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ORIGINAL RESEARCH |
From the Laboratoire Pasteur Cerba, Cergy-Pontoise and Paris; Unité d’Épidémiologie, Institut Pasteur, Paris; and Unité des Papillomavirus, INSERM U190, Institut Pasteur, Paris, France.
Address reprint requests to: Christine Bergeron, MD, PhD Laboratoire Pasteur Cerba 95066 Cergy-Pontoise France E-mail: bergeron{at}pasteur-cerba.com
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Abstract |
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Methods: We prospectively studied 378 women with atypical squamous cells of undetermined significance (ASCUS) (n = 111) or low-grade squamous intraepithelial lesions (SILs) (n = 267) demonstrated by referral cytology. We did repeat cytology, sampling for detection of HPV DNA by Hybrid Capture II, PCR, and Southern blot hybridization, and colposcopic evaluation with cervical biopsies.
Results: All participants underwent the Hybrid Capture II test and 320 underwent the three HPV tests. Sensitivities of Hybrid Capture II for detecting CIN and high-grade CIN (0.81 and 0.86, respectively) were similar to those of cytology (0.83 and 0.82, respectively) and PCR (0.77 and 0.95, respectively), and higher than those of Southern blot hybridization (0.48 and 0.45, respectively). Compared with cytology, combined triage with Hybrid Capture II improved sensitivities for detecting CIN (0.94 versus 0.83, P < .001) and high-grade CIN (0.96 versus 0.85), though the latter difference was not significant (P = .17). In women with ASCUS, sensitivities of combined triage and cytology for detecting CIN were 0.94 and 0.71, respectively (P = .01), and sensitivities of the two methods for detecting high-grade CIN were 0.92 and 0.66, respectively (P = .13). The increase in sensitivity was lower among women with low-grade SILs; for these women, cytology had high sensitivity (0.86 for CIN and 1.00 for high-grade CIN). The specificity of combined triage was significantly lower than that of cytology in both groups.
Conclusion: Compared with repeat cytology, combined triage with HPV testing markedly improves sensitivity for detecting CIN in women with ASCUS, but at the expense of specificity.
Treating women with cytologic diagnoses of atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesions (SILs) remains difficult for clinicians.1–6 Some authors propose conservative treatment, involving repeat Papanicolaou tests, whereas others prefer to do colposcopies. Although most results of Papanicolaou tests indicating ASCUS or low-grade SILs spontaneously revert to normal, some persist and others later might indicate high-grade lesions. Cytology is a screening, not a diagnostic, tool. In 5–15% of cases, women with ASCUS or low-grade SILs might have histologically high-grade cervical intraepithelial neoplasia (CIN) already.2,3,7,8 Thus, it is important to identify women with ASCUS or low-grade SILs who are at risk of developing high-grade CIN.
An association has been shown between specific human papillomavirus (HPV) types and high-grade CIN and cervical cancer.9–12 Although there is no universally accepted method for HPV detection, polymerase chain reaction (PCR) and the Hybrid Capture HPV DNA assay (Digene Diagnostics Inc., Silver Spring, MD) recently have become the most frequently used tests for evaluating HPV-related diseases in large populations.13,14 Testing for HPV after abnormal cytologic finding, particularly if cytologic abnormalities are poorly defined or low-grade, has been evaluated as an objective marker for high-grade CIN, but the results were controversial.8,15–21 Whereas some investigators found HPV testing to be an alternative to immediate colposcopy or cytologic follow-up8,15–20 others found HPV testing to be of no prognostic value for detecting high-grade CIN in women reported as having ASCUS or low-grade SILs.3,21
The current study was designed to evaluate the efficiency of HPV testing by second-generation Digene Hybrid Capture assay, compared with PCR, Southern blot hybridization, and repeat cytology, with regard to predicting CIN or high-grade CIN in women with referral Papanicolaou smears showing ASCUS or low-grade SILs.
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Materials and Methods |
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At clinic visits within 2 months after referral cytology, Papanicolaou smears were collected immediately before colposcopies, with wooden spatulas (ectocervices) and cytobrushes (Medscand, Hollywood, FL) (endocervices). For HPV DNA analysis by Hybrid Capture II, samples were collected by rotating a cone brush (Medical Packaging, Camarillo, CA) in the cervical os and rubbing the brush across the entire transformation zone. Samples then were placed into a specimen transport medium provided by the manufacturer (Digene Diagnostics Inc., Silver Spring, MD) and stored at +4C. For PCR and Southern blot hybridization, another sample was obtained with a cone brush, placed in a cryotube in Eagle’s medium containing antibiotics, and stored at -80C.
All participants had colposcopies, and biopsy specimens were taken from the abnormal transformation zone seen in all but 20 women. Smears and biopsy specimens were evaluated by the same pathologist (CB), who was masked to results of other tests. Enrollment and repeat Papanicolaou smears were classified according to the Bethesda system. Smears first were screened by a cytotechnician and then were examined by the pathologist if findings were abnormal. Results were classified as negative (including normal biopsy results and no biopsy results because of a normal transformation zone), low-grade CIN, or high-grade CIN.
The Hybrid Capture II System was used for HPV screening according to the manufacturer’s instructions. It included low-risk and high-risk probes. The low-risk probe, designated A, detects HPV 6, 11, 42, 43, and 44. The high-risk HPV probe, designated B, detects types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Results of viral test were considered positive when high-risk or low-risk HPV types were present beyond a threshold level of 1.0 pg/mL of HPV DNA. That level corresponds to approximately 5000 copies of the HPV genome in the test. Light measurements produced by a luminometer were expressed as relative light units. The amount of light produced by the Hybrid Capture assay is proportional to the amount of target DNA in each specimen,13 so a score from 1 to 4 was given according to the luminometer values: 1 to 10, 10 to 100, 100 to 1000, and more than 1000 relative light units.
Total DNA extracted from the cell samples was analyzed for the presence of DNA sequences by PCR using the MY11 and MY09 primers and by low-stringency Southern blot hybridization as described previously.22,23
Statistical analysis of data was done using SAS software (SAS Institute, Cary, NC). Concordance between the Hybrid Capture II assay and PCR or Southern blot hybridization was assessed using the percentage of concordance and 
statistics. values express the proportion of possible concordance beyond chance. A estimate of less than 0.4 represents poor agreement, a estimate between 0.4 and 0.75 represents fair to good agreement, and a estimate of more than 0.75 represents excellent agreement. Test performances on matched pairs were compared using McNemar 
2 tests. Hybrid Capture II score medians (with a score of 0 for negative Hybrid Capture results) of cases of positive PCR and negative Southern blot hybridization results were compared using the Wilcoxon Mann-Whitney test. Sensitivities and specificities of repeat Papanicolaou tests, HPV testing, and combined triage were calculated using the biopsy results as the standard (CIN versus negative results, high-grade CIN versus low-grade CIN, and negative results). Proportions were compared using the McNemar 2 test or the Fisher paired test. All findings were considered statistically significant at P < .05.
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Results |
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The results of the three methods of HPV detection are compared in Table 1
. Hybrid Capture II and PCR yielded similar percentages of positivity (60.9% versus 62.5%). The percentage of concordance between the two tests was 83.6% and the statistic was 0.54. Hybrid Capture results were positive more often than Southern blot hybridization results (60.9% versus 35.3%) (McNemar test, P < .001). The percentage of concordance between the two tests was only 53.8% and the statistic was 0.40. Of the 103 women whose Southern blot hybridization results were positive, 41 (36.3%) had HPV types identified, including HPV 16 (11 cases), HPV 42 (ten cases), HPV 31 (nine cases), HPV 6 (three cases), HPV 18, 35, and 39 (two cases each), and HPV 11 and 33 (one case each). Seventy-two women (63.7%) had uncharacterized HPV DNA, including HPV related to type 16, 18, or 33 (35 cases), type 31, 35, or 39 (19 cases), and type 6, 11, or 42 (eight cases).
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The efficiency of the three methods of HPV DNA testing with regard to detection of CIN and high-grade CIN was compared with that of repeat cytology (Table 2). The sensitivities of Hybrid Capture II using both probes for detecting CIN and high-grade CIN (0.81 and 0.86, respectively) were equivalent to the sensitivities of cytology (0.83 and 0.82, respectively) and PCR (0.77 and 0.95, respectively), but the sensitivities of Southern blot hybridization for detecting CIN (0.48) and high-grade CIN (0.45) were significantly lower than those of cytology (P < .001).
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). The sensitivity for detecting high-grade CIN was identical to that of Hybrid Capture II using both high-risk and low-risk probes (0.88). It was significantly lower for detecting CIN (0.77 versus 0.82, P = .004). Specificities were significantly better with the high-risk probe than with both probes for detecting CIN (0.66 versus 0.58, P < .001) and high-grade CIN (0.49 versus 0.42, P < .001). Sensitivities of Hybrid Capture II using the high-risk probe were similar to that of cytology for detecting CIN (0.77 and 0.83, respectively) and high-grade CIN (0.88 and 0.85, respectively). Combined triage improved sensitivity for detecting CIN compared with cytology (0.94 versus 0.83, P < .001). It also increased sensitivity for detecting high-grade CIN (0.96 versus 0.85), but the difference was not significant (P = .17). Specificities were decreased significantly for detecting CIN (0.57) and high-grade CIN (0.36) (P < .001). Considering 203 women with positive Hybrid Capture test results (high-risk probe only), a score equal to 3 or 4 was found as frequently in the 71 women with negative biopsy results (33.8%) as in the 23 women with high-grade CIN (21.7%).
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). The sensitivity of Hybrid Capture II for detecting high-grade CIN was higher (0.83 versus 0.66 for cytology), but that difference did not reach statistical significance (P = .31). The specificity of cytology for detecting CIN was higher than that of Hybrid Capture II (0.84 versus 0.66, P = .01) but not significantly different for detecting high-grade CIN. When both methods were combined, the sensitivity for detecting CIN was improved significantly compared with that of cytology (0.94 versus 0.71, P = .01). The sensitivity for detecting high-grade CIN (0.92) was higher than that of cytology (0.66) although not significantly different (P = .13). Compared with cytology, specificities of combined triage for detecting CIN (0.58 versus 0.84) and high-grade CIN (0.46 versus 0.71) were decreased significantly (P < .001).
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). Specificities of cytology and Hybrid Capture II for detecting high-grade CIN were similar (0.45 and 0.44, respectively), whereas the specificity of cytology (0.74) was better than that of Hybrid Capture II (0.65) for detecting CIN (P = .06). When both methods were combined, the sensitivity for detecting high-grade CIN was not improved over each approach alone, whereas that for detecting CIN was significantly improved compared with that of cytology (0.94 versus 0.86, P = .001). Compared with cytology, specificities were decreased for detecting CIN (0.56 versus 0.74, P < .001) and high-grade CIN (0.32 versus 0.45, P = .001).
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Discussion |
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Most studies compared sensitivity and specificity of HPV testing using Hybrid Capture I or PCR with those of repeat cytology for detecting CIN.8,15–21 Follow-up with HPV testing was not better than repeat cytology, but all studies showed increased sensitivity when both methods were combined, though at the expense of specificity.8,15–21 In our participants with referral Papanicolaou smears that showed mild atypia, triage based on repeat cytology for detecting CIN or high-grade CIN was equivalent to HPV testing alone using the Hybrid Capture II test. When both methods were combined, sensitivity for detecting CIN was improved significantly compared with that of repeat cytology. The improvement was more pronounced in women with ASCUS than in those with low-grade SILs, because of the high sensitivity of cytology in the latter group. An improvement of similar magnitude for detecting high-grade CIN was found in women with ASCUS, but the difference did not reach statistical significance (P = .13). The power to find significance might have been limited by the small number of cases of high-grade CIN, as in a similar study.30 An ongoing randomized trial involving 7200 women and funded by the National Cancer Institute compared the three treatments of ASCUS and low-grade SIL abnormalities (ie, immediate colposcopy, repeat Papanicolaou testing with liquid-based cytology, and HPV testing). That trial should yield more definite data on how efficient and cost-effective each option is. Cellular residues of collection media used for preparing monolayer cytology could be used for HPV testing,18,27,30,31 which would obviate second office visits, saving money. However, liquid cytology for initial cervical screening is used only in a few settings, mostly because of high cost.31
According to our study, HPV testing alone does not represent alternative to immediate colposcopy or cytologic follow-up in women with mild cytologic atypia. Combining HPV testing and cytology is more efficient than repeat cytology for detecting CIN and, most likely, high-grade CIN, especially in women with ASCUS. The cost-effectiveness of HPV testing combined with repeat conventional cytology or performed with samples collected for initial liquid-based cytology screening should depend on availability, reliability, and cost of colposcopy, biopsy, and HPV testing in each country. The rate of ASCUS, shown to be highly variable in an interlaboratory comparison survey,32 also should be considered. In our study, HPV testing was done to ascertain diagnoses after first screenings, introducing a verification bias, and only a part of the HPV infection spectrum was represented. The sensitivity and specificity estimates reported should be reevaluated in cases of primary screening for cervical cancer.
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Footnotes |
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Received September 24, 1999. Received in revised form December 23, 1999. Accepted January 10, 2000.
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