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Carcinogenic or “high-risk” human papillomavirus (high-risk HPV) testing has become the standard triage in the US for women with atypical squamous cells of undetermined significance (ASC-US) cytology and is designated as a stand-alone follow-up option in a number of postcolposcopy and posttreatment clinical management scenarios.1,2 Primary cervical screening guidelines now also include the option of cotesting women aged 30 years and older with the Papanicolaou (Pap) test and a high-risk HPV test and extending screening intervals to 3 years if both are negative.1–3 Additionally, some are advocating moving to screening women in this age group with the more sensitive high-risk HPV test alone, followed by a more specific test (eg, cytology) for women testing high-risk HPV positive.4 This paradigm shift to molecular testing in primary cervical screening and abnormal Pap test management has been based on the nearly absolute requirement of persistent infections by high-risk HPV genotypes in the carcinogenesis process leading to cervical cancer and its immediate precursor lesion, cervical intraepithelial neoplasia grade 3 (CIN-3).5
The ASCUS LSIL Triage Study (ALTS), a multicenter, randomized clinical trial designed to compare management strategies for women with low-grade squamous intraepithelial lesion (LSIL) and equivocal (ASC-US) cytologic abnormalities,6–9 confirmed findings of previous studies that high-risk HPV testing has a high sensitivity (>90%) to identify women with CIN-3 and a corresponding high level of reassurance of the absence of CIN-3 among high-risk HPV-negative women.10,11
Dependence on a single test result as the only determinant of screening interval and follow-up options requires confidence that the test will accurately reflect risk. Cervical intraepithelial neoplasia 3 is almost invariably caused by high-risk HPV, and women are generally high-risk HPV positive several years before a CIN-3 diagnosis. However, no test has 100% accuracy, and HPV testing is no exception. Increasing use of HPV testing has resulted in occasional reports of “false-negative” HPV test results for women with CIN-3.12,13 If testing high-risk HPV positive supposedly identifies the subset of women who are at risk of CIN-3 and cancer, and testing high-risk HPV negative provides reassurance against CIN-3 and cancer, what is the meaning of a high-risk HPV-negative test occuring within a couple of years before a CIN-3 diagnosis? What are the possible explanations for these HR-HPV negative CIN-3? Most importantly, how clinically relevant are these relatively rare occurrences?
In ALTS, the 2-year risk of CIN-3 among high-risk HPV-negative women was quite low,11 less than 2%. To better understand high-risk HPV-negative CIN-3, we described the occurrence of such cases in ALTS. We combined the test results of two well-validated HPV tests to minimize simple testing errors, thereby focusing our analysis on other sources of error. We compared CIN-3 diagnosed throughout the 2-year duration of ALTS among participants who at baseline tested high-risk HPV negative to those who tested positive to characterize differences by high-risk HPV status and to elucidate what percentage of cases might be 1) incident cases, 2) due to non–high-risk HPV genotypes, 3) false-positive pathology, and 4) false-negative for high-risk HPV (despite dual HPV testing). The goal of the study was to provide insight into the cause of many of the cases of high-risk HPV-negative CIN-3 and their clinical relevance.
PARTICIPANTS AND METHODS
ALTS was a randomized trial comparing three management strategies for 5,060 women with ASC-US (n=3,488) or LSIL (n=1,572): 1) immediate colposcopy arm (referral to colposcopy regardless of enrollment test results); 2) HPV triage (referral to colposcopy if enrollment HPV result by Hybrid Capture 2 [Digene Corporation, Gaithersburg, MD] was positive or missing, or if the enrollment cytology was high-grade squamous intraepithelial lesion [HSIL]); or 3) conservative management (referral to colposcopy if enrollment cytology was HSIL). At enrollment, all women underwent a pelvic examination with collection of two cervical specimens; the first specimen in PreservCyt for ThinPrep cytology (Cytyc Corporation, Marlborough, MA) and the second in specimen transport medium (Digene Corporation). Women in all three arms of the study were reevaluated by cytology every 6 months for 2 years and sent to colposcopy if cytology was HSIL. An exit examination with colposcopy was scheduled for all women, regardless of study arm or prior procedures, at the completion of the follow-up. We refer readers to other references for details on randomization, examination procedures, patient management, and laboratory and pathology methods.6–9 The National Cancer Institute and local institutional review boards approved the study and all participants provided written informed consent.
Testing was blinded to all clinical data, and testing data were not used in making diagnoses. Two HPV DNA tests were performed on residual clinical specimens collected at enrollment and follow-up.
Hybrid Capture 2 using probe set B, a pooled probe DNA test for one or more high-risk HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), was performed on residual Preserv-Cyt specimens after cytology slides were prepared. A positive test does not identify which high-risk HPV genotype(s) are present. Hybrid Capture 2 is also well-known to cross-react with untargeted HPV genotypes14,15 including HPV 66, recently classified as a high-risk HPV genotype.16
Human papillomavirus genotyping was performed using Line Blot Assay (Roche Molecular Systems, Alameda, CA), which is an L1-based polymerase chain reaction assay that uses a primer set designated PGMY09/11. Line Blot Assay was conducted on the specimen transport medium specimen for detection of 27 (HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51–59, 66, 68, 73, 82, 83, and 84) or 38 HPV (the 27 HPV genotypes listed plus non-high-risk HPV genotypes 61, 62, 64, 67, 69–72, 81, 82 variant, and 89) genotypes at enrollment and during follow-up.15
Enrollment specimens collected from women referred because of an ASC-US Pap test were retrospectively tested using Linear Array (Roche Molecular Systems), a commercialized, research-use-only version of Line Blot Assay that tests for 37 HPV genotypes (excluding HPV 57) as previously described.17 Of note, among those with a single high-risk HPV genotype detected by Linear Array, increasing band intensity (categorized as 1 [low] to 4 [high]), as judged subjectively from the line blot, was strongly associated with increasing Hybrid Capture 2 signal strength (P<.001, nonparametric test of trend18) (data not shown), a semi-quantitative measure of viral load.19 We therefore considered band intensity a qualitative measure of HPV viral load.
HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 were considered the primary high-risk HPV genotypes. Line Blot Assay and Linear Array results were categorized into an HPV risk group according to a priori established cervical cancer risk: 1) positive for HPV 16; or 2) positive for any high-risk HPV genotype(s) excluding HPV 16; or 3) positive for any non–high-risk HPV genotypes and negative for all high-risk HPV genotypes; or 4) negative.
Clinical management was based on the clinical center pathologists’ cytologic and histologic diagnoses. In addition, referral smears, ThinPreps, and histology slides were sent to the Pathology Quality Control Group (Quality Control pathology) based at the Johns Hopkins Hospital for review and secondary diagnoses. Cervical intraepithelial neoplasia 2 or more severe (CIN-3, adenocarcinoma in situ, carcinoma in situ, adenocarcinoma, and squamous cell carcinoma) (≥CIN-2) histologic diagnoses based on the clinical center pathology or a CIN-3 or more severe (≥CIN-3) histologic diagnoses based on the Quality Control pathology review triggered treatment by loop electrosurgical excision procedure. In addition, women with persistent LSIL or high-risk HPV-positive ASC-US at the time of the exit from the study were offered loop electrosurgical excision procedure. There were 621 women who had a diagnosis of CIN-3 by either pathology group and this was the subset of women included in this analysis. (nb, seven cases of squamous cell cancer, all high-risk HPV positive, were included in this analysis. Exclusion of these cases did not appreciably change the results of the study.)
We conducted a blinded, retrospective analysis of the visual appearance of the cervix of high-risk HPV-negative and HPV-positive CIN-3 by reviewing Cervigrams (National Testing Laboratories Worldwide, Fenton, MO) of high-risk HPV-negative cases matched 1:2 to high-risk HPV-positive cases on study arm, clinical center, time of diagnosis (enrollment, follow-up, or exit), and referral Pap test. Cervigrams were digitized using standard features20 to obtain high-resolution images equivalent to 6-megapixel pictures. One of us (J.J.) reviewed both baseline and time-of-diagnosis Cervigrams for the cases diagnosed during follow-up. Baseline Cervigrams from 29 high-risk HPV negative and 57 high-risk HPV-positive cases (one high-risk HPV-negative case had only one matching high-risk HPV-positive case) and time-of-diagnosis Cervigrams (nb, some baseline Cervigrams were also time-of-diagnosis Cervigrams) from 27 high-risk HPV-negative and 50 high-risk HPV-positive cases (four high-risk HPV-negative cases had only one matching high-risk HPV-positive case) were evaluated; other high-risk HPV-negative cases were either missing Cervigrams or no match could be found. Evaluations were done using a Web-based software that presented the digitized Cervigram and masked the evaluator to any clinical data.21
We defined women with CIN-3 as high-risk HPV negative if Line Blot Assay and Hybrid Capture 2 were negative for high-risk HPV at baseline or if one test was negative for high-risk HPV and the other test result was missing (Table 1); we designated 588 cases (94.7%) as high-risk HPV positive and 33 cases (5.3%) as high-risk HPV negative. We considered women who were Hybrid Capture 2–positive, Line Blot Assay–positive for non–high-risk HPV genotypes but negative for high-risk HPV genotypes (n=11) as high-risk HPV-positive cases. However, we could not ascertain whether these results were the consequence of false-negative Line Blot Assay result or false-positive Hybrid Capture 2 result due to cross-reactivity with non–high-risk HPV genotypes (eg, HPV 53 and HPV 67).14,15 Exclusion of these cases did not appreciable alter our findings. We did not consider the Linear Array results in our primary definition of HPV-negative CIN-3 cases because we had Linear Array testing only on ASCUS referrals.
We first described our cases of CIN-3 by the distribution of paired diagnoses rendered by both pathology groups for all women and stratified by HPV status (positive compared with negative) as described above. We also compared the timing of diagnosis (enrollment, follow-up, or exit) of CIN-3 by HPV status for women enrolled in the immediate colposcopy and conservative management arms. Women enrolled in the HPV arm of ALTS were excluded from this analysis because of the inherent bias of referral based on HPV testing results by Hybrid Capture 2.
Standard contingency table methods, with Fisher exact tests, were used to assess possible univariate associations of categorical variables with HPV status. Odds ratios (ORs) and 95% confidence intervals (95% CIs) adjusted for relevant parameters (eg, identified as part of preliminary data analysis and stepwise modeling) were calculated using multivariate logistic regression.
Finally, we considered the results of Linear Array testing conducted in the ASC-US referral population. There are several studies to suggest that Linear Array may be more analytically sensitive than Line Blot Assay for the detection of HPV genotypes. We compared the distribution of HPV-negative and HPV-positive cases for HPV risk group status as determined by Linear Array.
Fisher exact test was used to test for statistical significance (P<.05) for categorical variables; Kruskal- Wallis test was used to test for statistical significance for continuous variables. Stata 8.2 (Stata Corporation, College Station, TX) was used for all statistical analyses.
In Table 2, we show the paired diagnoses by the two pathology groups at the time of the CIN-3 histologic diagnosis for anyone with at least one CIN-3 diagnosis during ALTS. Overall, 55% of women with a CIN-3 diagnosis had a CIN-3 diagnosis by both pathology groups, and 93% had CIN-3 diagnosis by one pathology group and CIN-2 diagnosis or worse by the other group. Hence, only 7% with a >CIN-3 diagnosis by one group were interpreted as normal or low grade (CIN-1) by the other group. The distribution of paired diagnoses was very different by HPV status (P<.001). High-risk HPV-negative CIN-3 were less likely to have a concordant CIN-3 diagnosis by both groups (24% compared with 56%) (P<.001) and were less likely to have a second diagnosis of at least CIN-2 (76% compared with 94%) (P =.001) than the high-risk HPV-positive CIN-3.
Table 3 shows the time of detection of CIN-3. In both the immediate colposcopy arm (P =.001) and the conservative management arm (P =.02), HPV-negative CIN-3 was found later than HPV-positive CIN-3 and predominantly at the exit visit. In the immediate colposcopy arm, in which all women underwent colposcopy at baseline, a greater proportion of high-risk HPV-negative CIN-3 (55.0%) was diagnosed at exit than the proportion of high-risk HPV-positive CIN-3 (17.8%). In the conservative management arm, in which women went to baseline colposcopy only for enrollment HSIL cytology, a greater proportion of high-risk HPV-negative CIN-3 (75.0%) were diagnosed at exit than the proportion of high-risk HPV-positive CIN-3 (31.5%).
We also examined what other factors might be associated with case HPV status using contingency tables and then multivariate modeling to mutually adjust for all relevant covariates (Table 4). Cases of high-risk HPV-negative CIN-3 (compared with high-risk HPV-positive CIN-3) were: 1) less likely to be referred into ALTS because of an LSIL Pap test (compared with ASC-US Pap test) (OR 0.44, 95% CI 0.18–1.1); 2) far less likely to have an enrollment cytology interpreted by the clinical center pathologist as HSIL (compared with <HSIL) (OR 0.10, 95% CI 0.046–0.22); 3) more likely to be enrolled at the clinical center 3 (OR 2.7, 95% CI 0.99–7.6) (nb, CIN-3 diagnosed by clinical center 3 were also less likely to be confirmed CIN-3 by Quality Control pathology [data not shown].); and 4) less likely to report having abnormal discharge in the last year (compared with no discharge) (OR 0.33, 95% CI 0.12–0.91).
Enrollment specimens from high-risk HPV-negative cases of CIN-3 were much more likely to again test negative for high-risk HPV by a third test, Linear Array, (64.0%; 28.0% negative for all genotypes and 36.0% for non–high-risk HPV genotypes) than high-risk HPV-positive cases (2.7%; 1.5% negative for all genotypes and 1.2% for non–high-risk HPV genotypes)(P<.001) (Table 5). Among those cases in which Linear Array was positive for only one high-risk HPV genotype (n=84), the band intensity was lower among the cases of CIN-3 originally identified as high-risk HPV negative (n=9) than high-risk HPV positive (n=75) (P<.001).
Cervigrams collected at the time of diagnosis from high-risk HPV-negative cases were marginally less likely to be called high-grade or cancerous than those from high-risk HPV-positive cases (52% compared with 70%, P =.1). Cervigrams from high-risk HPV-negative cases had marginally smaller areas of acetowhitening than those from high-risk HPV-positive cases (P =.08).
Considering all measurements of HPV throughout the duration of ALTS, only 82% of the CIN-3 initially high-risk HPV-negative cases at enrollment were positive for any HPV genotype on any occasion. Only 64% of these cases were ever positive for any high-risk HPV genotype, and 30% were ever positive for HPV 16. All percentages were lower than the percentages observed for cases of CIN-3 diagnosed in cross-sectional studies22,23 (see Appendix online at www.greenjournal.org/cgi/content/full/111/4/847/DC1). Among cases of CIN-3 diagnosed during follow-up that tested HPV-negative at enrollment (n=26), 19 (73%) were positive for high-risk HPV by either Line Blot Assay or Hybrid Capture 2 at the time of diagnosis, and only 10 of 19 of those that were positive by either test were positive by both assays (53% compared with 93% for the baseline high-risk HPV-positive cases, P<.001).
In ALTS, it seemed that the few high-risk HPV-negative CIN-3 cases diagnosed were less definitively “CIN-3.” Based on these data, we suggest four possible causes of HPV-negative CIN-3: 1) incident (new) cases of CIN-3 that developed during follow-up; 2) “non–high-risk HPV” HPV genotypes that rarely if ever cause cancer but may cause CIN-3; 3) histologic false-positives, classified incorrectly (misclassified) as CIN-3; and 4) false-negative high-risk HPV test results. To help illustrate examples of each, we have included case histories for all 33 HPV-negative CIN-3 (see the Appendix online at www.greenjournal.org/cgi/content/full/111/4/847/DC1). Although it is difficult to ascribe with certainty any one cause for these high-risk HPV-negative cases, upon independent review of the case histories by four of the authors (P.C., T.C., J.J., and M.S.), we suggest that of the 621 CIN-3 cases overall there were 12 incident cases (1.9%), five cases (0.8%) due to non–high-risk HPV, and eight cases (1.3%) due to incorrectly classified CIN-3, leaving only eight cases (1.3%) with false-negative high-risk HPV test result(s) (nb, based on using only Hybrid Capture 2 test results, the 37 Hybrid Capture 2–negative cases could be ascribed to 10 incident cases (1.7%), five cases (0.8%) due to non–high-risk HPV, and seven cases (1.2%) due to incorrectly classified CIN-3, leaving 15 cases (2.5%) with false-negative Hybrid Capture 2 test results.).
The patterns of some high-risk HPV-negative cases were suggestive of incident CIN-3 (nb, our a priori assumption was that cases diagnosed during follow-up that were high-risk HPV positive at baseline were missed prevalent disease. This assumption was based on the less-than-perfect sensitivity of colposcopy and the very similar number of cases of CIN-3 in the different arms of the trial despite the different sensitivity for detection of disease at baseline, which resulted in different timing of disease detection.9 Some CIN-3 likely developed from a baseline, prevalently detected high-risk HPV infection, but distinguishing between incident and missed prevalent CIN-3 is not possible.). Most compelling were 10 of 12 (83%) cases called incident in this analysis that were diagnosed at exit from ALTS—approximately 24 months after enrollment. All 12 of these cases had a second diagnosis of at least CIN-2.
There were clear examples of CIN-3 that seemed to be caused by genotypes other than those genotypes included in our definition of high-risk HPV. The CIN-3 in Participant 4 seemed to be caused by HPV 26, which has been described as possibly high-risk HPV.24 The CIN-3 in Participant 32 seemed to be caused by HPV 67, which is not considered a high-risk HPV genotype but shares the same phylogenetic grouping as HPV 16 and related high-risk HPV genotypes.25 Thus, some HPV genotypes closely related to high-risk HPV genotypes might occasionally cause CIN-3, a full epithelial thickness lesion indicative of precancer, but very rarely progressing to invasive cancer.
Some cases of high-risk HPV-negative CIN-3 were also attributable to misclassified (false-positive) histology, ie, morphologic changes that mimic CIN-3. One exemplary case was Participant 20, who tested high-risk HPV negative by both Hybrid Capture 2 and Line Blot Assay at all five visits. Yet, at the exit visit that included colposcopy, a biopsy was taken and diagnosed as CIN-3 by Quality Control pathology and CIN-2 by CC pathology. Interestingly, the colposcopic impression at that time was atypical metaplasia, which may be difficult to differentiate from CIN-3 histologically because both may display a full-thickness intraepithelial basaloid appearance.26
Finally, there were a few examples of false-negative high-risk HPV cases, ie, HPV testing errors, despite the use of dual testing. Participant 5 tested negative at baseline by Hybrid Capture 2 and Line Blot Assay but tested positive by Hybrid Capture 2 and positive for HPV 16, HPV 51, and HPV 59 by Line Blot Assay at the 12-month and exit visits (there were no test results at 6 and 18 months because of missing visits). Subsequently, HPV 51 was detected in the baseline specimen by Linear Array, consistent with persistent HPV 51 eventually leading to a CIN-3 diagnosis. Figure 1 shows the Cervigrams collected from this case at enrollment, 6-month, 12-month, and 24-month (exit) visits and shows that the lesion was distant from the cervical os, where cervical sampling occurs, at the enrollment visit, which may have resulted in poor sampling of the lesion and testing high-risk HPV negative by Line Blot Assay and Hybrid Capture 2 but positive by the more analytically sensitive Linear Array. Participant 33 (Fig. 2) might be either a false-negative high-risk HPV or incident case diagnosed at the 12-month visit, depending on whether a small, equivocal-appearing change in the epithelium at baseline that was unapparent at 6 months was a small focal lesion or not.
Eltoum and colleagues27 also evaluated cases of HPV-negative high-grade cervical precancer (≥CIN-2) in their population of women with ASC-US, determining that most lesions were small and shed few abnormal cells. Another possibility is poor exfoliation of cells due to altered adhesion molecule distribution.13 The authors concluded that these lesions were either early in development or were regressing, questioning their clinical significance. Another likely and clinically important explanation, as observed in this analysis, is that some false-negative tests are the result of poor sampling of the lesion.
So what do these cases mean clinically? First, the high-risk HPV-negative CIN-3 cases that are the result of misclassified histology do not truly represent CIN-3 and therefore do not present a risk to women. These are clinically relevant only with respect to possible overtreatment. Similarly, cases due to low-risk HPV genotypes are unlikely to develop into invasive cancer. Of the remaining high-risk HPV-negative CIN-3 cases, five were attributable to false-negative high-risk HPV and 12 to incident cases. Most women would have been diagnosed during follow-up with adherence to current guidelines1,2,28 for the management of abnormal cervical cytology.
The presence of these few false-negative high-risk HPV cases of CIN-3 emphasizes that no test, or even combinations of tests, will achieve 100% sensitivity for identifying women with cervical precancer. However, we caution against misguided attempts to achieve 100% clinical sensitivity for HPV assays by either increasing the analytic sensitivity and/or targeting additional HPV genotypes for detection, because such small gains in sensitivity will result in dramatic increases in the number of false positives (ie, high-risk HPV positive but no CIN-3).29,30 Changes in analytic sensitivity or targeting additional HPV genotypes for detection to improve clinical sensitivity require formal evaluations and validation, using receiver operating characteristic curve or other analytic approaches and thoughtful consideration of the balance between true and false positives, or more apropos to the clinical setting, negative and positive predictive values.
We conclude that in any sizeable population, even among women with evidence of cytologic abnormalities, there will be a few cases of cervical precancer that will test high-risk HPV negative for one or more of a number of possible reasons. In addition to understanding their meaning clinically, it is important to recognize that high-risk HPV-negative cases will occur in the clinical validation studies of new tests, which does not necessarily imply failure of the new test. On the whole, these high-risk HPV-negative CIN-3 cases were rare even over a 2-year period and differed substantially from the typical, HPV-positive CIN-3. About one half of the high-risk HPV-negative cases were the result of misclassification, either of enrollment high-risk HPV status or histopathologic diagnosis, and most of the rest were due to new incident HPV detection. As expected in a short window of observation time, there were a relatively small number of incident cases caused by a newly acquired infection leading to a characteristically small precancerous lesion within 24 months.
Although we took advantage of dual HPV testing to minimize testing error, we found very similar results relying only on Hybrid Capture 2 test results, naturally with a small increase in the false-negative test results. Eight of the 37 (22%) Hybrid Capture 2 negatives, or one half of the false Hybrid Capture 2 negatives, were called high-risk HPV positive by Line Blot Assay. Failure due to error by the test itself is rare, giving us confidence that a negative high-risk HPV result provides significant reassurance against clinically relevant CIN-3, and more importantly against cancer, over at least a couple of years. Of note, none of 1,796 (0%, 95% CI 0–0.2%) women who tested Hybrid Capture 2 negative at enrollment were diagnosed with cancer in 2 years, whereas all seven cases of cancer diagnosed during ALTS tested Hybrid Capture 2-positive at enrollment.31 Even when an HPV test misses a bona fide CIN-3, the lesion is most likely small or significantly peripheral to the cervical os; lacking the typical features associated with potential invasion, such as large size, gland duct involvement, and location proximal to or within the cervical canal.32 Additionally, fewer than one half of CIN-3 lesions evaluated in this study as high-risk HPV negative at enrollment remained negative in follow-up, further reducing the risk that a false-negative high-risk HPV test or an incident lesion will continue to go undetected in women managed according to clinical guidelines. Thus, these results confirm that HPV testing as a management option is significantly reassuring to be clinically useful in the follow-up of women initially referred for the evaluation of ASC-US or LSIL and not found to have CIN-2/3 at colposcopy.
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© 2008 by The American College of Obstetricians and Gynecologists.
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