HPV 16 Is Related to the Progression of Cervical Intraepithelial Neoplasia Grade 2: A Case Series

Purpose. To describe the acquisition, persistence, and clearance of HPV infection in women with CIN 2 followed up for 12 months. Methods. Thirty-seven women with CIN 2 biopsy, who have proven referral to cervical smear showing low-grade squamous intraepithelial lesions or atypical squamous cells of undetermined significance and tested for HPV, were followed up for one year with cervical smear, colposcopy, and HPV test every three months. HPV DNA was detected by the polymerase chain reaction and genotyping by reverse line blot hybridization assay. Results. CIN 2 regression rate was 49% (18/37), persistence as CIN 1 or CIN 2 was 22% (8/37), and progression to CIN 3 was 29% (11/37). Multiple HPV types were observed at admission in 41% (15/37) of cases. HPV 16 was detected at admission in 58% (11/19) of the cases that persisted/progressed and in 39% (7/18) of the cases that regressed. HPV 16 was considered possibly causal in 67% (10/15) of the cases that persisted or progressed and in 10% (1/10) of the cases that regressed (P = 0.01). Conclusion. Multiple HPV infections were frequently detected among women with CIN 2 at admission and during the followup. The CIN 2 associated with HPV 16 was more likely to persist or to progress to CIN 3.


Introduction
Cervical intraepithelial neoplasia (CIN) and cervical cancer derive from cellular transformations of the cervix epithelium after HPV infection. High-risk HPV (HR-HPV) persistent infections represent a necessary cause of cervical cancer, but not sufficient [1]. Additional conditions and events are required for the neoplastic transformation.
Firstly, it was thought that cervical squamous cell carcinomas (SCC) would always evolve from HPV infected normal cervical epithelium via a continuum, long-lasting consecutive CIN 1, CIN 2, and CIN 3 lesions [1]. However, it has been shown that clinically relevant CIN 2 or CIN 3 may be induced within 2-3 years following HPV high-risk infection, and another 10-12 years may pass until invasive cervical carcinoma would develop [2]. Most CIN 1 lesions that are associated with HR-HPV, and some CIN 2 lesions, should not be considered as true precursor stages of cervical cancer but rather the cytopathic effect of a productive viral infection [3]. Moreover, CIN 1 regression rate is high [4], as is the regression rate of CIN 2 [5][6][7], and both display viral expression patterns suggestive of productive viral infections. In contrast, some CIN 2 lesions and CIN 3 lesions show different viral gene expressions, leading to atypical proliferation and cell transformation. The development of CIN 3 arises in women who cannot resolve the HPV infection, and therefore the infections can persist for years or decades following initial exposure [8].
CIN 2 thus is a borderline lesion, can show clinical behavior similar to CIN 1 or to CIN 3, and occurs frequently in young women [6]. The 2012 Updated Consensus Guidelines of the American Society for Colposcopy and Cervical Pathology (ASCCP) [9] recommend that when a histological diagnosis of CIN 2 is specified for a young woman, observation is preferred, provided colposcopy is adequate.
The pattern of HPV infection and reinfection in women and the outcome of CIN 2 are not well known. A better discrimination of CIN 2 that trends to progression or regression could help in choosing the best clinical management. This study therefore aimed to describe the acquisition, persistence, and clearance of HPV infection in women with CIN 2 confirmed by biopsy followed for 12 months under expectant management.

Selection and Followup of the Women Studied.
Women aged 18 to 46 years old were considered eligible for the study if they fulfilled the following criteria: (1) referral cervical smear showing atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL); (2) biopsy showing CIN 2 reviewed by senior pathologist who was unaware of the DNA HPV test results [10]; (3) lesion completely visualized by colposcopy and squamocolumnar junction totally visible; (4) not being pregnant; (5) showing no evidence of any immunodeficiency diseases; (6) no history of previous invasive neoplasia; (7) HPV test result. The selection of patients was fully described in a previous publication (5). Fifty women consecutively fulfilled these criteria, agreed to participate in the study, and signed the informed consent.
The planned followup was visits every three months during one year with cervical smear, colposcopy, and sample collection for HPV detection. The colposcopic image showing a worsening of the lesion margem, color, and vascular pattern was submitted to biopsy. When the biopsy revealed CIN 2 or a less severe diagnosis, the woman was maintained in the follow-up plan. When the biopsy revealed CIN 3, immediate treatment by excision of the lesion was performed.
At one year of followup, all the women who still showed cytological or colposcopy abnormalities were submitted to complete evaluation, and they were treated according to the final diagnosis. No ablative procedures were carried out.
During followup, 13 women were discontinued due to the following: five women diagnosed with persistence in the intermediate visit did not show up for the final visit; one woman tested positive for HIV; one woman missed three consecutive visits; one woman became pregnant; five women were without at least one HPV test during followup. After 12month followup, 37 women had final diagnosis established. CIN 2 Followup. The final outcome of CIN 2 was classified as progression, persistence, or regression, according to the following criteria. (ii) Persistence: biopsy showing CIN 1 or CIN 2 at twelvemonth followup.

Outcome of
(iii) Regression: concomitant negative cervical smear and negative colposcopy or negative biopsy observed at any time during the followup and confirmed at twelve-month followup. If the patient had a HLSIL cervical smear and negative biopsy, it was not considered regression and the patient was submitted to excisional treatment.

Possible Causal HPV Type.
The HPV type possibly associated with the CIN 2 lesion was considered the possibly causal type. For the CIN 2 lesions that regressed, possibly causal HPV was considered when HPV type was detected at admission and persisted up to the follow-up visit immediately before the regression. If the lesion persisted, either as CIN 1 or CIN 2, possibly causal HPV was considered when detected at admission and during the follow-up visits. Among the cases that progressed, possibly causal HPV was considered when detected at admission and at least until the follow-up visit when progression was detected. The possibly causal HPV type was considered undefined if the test was negative at admission or if it was not possible to associate the HPV type with the CIN 2 lesion as described previously.

Sample Processing and DNA Extraction. Aliquots of 200 L of Universal Collection Medium UCM (QIAGEN Sample and Assay Technologies, QIAGEN Biotechnology
Brazil Ltda) were taken for polymerase chain reaction (PCR) testing and were centrifuged for 10 minutes at 13,000 g. The supernatants were immediately removed, and split cellular pellets were stored at −80 ∘ C before nucleic acid extraction and HPV detection. The cellular pellets were resuspended in 200 L of digestion solution (1 mM Tris, 200 mg of proteinase K/mL, 0.5% sodium dodecyl sulfate) and digested at 55 ∘ C for 2 hours. The digestion was followed by 5-minute incubation at 95 ∘ C to inactivate the proteinase K. Nucleic acids were purified by phenolchloroform extraction followed by ethanol precipitation. The DNA pellet was dried and was dissolved in 100 L of TE. Nucleic acids were stored at −80 ∘ C before HPV detection.    Eleven cases (29%) progressed to CIN 3 up to twelvemonth followup. HPV 16 was detected at admission in 64% (7/11) of the cases, HPV 52 in 27% (3/11), and HPV 68 in 18% (2/11). Multiple HPV types were detected at admission in 54% (6/11) of cases and new HPV type was detected during followup in 45% (5/11). Among the cases that progressed there was no negative HPV test. HPV 16 was considered possibly causal type in 64% (7/11) of cases. Table 2 shows the association of HPV 16 with the CIN 2 clinical outcome. Cases that persisted as CIN 1 and CIN 2 were grouped together with the cases that progressed to CIN 3. HPV 16 was detected at admission in 58% (11/19) of the cases that persisted or progressed and in 39% (7/18) of the cases that regressed, but this difference was not statistically significant ( = 0.20). HPV 16 was considered possibly causal in 67% (10/15) of the cases that progressed or persisted and in 10% (1/10) of those that regressed, and the difference was statistically significant ( = 0.01). The cases with undefined HPV possibly causal type were not considered for the latter analysis.

Discussion
According to this cohort study, multiple HPV infections were frequently detected in women with CIN 2, as new HPV types were also frequently detected during twelve-month followup. The CIN 2 lesions associated with HPV 16 were more frequent among those lesions that progressed to CIN 3.
Cuschieri et al. [11] showed that multiple high-risk HPV infections were prevalent in young women, in high and lowgrade cervical intraepithelial neoplasia, reflecting common sexual transmission of multiple high-risk HPV. Sometimes the lesion became HPV negative and at the next visit new infections were acquired. In this scenario, most of the HPV infections are probably productive, as pointed by Snijders et al. [1], that is, transitory infections, not inducing transformation process in the host cell.
Castle et al. [12] found that the presence of HPV 16 was positively associated with CIN 3 and they reported that CIN 2 caused by HPV 16 may be more likely to progress than CIN 2 caused by other high-risk HPV types [13]. Wentzensen et al. [14] also showed that CIN 2 related to HPV 16 was more likely to persist. The absolute risk of precancer diagnosis can approach 40% after 3-5 years of persistent HPV 16 infection [15]. Kjaer et al. [16], in a population study, found that HPV 16 persistent infection was associated with high absolute risk for progression to high-grade cervical lesions.
HPV infection can be considered productive when the expression of viral gene products remains regulated, not leading to significant host cell changes. Transformation infections arise where the productive infection cannot be properly supported due to increased activity of viral protein E6 and E7, which leads to genomic instability in the infected cell, accumulation of oncogene mutations, further loss of cellgrowth control, and ultimately precancer lesions and cancer [8,17].
There was no negative HPV test in those cases that progressed, regardless of the HPV type. Among the eight cases that persisted, three cases presented negative HPV during followup, but in two cases the lesion persisted as CIN 1. Among the 18 cases that regressed, 11 presented negative HPV during followup, indicating transient infections. Only 4 out of 37 cases were HPV negative at admission, which could be a false negative or a true negative test, but none progressed.
The regression rate found is not different from the reported regression rate of CIN 2 in adult women, which ranges from 15 to 55% regression over 4-6 years of followup [13,18]. Moscicki et al. reported CIN 2 regression rate of almost 70% among adolescents and young women and progression rate of 15% at three years [7]. This greater regression rate might be due to the younger age of the women studied (13 to 24 years old), which could reflect a shorter time of HPV persistent infection, and to the longer followup of three years. In the present study, the women's age was not used to select the population, the followup was twelve months, and the progression rate was similar to others that showed greater risk of CIN 2 progression when the lesion was associated with HPV 16 infection [1,14].
This study has limitations due to small sample subject. The inclusion criteria were women with cervical smear referral showing ASCUS or LSIL, CIN 2 proven biopsy, and HPV test at admission and at least at one follow-up visit, which limited the number of selected women. Besides, multiple HPV infections were frequent in these women and, therefore, the indication of possibly casual HPV type for the CIN 2 was not always obvious. Despite the lack of reproducibility of CIN 2, the HPV types did not differ significantly between CIN 1, CIN 2, and CIN 3 [11]. False negative results with colposcopy and guided biopsy were expected, but this effect is minimized using cytology as cotesting.
Obstetrics and Gynecology International 5 For clinical practice, the findings of this study suggest that the HR-HPV testing would have limited utility to indicate those women with greater risk of CIN 2 progression since the rate of HR-HPV positive was high among women with CIN 2 regression. Nevertheless, expectant management, which may be proposed for young women, should be considered cautiously when HPV 16 infection is detected. In conclusion, infections with multiple HPV types were frequently detected at admission and follow-up visits as well as infections with new HPV types during followup. CIN 2 lesions with HPV 16 alone or in combination with other HR-HPV types are more prone to progression to CIN 3.