We studied the clinical files of 183 patients who, from April 2016 to April 2020, had undergone LEEP for CIN3 at our Colposcopy Clinic of the Gynecology Unit, University hospital, Catania, Italy. We selected the women of whom we were able to reconstruct their entire diagnostic therapeutic process from colposcopy, to biopsy, to LEEP and who met the following inclusion criteria: patients with hr-HPV positive test, histological diagnosis of CIN3 to biopsy, positive for p16 protein, a visible lesion on the portio after biopsy, and had been subjected to LEEP. Patients without pathologies of the immune system and not pregnant.
All the data of the women in our database who met the inclusion criteria were retrospectively analyzed in an observational cohort study. The data were analyzed anonymously. This study is conforms to the provisions of the Helsinki Declaration, as revised in 2013. The study protocol was notified, according to the current legislation on observational studies provided by AIFA, to the Ethics Committee of the Catania University Hospital which did not request additions or changes to the protocol. Furthermore, the Ethics Committee of the Catania University Hospital found the consent of the study participants unnecessary as the study concerned only the retrospective review of the medical database.
Only 171 patients met the inclusion criteria and their clinical data were collected, of which: age, HPV genotype, lesion size, histological result of the targeted biopsy and date, date of LEEP and histological examination of the cone and the biopsy- cone interval were calculated. All these patients had the lesion visible on the portio after the diagnostic biopsy to rule out the possibility that the lesion had been completely removed by the biopsy. Each patient underwent colposcopy, targeted biopsy and HPV testing. The histological examination was supported by the search for the p16 protein, which is performed routinely. Histopathological diagnoses were made using WHO criteria. All the cases included in this study showed a p16 positivity at the diagnostic biopsy and were positive for the p16 protein, an immunohistochemical technique that we use routinely. We selected all the cases of CIN3, histological diagnosis that we chose as an entry criterion, compared to the cytological diagnosis [13] of HSIL.
The patients were examined by conventional Pap smear test and colposcopy, in addition, cytological exocervical samples were taken and placed in Thin Prep solution. Samples were sent to the laboratory for DNA extraction and viral DNA genotyping by genetic amplification followed by hybridization with genotype-specific probes capable of identifying most of the HPV genotypes of the genital region [28 high-risk HPV genotypes (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82), low risk (6, 11, 40, 43, 44, 54, 70) and undefined risk (69,71,74)]. The commercial method used was the MAG NucliSenseasy system (bioMerieux SA, Marct l’Etoile, France).
Colposcopy was performed using a Zeiss OPM1F colposcope (Carl Zeiss, Jena, Germany) and applying acetic acid and a Lugol iodine solution. Any colposcopic anomaly was classified according to the nomenclature proposed by the International Federation for Colposcopy and Cervical Pathologies (IFCPC) into 3 degrees of increasing abnormalities according to severity: Abnormal Transformation Zone (ATZ) grade 1 (ATZ1) and grade 2 (ATZ2) or cancer. We evaluated the visibility of the squamous-columnar junction and targeted biopsies were taken from the portio. 72.5% (124 cases) of CIN3 lesions involve the three quadrants of the portio, 17.5% (30 cases) two quadrants of the portio, in 13 (7.6%) cases the lesion extends to the entire portio, only 2.3% (4 cases) affects one quadrant of the portio.
The targeted biopsy is performed according to protocol by the colposcopist on the atypical transformation area. In 56% of cases more biopsy samples were performed (2–3) as ATZ2 often affects the three quadrants of the utero portio. The biopsy specimens had an average diameter of about 5 mm. The hemorrhage was not important. The biopsy piece was sent in formalin to the dedicated pathologist. Then it was fixed in paraffin blocks. Doubtful cases were studied by two pathologists, also resorting to a further p16.
In our study the biopsy/cone were revised by two pathologists.
P16 immunohistochemistry was performed and evaluated using the CINTec histological kit (CINTec © INK4a. Roche Diagnostics) according to the manufacturer’s protocol. A detailed description of the p16 technique has been published [18].
For the results of p16 we considered two factors: intensity and distribution. The intensity is considered diffuse block, patchy or focal; the distribution may be limited to a lower third, up to the middle third or up to the upper third.
p16 was positive if the samples showed a continuous nuclear or nuclear and cytoplasmic staining of the cells of the basal and parabasal layer of the epithelium, extended to more than a third of the entire epithelial thickness, and exclusively intense localization.
p16 was negative if the samples showed the absence of staining in the epithelium, cytoplasmic staining of isolated cells or small focal cell clusters, and an extent of less than one third of the epithelial thickness.
As per protocol, women diagnosed with CIN3 were given LEEP within 3–4 weeks of the biopsy and immediately after the end of menstruation so that the woman was not pregnant, this also avoids cases of secondary endometriosis, as well as cases of excessive bleeding during surgery that is carried out in day hospital on an outpatient basis, by experienced personnel. The obtained cone or biopsy was examined by the pathologist. The diagnosis of CIN3 or higher was considered as persistence or progression, the diagnosis of CIN1 or lower was considered as regression of the lesion.
We calculated the time interval in weeks between the diagnostic biopsy and LEEP and between the diagnostic biopsy and the second biopsy. Both intervals will be referred to as the biopsy-cone interval.
In accordance with the journal’s guidelines, we will provide our data for the reproducibility of this study in other centers if such is requested.
Statistical analysis
The analysis was implemented with the Rstudio software (RStudio Team, 2015. RStudio: Integrated Development Environment for R. Boston, MA. Retrieved from http://www.rstudio.com/).
We used out a logistic model to study the probability of spontaneous regression of CIN3 as a function of the patient’s age, the time elapsed between the biopsy and the cone (in weeks) and the HPV genotype, considering the week and age variables as continuous: OUTPUT (log transform coefficients). To better assess the dependence between regression and age, we then considered three age groups (divided according to the 33rd and 66th percentile: “20–29”, “30–34” and “34 + ”), thus considering the age variable as a category.
Therefore, we carried out the logistic model again with the variable “weeks”, we considered two age classes, under 25 years (including 25) and over 25 years.
Subsequently, the presence of a cut-off of the biopsy-cone interval was identified, after which the probability of regression was greater. The logistic model was calculated several times with the only explanatory variable “weeks” considered as categorical. Each implementation of the model was performed by dividing the sample according to the variable “weeks” increasing the cut-off by one week at a time.