Introduction
Cervical intraepithelial neoplasia grade 1 (CIN1) is a frequently diagnosed condition following a colposcopy referral triggered by abnormal cervical cancer screening results. While CIN1 is understood to be the visual manifestation of a human papillomavirus (HPV) infection, it is often grouped with more severe cervical abnormalities, CIN2 and CIN3, under the umbrella term “CIN” or cervical neoplasia, suggesting a precancerous state. This categorization is partly due to the challenges in accurately and consistently distinguishing CIN1 from CIN2 and normal cervical tissue.
However, the clinical significance of a Cin 1 Diagnosis is not fully clear, as up-to-date prospective studies exploring the subsequent risk of cervical precancer are limited. Earlier research suggested that around 10% of women with CIN1 might develop CIN3 within a decade. The Atypical Squamous Cells of Undetermined Significance (ASCUS) and Low-Grade Squamous Intraepithelial Lesion (LSIL) Triage Study (ALTS) reported a two-year CIN3 risk of 9% for women with CIN1. Other studies have indicated lower risks. The variation in reported risks across studies may stem from factors such as diagnostic inconsistencies, differing diagnostic criteria for CIN grades, population variations, follow-up protocols, and the duration of follow-up.
Given the established role of HPV infection in cervical carcinogenesis, it’s crucial to understand the implications of a histologic cin 1 diagnosis compared to not finding CIN1 (i.e., negative biopsies or no biopsies due to normal colposcopic findings). Does a cin 1 diagnosis, considering HPV infection status, genuinely indicate a higher risk of CIN3 than not having this diagnosis? To investigate this, we conducted a retrospective analysis of data from the ALTS study, focusing on women, primarily younger than 30, diagnosed with CIN1 at enrollment, as a continuation of prior research.
Methods
Study Design and Participants
The ALTS study, conducted between 1997 and 2001, was a multi-center clinical trial that randomized women referred for ASCUS or LSIL cytology to one of three management strategies: Immediate Colposcopy (IC), HPV Triage, or Conservative Management (CM). The “ASCUS” category used in the 1991 Bethesda system was broader than the “ASC-US” category in the 2001 system, encompassing more reactive changes and ASC-H (atypical squamous cells, cannot rule out high-grade intraepithelial lesion). The study was approved by the National Cancer Institute and local institutional review boards, and all participants provided informed consent.
During enrollment and follow-up visits over two years, all women underwent pelvic exams and provided two cervical specimens. The first was for ThinPrep cytology and clinical HPV testing (Hybrid Capture 2), and the second for HPV genotyping via polymerase chain reaction (PCR). Women in all study arms had cytology evaluations every six months for two years and were referred to colposcopy if high-grade squamous intraepithelial lesion (HSIL) cytology was detected. An exit colposcopy exam was scheduled for all participants. Detailed information on randomization, procedures, management, and laboratory methods can be found in cited references.
Clinical center (CC) pathologists’ diagnoses were used to determine baseline histologic status: CIN1, negative histology, or no biopsy. The analysis focused on women in the IC or HPV Triage arms referred to colposcopy for HPV-positive ASCUS Pap tests or LSIL Pap tests, aligning with current colposcopy referral practices. It’s important to note that in the HPV Triage arm, a small portion of women referred with LSIL Pap tests were excluded from this analysis due to negative HPV results by HC2 at enrollment (14.7%). Current guidelines recommend colposcopy for all women with LSIL, regardless of HPV status. CIN1 cases from the CM arm were excluded because colposcopy referral in this arm was limited to enrollment HSIL cytology.
Initially, there were 738 women with CIN1 diagnoses, 346 with negative histology, and 346 with no biopsy. The analysis further focused on women who completed the study (exit visit at 24 months or treated and censored): 594 with CIN1, 289 with negative histology, and 281 with no biopsy. The percentage of women who exited the study did not significantly differ across biopsy outcome groups. Over the study period, quality control (QC) pathology diagnosed 100 CIN3 cases, the primary endpoint. A secondary endpoint was CIN2/3 (n = 138) as diagnosed by CC pathologists, clinically relevant as these women would be treated under current guidelines.
HPV Testing Procedures
Hybrid Capture 2 (HC2), a DNA test targeting 13 carcinogenic HPV genotypes, was performed on residual PreservCyt specimens. STM specimens were tested for 27 or 38 HPV genotypes using PCR and line blot assay (LBA). HPV16 and HPV18 results from LBA among HC2-positive samples were used to categorize women into hierarchical HPV risk groups: HPV16 positive, HPV18 positive (if HPV16 negative), other carcinogenic HPV positive (if HPV16/18 negative), or non-carcinogenic/HPV negative (if negative for all carcinogenic HPV). HPV data was missing for 4 women with no biopsy, 6 with negative biopsy, and 20 with CIN1.
Statistical Analysis
Enrollment characteristics were compared across CIN1, negative histology, and no biopsy groups using Pearson’s chi-square tests. Two-year risks of QC pathology CIN3 and CC pathology CIN2/3 were calculated overall and stratified by baseline status, screening results, and HPV risk group. Fisher’s exact or Pearson’s chi-square tests were used to assess statistical differences in risk. Binomial exact 95% confidence intervals (95%CI) were calculated for CIN3 risks.
Logistic regression was used to calculate odds ratios (OR) and 95%CI to model the association of risk factors with QC pathology CIN3 and CC pathology CIN2/3. P-values < 0.05 were considered statistically significant.
Results
Slight but statistically significant age differences at enrollment were noted across the groups (median age: CIN1 – 23 years, negative histology – 24 years, no biopsy – 23 years, p = 0.04). Table 1 details the study and behavioral characteristics of the three groups. Significant differences were found for enrolling clinical center and referral Pap test. Women with cin 1 diagnosis were more likely to have riskier HPV genotypes (p = 0.02) and a smoking history (p = 0.02). No significant differences were observed for race, pregnancy history, or oral contraceptive use. Comparing negative histology to no biopsy groups, differences were limited to enrolling clinical center (p < 0.001) and referral Pap test (p = 0.05).
Two-year CIN3 risks were 10.3% (95%CI: 7.9–13.0%) for cin 1 diagnosis, 7.3% (95%CI: 4.6–10.9%) for negative histology, and 6.4% (95%CI: 3.8–9.9%) for no biopsy (p = 0.1) (Table 2). No clear patterns emerged in the small CIN3 risk differences between women referred for ASCUS Pap and HPV positive versus LSIL Pap. Most women referred for LSIL Pap and diagnosed with CIN1, negative histology, or no biopsy tested HC2 positive (90.2%, 85.6%, and 80.9%, respectively).
Risks following ASCUS and LSIL Pap tests for women in the IC arm were presented as an unbiased reference. No significant differences in CIN3 or CIN2/3 risks were found between negative histology and no biopsy groups (p = 0.7 for both), so these were combined into a “no CIN1” group (n = 570), with a 6.8% two-year CIN3 risk (95%CI: 4.9–9.2).
Initially, without considering HPV status, women with cin 1 diagnosis were slightly more likely to have high-grade cytology at enrollment (HSIL or atypical squamous cells cannot rule out HSIL) than those without cin 1 diagnosis (8.0% vs. 5.8%, p = 0.2). Among women with cin 1 diagnosis, those with high-grade cytology at enrollment had double the CIN3 risk (19.2% vs. 9.6%, p = 0.05). Similarly, women without cin 1 diagnosis with high-grade cytology had approximately double the CIN3 risk (12.1% vs. 6.6%, p = 0.2).
Before HPV status stratification, CIN3 risk was higher for women with cin 1 diagnosis than those without (p = 0.04). Stratification by HPV risk group (Table 3) showed a risk gradient based on HPV risk group (ptrend<0.001): 19.1% for HPV16 positive, 13.9% for HPV18 positive, 5.7% for HC2 positive (HPV16/18 negative), and 5.2% for HC2 negative. No significant difference in two-year CIN3 risk was observed by baseline histology status when stratified by HPV risk group and screening result (HC2-positive ASCUS or LSIL), although no CIN1 tended to have slightly lower risks. Similar patterns were seen for CC pathology CIN2/3 endpoint.
Logistic regression analysis (Table 4) indicated that HPV16 positivity (OR = 3.2, 95% CI = 1.6–6.7) and high-grade cytology at enrollment (OR = 2.4, 95% CI = 1.2–4.9) were independently associated with elevated CIN3 risk. HPV18 positivity was not independently associated with CIN3 (OR = 1.2, 95%CI 0.26–5.5). Cin 1 diagnosis (vs. no CIN1) was not associated with CIN3 (OR = 0.99, 95%CI 0.54–1.8). Similar associations were found for CC pathology CIN2/3 endpoint.
Other enrollment variables, including the number of biopsies and HPV genotypes, and non-normal colposcopic impression were not significantly associated with CIN3 or CIN2. Excluding cases upgraded to CIN3 by QC pathology (18 from CIN1, 4 from no CIN1), two-year CIN3 risks were 7.5% and 6.2% (p = 0.2) for cin 1 diagnosis and no CIN1, respectively. Logistic regression results remained similar after excluding these cases.
Discussion
This study investigated whether a cin 1 diagnosis represents an independent risk factor for CIN3 development beyond HPV infection. After controlling for HPV risk group and high-grade cytology at enrollment, no evidence was found that cin 1 diagnosis significantly increased the two-year CIN3 risk compared to negative histology or normal colposcopy. The observed differences in CIN3 risk between cin 1 diagnosis and no CIN1 groups were primarily attributed to variations in HPV genotype distribution and the proportion of women with missed enrollment CIN3 indicated by high-grade cytology. Therefore, HPV genotype, rather than the cin 1 diagnosis itself, is the primary etiological factor for CIN3.
Clinically, women with cin 1 diagnosis had a 10% two-year CIN3 risk, compared to 7% for negative histology and 6% for no biopsy – statistically significant but small absolute differences. Given these risk similarities and limited management options (routine screening, surveillance, or follow-up colposcopy), differential management based solely on histologic status in this population might be difficult to justify, depending on risk thresholds for each option. The CIN3 risks reported here are consistent with previous ALTS analysis on CIN1.
HPV16- or HPV18-positive cin 1 diagnosis or no CIN1 increased the two-year risk 3-fold and 2-fold, respectively, compared to other carcinogenic HPV genotypes. Similar patterns were observed for CIN2/3.
All 13 CIN3 cases in the HC2-negative group tested negative for carcinogenic HPV, suggesting new HPV infections or misclassification. Fewer QC-pathology diagnosed CIN3 were confirmed by CC pathologists following HC2-negative results compared to HC2-positive.
Study limitations include: 1) limited CIN3 cases, 2) young study population not fully representative of older women with cin 1 diagnosis, 3) lack of long-term follow-up, 4) potential missed CIN3 due to colposcopy sensitivity limitations, 5) exclusion of women with high-grade Pap, and 6) exclusion of HPV-negative LSIL cases in the HPV arm, potentially slightly overestimating CIN3 risk after LSIL Pap and ≤CIN1 histology.
Study strengths include the use of consensus CIN3 diagnosis as the primary endpoint, avoiding issues with CIN2’s equivocal nature and diagnostic variability. The inclusion of CC pathology CIN2/3 endpoint confirmed similar risk patterns.
In conclusion, this study found no independent risk elevation for CIN3+ associated with cin 1 diagnosis beyond HPV infection itself. Women with cin 1 diagnosis were more likely to have HPV16, increasing CIN3 risk. While risk differences between HPV16-positive and HPV16-negative CIN1 or no CIN1 may not warrant different clinical management, awareness of elevated risk in HPV16-positive women is important. Categorizing histologic diagnoses simply as “CIN” is a misclassification, as CIN1, CIN2, and CIN3 are distinct biological entities with varying cancer risks.
Acknowledgments
Supported by the Intramural Research Program of the NIH, National Cancer Institute. ALTS was supported by contracts from the National Cancer Institute, National Institutes of Health Department of Health and Human Services. Equipment and supplies were donated or provided at reduced cost by various corporations.
The authors thank the ALTS Group Investigators.
Footnotes
Financial Disclosure: Dr. Wheeler has received funding for vaccine studies and HPV genotyping studies from pharmaceutical and diagnostics companies.
Reference List
(Simplified reference list – original list is very long, include representative examples if needed for length or relevance)
(Example references, if needed to expand length – can be shortened or omitted if length is sufficient)
- Stoler MH, Schiffman M; ALTS Group. Interobserver reproducibility of cervical cytologic and histologic interpretations: realistic estimates from the ASCUS-LSIL triage study. JAMA. 2001;285(11):1500-5.
- Castle PE, Stoler MH, Wright TC Jr, Sharma A, Colgan TJ, Katki HA,等人. Performance of carcinogenic human papillomavirus (HPV) testing and cervical cytology for women with atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesions: a systematic meta-analysis. Am J Clin Pathol. 2007 Apr;127(4):528-48.
- Ostor AG. Natural history of cervical intraepithelial neoplasia: a critical review. Int J Gynecol Pathol. 1993;12(2):186-92.
… (and so on, representative references from the original)
