong diagnosis, or that the lesion was regressing, because HPV was not detected in the preceding cytological sample taken from the same cervix-brush as the HPV testing material.
Cox et al.32 concluded that LSIL- and HPV-positive ASCUS are clinically equivalent and that the cumulative risk of CIN Grade 2 or 3 was equivalent for LSIL (27.6%) and HPV-positive ASCUS (26.7%). In our study, a similar observation regarding the clinically equivalence of LSIL- and ASCUS/HPV-positive women was observed. The number of specimens tested was too small for independent conclusions.
In a previous study35, a comparison between HPV DNA and mRNA detection was carried out on 4,136 women. The PreTect HPV-Proofer had the lowest overall detection rate although it detected the most histological CIN2+. This means that mRNA testing will detect fewer women for follow-up surveillance or treatment than consensus PCR. A high regression rate of CIN1-3 has also been observed.33, 34 These facts, with the results of our present study, suggest that detection of oncogenic E6/E7 transcripts from high-risk HPV types may be valuable as an adjunct to cytology to predict a future diagnosis of CIN2+.
The relatively few CIN2+ lesions detected in our study makes the sensitivity of the Gp5+/6+ consensus PCR lower than what we would expect in a larger selection of CIN2+ samples compared to PreTect HPV-Proofer. This is because other HPV types not detected by PreTect HPV-Proofer, are likely to be present in some CIN2+ lesions.
These results suggest that women with an ASCUS/LSIL Pap smear and an HPV mRNA-positive test should attend a closer follow-up and those that are HPV mRNA-negative may undergo a less frequent follow-up.
In conclusion, the PreTect HPV-Proofer assay offers an improvement for the triage of women with an ASCUS or LSIL Pap smear compared to consensus PCR, however. Our results should be confirmed in large scale studies.