In cervical cancer screening programs, women are sent to colposcopy for a diagnostic evaluation and biopsy of evident lesions if they screen positive by cytology or, increasingly, by various combinations of cytology and human papillomavirus (HPV) test results.1 Women diagnosed with a high-grade cervical lesion, defined in the United States as cervical intraepithelial neoplasia grade 2 (CIN 2) or more severe, are then treated, primarily by excision. However, there is increasing awareness that not all CIN 32 and especially not all CIN 2 is “precancer.”3 In fact, a large proportion of CIN 2 lesions may resolve without treatment, leading to recommendations not to treat CIN 2 immediately in young women.4 The natural history of CIN 2 is not clear, partly because most recent studies that have examined regression of histologically confirmed high-grade lesions did not differentiate between CIN 2 and CIN 3 diagnoses, had sample sizes of 100 or fewer cases,5 and did not use rigorous pathology review to rule out misclassification of CIN 1 and CIN 2.
To examine the specific issue of CIN 2 regression, data from the Atypical Squamous Cells of Undetermined Significance (ASCUS) and Low-Grade Squamous Intraepithelial Lesion (LSIL) Triage Study (ALTS) were used. Different algorithms for referring women to colposcopy at baseline in ALTS were used analytically to compare how the aggressiveness of referral to colposcopy influenced the cumulative 2-year incidence of CIN 2.
Atypical Squamous Cells of Undetermined Significance (ASCUS) and Low-Grade Squamous Intraepithelial Lesion (LSIL) Triage Study (ALTS) was a multisite, randomized trial comparing three management strategies for women referred for ASC-US (n=3,488) or LSIL (n=1,572) conventional cytology.6–10 The arms were 1) immediate colposcopy (IC arm) (referral to colposcopy regardless of enrollment test results); 2) HPV triage (HPV arm) (referral to colposcopy if the enrollment HPV result was positive by Hybrid Capture 2 (hc2; Qiagen Corporation, Gaithersburg, MD) or missing or if the enrollment cytology was high-grade squamous intraepithelial lesion [HSIL]); or 3) conservative management (CM arm) (referral to colposcopy only if the enrollment cytology was HSIL). Among women with LSIL randomly assigned to the CM arm (n=675), seven women (1.0%) during enrollment and 136 women (20.1%) during follow-up were sent to colposcopy on the basis of a protocol modification initiated as a safety intervention8; if this happened to be the 18-month follow-up visit and colposcopy was performed, the 18-month visit became the participant’s “exit” visit (see below).
At enrollment, all women received a pelvic examination with collection of two cervical specimens; the first specimen in PreservCyt for ThinPrep cytology (Cytyc Corporation, Marlborough, MA) and hc2 pooled testing (Qiagen Corporation) and the second in specimen transport medium (Qiagen Corporation) for HPV genotyping. Women in the three arms of the study were reevaluated by cytology every 6 months for 2 years of follow-up and sent to colposcopy if cytology was HSIL. A CIN 2 or more severe diagnosis by the clinical center pathologists, or CIN 3 or more severe by the Pathology Quality Control Group (QC Pathology), was the treatment threshold. An exit examination with colposcopy was scheduled for all women, with loop electrosurgical excision offered for CIN 2 or more severe or persistent low-grade lesions. The National Cancer Institute and local institutional review boards approved the study and all participants provided written informed consent.
Clinical management was based on the clinical center pathologists’ cytologic interpretations and histologic diagnoses as previously described.6–10 Referral smears, ThinPreps, and histology slides were also sent to the QC Pathology based at the Johns Hopkins Hospital for review, including computer-assisted review and secondary diagnoses as previously described.6–10
Two HPV DNA tests were performed on clinical specimens collected at enrollment. Hybrid Capture 2 using probe set B, a pooled probe DNA test for one or more carcinogenic or high-risk–HPV genotypes (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68), was performed on PreservCyt specimens.10 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 genotypes,11,12 including HPV 66, recently classified as a high-risk–HPV genotype.13 An L1 consensus primer-based polymerase chain reaction (PCR) assay14,15 was performed on the specimen transport medium specimen to test for 27 or 38 HPV genotypes.
HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68 were considered primary high-risk–HPV genotypes. An a priori HPV risk grouping was established according to cervical cancer risk as follows: 1) positive for HPV-16; 2) HPV-16 negative, but positive for other high-risk–HPV genotypes by PCR or by hc2 if PCR was negative or missing; 3) negative for all high-risk genotypes but positive for non–high-risk–HPV genotypes (nb, specimens that tested positive for non–high-risk HPV by PCR and positive by hc2 were conservatively called non–high-risk–HPV positive because hc2 sometimes cross-reacts with non–high-risk–HPV genotypes11,12; or 4) PCR negative.
We used the worst histological diagnosis by the QC Pathology over the 2-year duration of ALTS for these analyses. To measure the regression of CIN 2 (n=397), we relied on the fact that different study arms of ALTS were less or more aggressive in colposcopic referral and detection of CIN 2 or more severe: The CM arm sent 188 of 1,839 women (10.2%), the HPV arm sent 768 of 1,385 women (55.5%), and the IC arm sent 1,317 of 1,836 women (99.0%) women to colposcopy at enrollment.8,9 Forty women in the HPV arm, one with CIN 2 and six with CIN 3, were referred to colposcopy because of missing hc2 results. We used CIN 3 or more severe (n=542; 535 CIN 3 and 7 cancers) diagnoses as a reference group under the assumptions that 1) CIN 3 was much less likely to regress than CIN 2 and 2) the majority of CIN 3 or more severe diagnosed, regardless of the timing of the diagnosis, was already present at the time of enrollment.
An extension of the Wilcoxon rank sum test was used as a nonparametric test for trend across study arms.16 Binomial exact confidence intervals were calculated for proportions. A P<.05 was considered statistically significant. Stata 8.2 (Stata Corporation, College Station, TX) was used for all statistical analyses.
We included in our analysis the 5,052 of 5,060 women (99.8%) for whom we had HPV data (hc2 and/or PCR). Table 1 shows the demographics and risk factors for women by their worst 2-year histologic diagnosis (better than CIN 2, CIN 2, or CIN 3 or more severe) as rendered by QC Pathology. As previously noted, smokers (compared with nonsmokers)17 and the less educated18 were more likely to be diagnosed with CIN 3 or more severe.
Table 2 shows that there was little difference (Ptrend=.8) in the proportion of women diagnosed with CIN 3 or more severe over the 2-year duration of ALTS in the populations randomized to each study arm (10.9%, 10.3%, and 10.9% in the CM, HPV, and IC arms, respectively). However, the proportion of women diagnosed with CIN 2 differed by study arm: 5.8% (95% confidence interval [CI] 4.7–6.9%) in the CM arm, 7.8% (95% CI 6.5–9.4%) in the HPV arm, and 9.9% (95% CI 8.6–11.4%) in the IC arm. That is, there were more diagnoses of CIN 2 with strategies referring larger proportions of women to baseline colposcopy in the trial (CM less than HPV less than IC), with 41.6% (95% CI 24.3–59.0%) fewer cases of CIN 2 diagnosed in the CM arm compared with the IC arm. Given that follow-up and exit screening and management did not differ by study arm, the differences in proportion of women diagnosed with CIN 2 by the study arm were due to the differences in the number of women sent to colposcopy at baseline in each study arm. There was a significant trend (Ptrend<.001) of fewer 2-year cumulative CIN 2 histologic diagnoses by study arm relative to 2-year cumulative CIN 3 or more severe. Exclusion of women in the CM, referred for an LSIL Pap, and sent for an early colposcopic evaluation for safety reasons did not alter our findings (data not shown).8
Similar trends for CIN 2 were observed in the ASC-US referral population (4.7% in the CM arm and 7.9% in the IC arm), the ASC-US referral population restricted to those who tested hc2 positive (8.2% in the CM arm and 12.8% in the IC arm), and the LSIL referral population (7.6% in the CM arm and 13.8% in the IC arm). The differences in proportion of CIN 2 diagnosed in the CM compared with the IC arms were less pronounced in women aged 30 years and older (4.2% in the CM arm and 5.5% in the IC arm) than in women aged younger than 30 years (6.4% in the CM arm and 11.7% in the IC arm), but this age-specific effect did not reach statistical significance (P=.3).
Stratifying on the HPV risk group, this trend (of relative increase in number of CIN 2 diagnoses by fraction of women sent to colposcopy within a study arm) was also observed among women with enrollment high-risk–HPV infections (excluding HPV-16) infections (Ptrend=.01); the trend (Ptrend=.1) was nonsignificant among women with HPV-16, although women in the IC arm had relatively more CIN 2 than in the other study arms. A similar pattern was observed for the clinical center histologic diagnoses, although the clinical center pathologists tended to call more CIN 2 and less CIN 3 or more severe in general than QC Pathology (Table 3).
The timing of the diagnosis of CIN 2 and CIN 3 or more severe by study arm is shown in Figure 1. Of note, there were significantly fewer cases of both CIN 2 and CIN 3 or more severe diagnosed at enrollment in the CM arm of the trial. There were significantly fewer 2-year cumulative CIN 2 histologic diagnoses by study arm (CM less than HPV less than IC) relative to 2-year cumulative CIN 3 or more severe at enrollment but not during follow-up (Ptrend=.5) and exit (Ptrend=.3). The CIN 2:CIN 3 or more severe in the CM arm at enrollment was 0.40, threefold lower than the ratio in the IC arm (Ptrend<.001 by study arm), further evidence that the aggressiveness of the study arm in the colposcopy referral at enrollment influenced the overall number of CIN 2 cases diagnosed over the 2 years within a study arm. There were no observed differences in the ratio of CIN 2:CIN 3 or more severe by study arm during follow-up (Ptrend=.5) and exit (Ptrend=.3), ie, similar increases in cumulative incidence CIN 2 and CIN 3 or more severe and the relatively stable CIN 2:CIN 3 or more severe ratio in each arm, reflecting that women in all arms were managed similarly after enrollment.
Cervical intraepithelial neoplasia 2 diagnoses by QC Pathology that were called less than CIN 2 or CIN 2 by the clinical center pathologists were the most likely to be underrepresented in the CM arm compared with the IC arm (Table 4). For example, the ratio of the number of QC pathology cases diagnosed CIN 2 in the CM arm compared with the IC arm among those diagnosed as less than CIN 2 by clinical center pathologists was 0.40. By comparison, the ratio was 0.74 among diagnosed as CIN 3 or more severe by the clinical center pathologists. Overall, there was a marginally significant trend of increasing differences in the number of CIN 2 between study arms by less severe diagnosis by the clinical center pathologists (Ptrend=.07). A similar pattern was observed when restricted to only high-risk HPV (excluding HPV-16)–positive CIN 2 (Ptrend=.07).
We previously have shown that a portion of CIN 2 diagnoses are also due to misclassification of disease3,19 and to HPV infections by non–high-risk–HPV genotypes.3 Here, we present data from ALTS showing that a significant portion of CIN 2 diagnosed by central panel review, even caused by high-risk–HPV genotypes, may represent only regressive lesions. Similar patterns were observed in the ASC-US referral population, in the ASC-US referrals that tested hc2 positive, and in the LSIL referral population. We estimated that approximately 40% of rigorously defined CIN 2 diagnoses in primarily young women (mean age 24.8 years, median age 23 years, range 18–62 years) were destined to resolve on their own by using a “subtractive” analytic approach that compared the fraction found in the least sensitive (CM) and most sensitive (IC) study arm (by virtue of the proportion of women in arm referred to colposcopy) for detection of cervical abnormalities. The differences in CIN 2 detection are due to the delay in the colposcopic evaluation of patients. It is important that the time difference in diagnostic evaluation was assigned by the randomization, which created comparable populations. For reference, 2720 and 1921 of histologically confirmed CIN 2 regressed over approximately 2 years of follow-up in the placebo arms of two chemoprevention trials.
We used CIN 3 or more severe as a reference to show that the study arms were roughly equivalent in cancer risk, and the differences in the criteria for enrollment colposcopy primarily affected the fraction of women diagnosed with CIN 2. However, we note that, in addition to some CIN 2 regressing, some HPV infections may have caused incident CIN 2, whereas some CIN 2 may have “progressed” to CIN 3. Thus, CIN 2 regression cannot be precisely determined, although reasonable estimations can be derived.
It is also worth mentioning that CIN 2 is the least reproducible of all cervical diagnoses,19,22 and it is possible that the regressive nature of CIN 2 will depend somewhat on the individual pathologist diagnosing it. As shown in Table 4, this lack of agreement is directly related to the regression of CIN 2: the likelihood of regression correlated with the severity of the second (original) diagnosis by the clinical center. For scientific rigor, we used the histopathologic diagnoses rendered by the Pathology Quality Control Group review in our analysis. However, we observed a similar difference in the fraction of CIN 2 as diagnosed by the clinical center pathologists between the CM and IC arms.
In real-world settings, some clinical pathologists equivocate on the use of a CIN 2 diagnosis, relying on nonstandard diagnoses such as “CIN 1–2” or “CIN 2–3.” Their use may reflect further the uncertain nature of CIN 2. It is plausible that each diagnosis will represent different likelihoods of regression, although it is unclear how reliable such distinctions are, given how generally unreliable a CIN 2 diagnosis is.19,22 The inclusion of lesions of an even more ambiguous nature in the definition of CIN 2 would be expected to increase the overall regressive potential of CIN 2. The use of equivocations like CIN 1/2 should probably be discouraged given the lack of evidence of utility, unproven reliability, and the possible increase in the number of women receiving unnecessary therapy.
We point out that the CIN 2 diagnosed at enrollment followed an ASC-US or LSIL Pap test result, which led to referral into the study. That is, women were not referred into ALTS because of an HSIL Pap test. In ALTS, we previously found that women with biopsy-diagnosed CIN 2 after the HSIL enrollment cytology (nb, women enrolled into ALTS had cytology repeated at the enrollment visit) were more apt to have an underlying CIN 3 than those with a less severe enrollment cytology.3 We therefore hypothesize that CIN 2 after an HSIL cytology may be on average less regressive than that observed for CIN 2 diagnosed in the ALTS population.
A CIN 2 diagnosis on biopsy is the clinical threshold for treatment in the United States, and its treatment provides a margin of safety against cancer risk. However, a greater recognition of the true equivocal nature of CIN 2 is needed. Aggressive clinical management of young women (median age 25 years, mean age 27.5 years in ALTS) who have ASC-US or LSIL cytology will lead to overdiagnosis of CIN 2 and overtreatment of a significant number of regressive lesions. This is an important clinical issue, because most CIN 2 is diagnosed in women after ASC-US or LSIL cytology.23
From these data, we suggest that strategies that send more women to colposcopy, such as immediate colposcopy for LSIL or ASC-US or HPV triage of ASC-US, offer the benefit of early detection of CIN 3 at the cost of increased detection of CIN 2, some proportion of which is destined to resolve, and its treatment translates into patient morbidity without benefit. Repeat cytology at an ASC-US threshold as recommended, rather than an HSIL threshold as used in CM arm of ALTS, would be expected to yield different results, with more women going to colposcopy and higher proportion of CIN 2, ie, more akin to the IC and HPV arms of ALTS. However, this is only speculative, because we did not evaluate this clinical algorithm in ALTS.
Human papillomavirus-16–positive CIN 2 seems less likely to regress, perhaps as the result of its greater tendency to persist and its greater oncogenic potential to progress to precancerous lesions than other HPV genotypes.24 When HPV genotyping from validated tests becomes routinely available, detection of HPV-16 may be a useful stratifier of risk25 for deciding the clinical management of CIN 2 diagnoses: HPV-16–negative CIN 2 diagnosed in young women with ASC-US or LSIL cytology could be managed less aggressively through increased surveillance rather than immediate treatment,1,4 whereas HPV-16–positive CIN 2, the most strongly linked with CIN 33 and least regressive, probably warrants inclusion with CIN 3 for management.
More generally, a clinical trial to determine the best management strategies for CIN 2, akin to ALTS for ASC-US and LSIL Pap tests, is needed. Several biomarkers, such as HPV genotyping and p16INK4A immunostaining,26–28 warrant evaluation for determining which women with CIN 2 need immediate treatment compared with surveillance. Key outcomes for any triage strategy for CIN 2 would be the timely detection of CIN 3 (misclassified as CIN 23 or progression of a carcinogenic HPV infection, diagnosed as CIN 2, to CIN 3), safety against cancer, and the reduction in the number of women with regressive CIN 2 treated as compared with the current standard of care4 of excising treatment for all women diagnosed with CIN 2.
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