In this analysis, we have compared clinical, virologic, and histologic characteristics of patients with biopsy-confirmed CIN2/3 over a short observational window before standard therapeutic resection. We found an overall spontaneous regression rate of 28% over a 15-week window. Women with CIN2/3 associated with infection with HPV16 were significantly less likely to have lesion regression compared with the rest of the cohort. However, HPV16-positive patients who resolved their lesions in general had lower HPV16 viral loads than those who failed to resolve their lesions. Moreover, those who did resolve their lesions had decreasing viral loads over the observation window, in contrast to patients who did not resolve their lesions. Finally, the interaction of host HLA class I type with the HPV types identified increased the effect of the HPV infection by an order of magnitude, supporting the hypothesis that the efficacy of MHC class I – restricted presentation of viral antigens plays an important role in disease outcome.
The strengths of this study include its prospective design, histologic diagnosis of CIN2/3 as an entry criterion, virologic data, and the standardization of colposcopy and histopathologic review. However, the relatively small sample size (n = 100) as well as our incomplete understanding of the potential effect of biopsy on lesion behavior limit interpretation of our data.
The effect of a diagnostic biopsy on the natural history of CIN2/3 may explain the discrepancy between our observations and those of several large studies based on cytologic diagnosis alone, as the local response to a diagnostic biopsy may be quite different than a response to a cytologic smear, which is atraumatic and noninvasive. HPV infects the basal cell layer of the cervical mucosal squamous epithelium without disruption of the basement membrane. Subsequent production of foreign or viral proteins occurs with cellular maturation and takes place in the most superficial layer of epithelium. Viral antigens are thus released in the setting of natural epithelial senescence and desquamation and are therefore not necessarily presented to host immune system in the context of signal two or danger. Therefore, aside from decreasing lesion size, a diagnostic biopsy may elicit enough of a local reparative or inflammatory milieu so that viral antigens previously undetected may be presented in a setting sufficient to induce an effective immune response and subsequent lesion clearance.
Holowaty et al., for example, found a regression rate of 6.9% over 2 years in a large cohort study of women with moderate dysplasia (2
). This study was based on cytologic diagnoses only and was not linked with diagnostic biopsies. In our patient cohort, 65 of 187 (34.8%) of the women referred for evaluation of high-grade cytology did not have histologically confirmed high-grade lesions. Narod et al. followed a cohort of 70,236 women, also using cytologic diagnoses for an end point of progression to carcinoma in situ
or worse (11
). Again, as there was no histologic confirmation, it is difficult to extrapolate these findings to compare their clinical significance with the findings in our cohort.
The observed rate of spontaneous regression of biopsied CIN2/3 in this cohort is within the range of that seen in control/placebo arms of published interventional clinical trials in similar patient cohorts (; refs. 12
). However, none of these studies included HPV type – specific correlation.
Clinical behavior of biopsy-confirmed CIN2/3
Previous studies have assessed risk for incident intraepithelial lesions in the setting of persistent HPV infections (15
). Duration of infection with HPV type 16 in particular has been shown to be longer than with other HPV types (1
). Ho et al. evaluated the natural history of HPV infection in a cohort of college women. These authors found that multiple infection conferred a 4-fold increase in risk for persistent HPV infection at >6 months. However, this effect was noted in women without incident lesions. They found that persistent infection with the same HPV types, particularly high-risk types, conferred the highest risk for subsequent incident intraepithelial lesions. The association in our cohort between women with single infection with HPV16 and persistent established high-grade lesions may reflect an inability on the part of these women to have resolved a persistent HPV16 infection in the first place. Coinfection with other HPV types may elicit an immune response capable of resolving an established lesion regardless of the HPV type causing it.
We made the assumption that in those patients infected with one HPV subtype only, the CIN2/3 arose from that HPV subtype. Integration of viral DNA into the host cell genome is a rare event; high-grade HPV cervical lesions are thought to be clonal processes (18
). Our data is consistent with earlier reports documenting infections with multiple types of HPV among both women with normal cytology and those with histologic CIN (20
). In the ALTS study, for example, Sherman et al. have reported that ~50% of CIN2 and CIN3 lesions were associated with multiple oncogenic HPV types. Our study suggests that HPV16 infection, compared with other HPV types, continues to confer an adverse effect in the setting of an established high-grade lesion.
A classic approach to determine whether a disease outcome has an immunologic component is to search for associations with specific HLA alleles. We therefore initially assessed whether natural regression was affected by the presence or absence of the most common HLA class I allele (A201
) expressed by roughly half of the population. We found that, whereas HLA-A201 expression had relatively little effect on regression in HPV16+ lesions, it displayed a pronounced effect in non-HPV16 lesions. HLA-A2+ individuals with non-HPV16+ lesions were 30-fold less likely to experience natural regression than HLA-A2– patients with HPV16. The specific role of MHC class I – restricted immune responses in natural regression of established but premalignant cervical HPV lesions remains to be elucidated. The simplest mechanism is an “Ir gene” mechanism in which certain HLA alleles fail to present peptides efficiently. The relatively small size of E6 and E7 proteins might indeed provide a limited number of epitopes available for presentation. Application of this model to the current data set would suggest that evolutionary pressure on the less oncogenic HPV types (i.e., non-HPV16) would have been to eliminate E6 and E7 epitopes efficiently presented by common HLA alleles such as HLA-A201
. Whereas reports of association between cervical HPV disease and HLA haplotypes have not been consistent, an increasing body of evidence supports increasing down-modulation of the MHC class I antigen presentation machinery with progression of premalignant lesions. Both HLA (I and II) allele association and abnormal HLA class I antigen presentation have been reported in other chronic human viral infections such as hepatitis B and C (24
). Discrete HLA supertypes have been shown to be associated with differential clinical responses to HIV infection (26
). Taken together, the emerging data on HLA allele association and down-modulation of antigen presentation provide evidence for a T-cell recognition component to the outcome of HPV-associated premalignant cervical disease.
Other immunologic mechanisms must also be considered besides a straightforward HLA-restricted antigen presentation model. In fact, an Ir gene mechanism predicts that a failed response (i.e., lack of regression) is recessive. However, the majority of HLA-A201 patients are heterozygous at this locus, suggesting that the decreased rate of regression in HLA-A201+ individuals with non-HPV16 lesions is due to a dominant effect. This dominant effect could be related to the generation of regulatory T cells. Regulatory T cells are typically thought to be CD4+ and would thus not recognize HLA-A201; however, CD8+ regulatory T cells have recently been described (27
). Clearly, it will be important to identify the HPV epitopes which are naturally processed in patients and to generate T-cell lines and clones grown from regressing and nonregressing patients. Finally, a role for HLA allele expression on natural killer responses to HPV-infected cells must also be considered, owing to the ability of certain class I alleles to inhibit natural killer responses via killer inhibitory receptors of the immunoglobulin superfamily. Matching between specific HLA C alleles and killer inhibitory receptor haplotypes has been recently reported to affect the outcome of HCV infection (30
). The role of natural killer responses in controlling premalignant HPV lesions has not yet been assessed but warrants serious study. The direct accessibility of HPV+ cervical lesions offers unique opportunities to analyze the phenotype and function of lymphocyte populations directly infiltrating regressing and nonregressing lesions.
Whereas fluctuations in the local humoral immunologic milieu in the cervix fluctuates with the menstrual cycle, pregnancy, and use of hormonal contraceptives have been described (31
), we did not observe any effect of current oral contraceptive use upon rate of spontaneous regression. As noted above, however, our sample size is relatively small and the 15-week window of observation is very short; therefore, only large effects would be obvious.
Tobacco smoking is known to increase the risk of cervical dysplasia, in a dose-response fashion, from low-grade lesions to frank invasive cancer (15
). Nicotine and nicotine metabolites have been found in cervical secretions of women who smoke (40
). We observed a nonsignificant trend in our cohort toward an increased risk for persistent disease among women who were current smokers.
The length of observation in this study was motivated by concern over patient safety. The risks of progression to invasive disease in the time period chosen (15 weeks) were believed to be virtually zero. Based on our observations, as well as those of others, an argument for a longer time period of study design could be made (12
). Nonetheless, such studies mandate careful patient selection, regular colposcopic follow-up, and aggressive efforts to ensure that patients are not lost to follow-up before definitive therapy has been done.
Appropriate intermediate end points are critical to the design of interventional trials in patients with preinvasive disease. To date, only cytologic and histopathologic diagnoses have been sufficiently validated to warrant use as end points (45
). The use of other biological markers and emerging noninvasive imaging technologies remain experimental. To be useful as potential intermediate end points, they must be quantifiable, reproducible, and be shown to correlate with known biological variables of HPV disease. We found that quantitative HPV16 viral load in cervical swabs correlated with clinical behavior. This measure should be further evaluated in future interventional trials. For the immediate future, colposcopically directed tissue biopsy remains the gold standard end point for design and analysis of interventional trials in this population of women.
This prospective observational study was designed to estimate spontaneous regression of biopsy-proven CIN2/3 in a short time period. In this trial, we found the overall rate of spontaneous regression of CIN2/3 to be 28%. We found that the rate of lesion regression was strongly inversely associated with infection with HPV16 and that the HLA*A201 allele was associated with disease persistence. Should our findings be validated in a larger data set, then future interventional trials in women with CIN 2/3 may require stratification for HPV as well as HLA alleles to take into account differing rates of spontaneous regression.