Abstract
Background/Aim: This study aimed to assess the role of the stromal CD25+/CD8+ (Cluster of Differentiation) lymphocyte ratio and other immunohistochemical markers in predicting the risk of recurrence and human papillomavirus (HPV) persistence after Loop Electrosurgical Excision Procedure (LEEP) conization in patients with grade 2-3 cervical intraepithelial neoplasia (CIN2-3).
Patients and Methods: A retrospective analysis was conducted on 72 patients who underwent LEEP for CIN2-3 in our Department. Criteria for enrollment included HPV genotyping before and after surgery and a follow-up time ≥18 months. Immunohistochemical analysis assessed CD8+ cytotoxic T cells and CD25+ regulatory T cells in the cervical stroma, and the CD25+/CD8+ ratio was computed. Recurrence of CIN2-3 and HPV persistence after LEEP were the endpoints of the study.
Results: CIN2-3 recurrence occurred in 13.9% of patients with smoking, HPV-16/18 infection, positive surgical margins, and CIN3 histology being significant risk factors. A CD25+/CD8+ ratio >1.25 was associated with a shorter disease-free survival (DFS) (p=0.033) and HPV persistence at 18 months (p=0.017) after LEEP at univariate analysis. Adjuvant HPV vaccination reduced the recurrence risk (p=0.004). A lympho-monocyte infiltrate consisting of at least 10% CD25+ T cells was significantly associated with HPV persistence at 18 months (p=0.0046).
Conclusion: The CD25+/CD8+ ratio is a promising biomarker for identifying patients at higher risk of CIN2-3 recurrence and HPV persistence post-LEEP. These findings highlight the role of immune profiling in the management of patients affected by CIN2-3 and support the integration of vaccination and personalized follow-up strategies.
- Cervical intraepithelial neoplasia (CIN2-3)
- CD25+/CD8+ lymphocyte ratio
- stromal lymphocyte infiltrate
- HPV persistence
- recurrence risk
- loop electrosurgical excision procedure (LEEP)
- cytotoxic T cells (CD8+)
- regulatory T cells (CD25+)
- HPV genotyping
Introduction
Cervical cancer is the fourth leading malignancy among women worldwide, with estimated 660,000 new cases and 350,000 deaths in 2022 (1). The persistent infection with high-risk human papillomavirus (HPV) is a critical etiological factor for the development of cervical intraepithelial neoplasia (CIN) and cervical cancer (2). Several cofactors, such as smoking, multiparity, long-time oral contraceptive use, and immunosuppression can modulate cervical carcinogenesis (3, 4). In fact the progression from an HPV infection to a high-grade CIN and invasive cervical carcinoma depends on a complex interplay of viral and host factors. The high-risk HPV genotypes, especially HPV-16 and HPV-18, are responsible for approximately 70% cervical cancers. The integration of the HPV genome into the DNA of the host causes an increased expression of the viral proteins E6 and E7, which degrade and inactivate p53 and Rb gene-encoded proteins, respectively, thus promoting malignant transformation (5).
High-grade CIN (CIN2-3) requires surgical treatment by conization (6-8). This can be accomplished through different techniques, such as laser therapy or loop electrosurgical excision procedure (LEEP). Conization has been very successful in minimizing the risk of disease progression to invasive cancer, although recurrence after treatment can occur up to 10% of the cases (9). Identifying predictive factors for persistent HPV infection and CIN2-3 recurrence is essential for optimization of management and follow-up strategies.
Immunological responses to HPV play a pivotal role in determining the outcome of infection and the risk of CIN progression or recurrence (10, 11). A robust cell-mediated immune response, characterized by cytotoxic Cluster of Differentiation (CD) 8+ T cells, is critical for lesion regression. Typically, the cytotoxic response mediated by CD8+ T cells is accompanied by an activation of regulatory CD25+ T cells. The latter have several functions, such as preventing an uncontrolled immune response against self-antigens and limiting an excessive cytotoxic response against non-self-antigens. However, over-activation of regulatory T cells could create an immunosuppressive microenvironment that promotes HPV persistence and dysplasia progression (12, 13).
Known risk factors for CIN2-3 recurrence after conization are immunodeficiency, smoking, higher-grade dysplasia, positive surgical margins, HPV-16 and HPV-18 infection, and noncompliance with post-treatment vaccination (9, 14). Recent evidence suggests that the immune microenvironment may play a decisive role in the risk of progression from CIN2-3 to cervical carcinoma (15). Also, the CD25+/CD8+ ratio in the stromal immune infiltrate could have a role in cervical carcinogenesis (16-18). However, limited data exist on the prognostic implications of stromal immune cell populations after surgery, particularly on the role of the immune response in determining the risk of CIN2-3 recurrence and HPV persistence after LEEP.
This study seeks to investigate the predictive value of the stromal CD25+/CD8+ ratio and other immunohistochemical factors in patients underwent LEEP for CIN2-3. By examining their association with CIN2-3 recurrence and HPV persistence, our work is aimed to contribute to more personalized and effective management strategies for women with high-grade cervical dysplasia.
Patients and Methods
Study design, study setting and participants. This was a retrospective study analyzing 72 patients with CIN2-3 treated with LEEP conization at the Department of Gynecology and Obstetrics of the Azienda Ospedaliera Universitaria Pisana (AOUP) from June 2015 to March 2023.
Inclusion criteria were as follows: availability of HPV genotyping test performed before and after treatment and a length of follow-up of at least 18 months. Patients with immune deficiency were excluded from the study. All patients included in the study had not performed prophylactic HPV vaccination.
Data collection. During follow-up, all patients underwent co-testing (HPV-test + Pap-test) and colposcopy at six and 18 months after LEEP. We also collected information on age at the time of surgery, smoking habits and eventual adjuvant vaccination with Gardasil 9.
The histological examination of the cervical cone was independently performed by two pathologists (S.P. and A.G.N.). The histological report included the grade of CIN, the status of the glandular lumen (negative or positive for dysplasia of any grade), the status of the exocervical, stromal, and endocervical margins (negative or positive for dysplasia of any grade). Margins were also considered positive when they were less than 1 mm away from the dysplastic lesion.
To identify the most representative cone specimen of higher-grade dysplasia and lympho-monocytic infiltrate, histologic slides that had already been prepared for post-LEEP diagnosis were reviewed. The selected cervical cone specimens were used to set up two new histological slides for each patient, which were used for immunohistochemical analysis carried out using anti-CD8 antibodies (Anti-CD8 Rabbit Monoclonal Primary Antibody, 10.2 mcg/ml, Roche Diagnostics SpA, Monza, Italy) and anti-CD25 antibodies (Anti-CD25 Mouse Monoclonal Antibody, 0.23 mcg/ml). Immunostainings were performed on Ventana BenchMark ULTRA System according to vendor protocols (Ventana Medical Systems, Oro Valley, AZ, USA).
Small (5×) and medium (20×) magnifications were used to determine the degree of lympho-monocytic infiltration (absent/low or moderate/intense) (Figure 1). High magnification (40×) was used to establish the percentage of CD8+ and CD25+ cells in the context of lympho-monocytic infiltrate and the possible presence of CD8+ cells in the context of dysplasia. We considered as positive for CD8+ and CD25+, histological slides with a lympho-monocytic infiltrate consisting of at least 10% of that specific cell line. We computed the CD25+/CD8+ ratio using the absolute value of the percentages of the two cell lines. Microscopic analysis was performed visually, by counting individual cells expressing that marker.
Example of a moderate-to-intense lympho-monocytic infiltrate, predominantly composed of CD8+ cells. Immunohistochemical analysis for CD8 (left) and CD25 (right) in a clinical case of grade 3 cervical squamous intraepithelial neoplasia. Magnification 5×.
Outcomes and statistical analysis. We considered as endpoints of the study any recurrence of CIN2-3, established by biopsy, conization, or hysterectomy, and the persistence of HPV after ≥18 months of follow-up. We used the two-tailed Fisher’s exact test to study the correlation between demographic, clinical, and anatomo-pathological variables and the above-mentioned endpoints.
Disease-free survival (DFS) was defined as the interval time between the LEEP and the detection of recurrence or last follow-up visit. The cumulative probability of DFS was estimated using the Kaplan–Meier method, and the long-rank test was used to compare the homogeneity of the DFS function across the strata defined by the prognostic variable categories. The relationship between the persistence of HPV after LEEP and the prognostic variables was assessed with Fisher’s exact test.
Statistical analysis was performed using IBM SPSS Statistics Software, Version 29.0 (IBM, Armonk, NY, USA). Statistical significance was set at a p-value lower than 0.05.
Ethical considerations. All data and diagnostic procedures considered in this study were part of the routine follow-up, conducted in accordance with standard clinical protocols. Patients were fully informed about the study’s objectives, the voluntary nature of participation, and the absence of additional risks beyond those inherent to standard care. All data, including anatomopathological information, were collected and processed anonymously using a unique identification code for each patient. No identifiable patient data was included in the analyses or shared in any form.
Results
Patient characteristics are shown in Table I. At the time of LEEP conization, the median age was 38 years (range=28-66 years). The median follow-up duration was 31 months (range=18-102 months). A total of 41 (56.9%) patients underwent adjuvant vaccination with Gardasil-9. Ten (13.9%) patients experienced CIN2-3 recurrence after a median time of 12.5 months. For women with recurrence, median follow-up lasted 30 months (range=30-90 months).
Patient characteristics (n=72).
Recurrence was significantly associated with smoking habit (p=0.0136), presence of HPV-16 and/or HPV-18 pre-LEEP (p=0.0432), higher degree of dysplasia at baseline (p= 0.0136), surgical margin positivity (p=0.0295), non-performance of adjuvant vaccination (p=0.0016), and persistence of HPV infection at six months (p=0.0048) and 18 months (p=0.0065; Table II).
Risk factors for CIN2-3 recurrence (Fisher’s exact test).
Using the ROC curve, the cut-off value of the CD25+/CD8+ ratio that optimizes sensitivity and specificity for predicting the risk of recurrence was found to be 1.25. Univariate analysis showed a trend of association (albeit not significant) between CD25+/CD8+ ratio >1.25 and recurrence risk, whereas the Kaplan–Meier method showed a significant correlation between CD25+/CD8+ ratio >1.25 and DFS (p=0.033) (Figure 2, Table III).
Kaplan–Meier curve for the primary outcome according to the stromal CD25+/CD8+ lymphocyte ratio.
Disease-free survival according to prognostic variables (Kaplan–Meier method).
Fisher test showed that persistence of HPV infection at six months correlated significantly with pre-LEEP HPV-16 and/or HPV-18 genotypes (p=0.0201), positivity for CD25 marker (p=0.0017), and adjuvant vaccination (p=0.0173). Moreover, persistence of HPV infection at 18 months after LEEP significantly correlated with smoking habit (p=0.002), CD25 positivity (p=0.0046), and a CD25+/CD8+ ratio >1.25 (p= 0.0278; Table IV).
Risk factors for HPV persistence (Fisher’s exact test).
Discussion
Main findings. This study assessed the predictive role of the stromal CD25+/CD8+ ratio and related immunohisto-chemical parameters in predicting CIN2-3 recurrence and HPV persistence following LEEP conization. Among 72 patients, ten (13.9%) experienced recurrence. Consistently with previous studies (9, 14), positive surgical margins, pre-LEEP infection with HPV-16 or HPV-18, smoking, a higher grade of dysplasia (CIN3), and persistent HPV at six and 18 months were significantly associated with higher recurrence rates. Conversely, adjuvant HPV vaccination significantly reduced recurrence risk.
A novel finding was the prognostic relevance of the CD25+/CD8+ ratio within the stromal infiltrate. A threshold value of 1.25 was identified, with ratios exceeding this value being significantly associated with higher risk of HPV persistence at 18 months. The Kaplan–Meier analysis showed that patients with a higher ratio experienced significantly shorter DFS. Furthermore, the presence of a lympho-monocyte infiltrate consisting of at least 10% CD25+ T cells was significantly associated with the risk of viral infection persistence at six and 18 months.
The role of lympho-monocytic infiltrate quality in affecting the risk of progression from low-grade to high-grade cervical dysplasia, and up to cancer, has been extensively studied in the literature. In fact, several studies showed that an abundant presence of regulatory T cells in the cervical stroma is related with an increased risk of progression (16-21). A similar pattern of regulatory T cell hyperactivation was observed even in peripheral blood samples from patients with CIN3 (22, 23). However, to the best of our knowledge, our study is the first investigation to highlight the relationship between the quality of the lympho-monocytic infiltrate and the risk of CIN2-3 recurrence after LEEP. Indeed, this innovative finding is the main strength of this work.
Weaknesses. The main weakness of our study is the small sample size, with only ten cases of CIN2-3 recurrence during follow-up. The small sample size did not allow us to perform multivariate analysis by Cox regression. Another limitation lies in the way the two pathologists calculated the number of CD25+ and CD8+ cells (i.e., visually). For such a purpose, it would have been interesting to employ automated and digitized methods (e.g., artificial intelligence tools).
Implications. The results of our study have several implications. The main implication is clinical, since a routine assessment of the quality of lympho-monocyte infiltrate on primary cone tissue could provide important information on the follow-up timing. As a matter of fact, the detection of an abundant presence of regulatory T lymphocytes might suggest the need for more frequent checks, thus tailoring the follow-up time according to patient’s features.
Our finding that the quality of the pre-conization lympho-monocyte infiltrate influences the risk of recurrence may suggest that there is a balance between the immune system and the virus such that surgical treatment is unable to completely break it. Consequently, these patients could benefit from additional treatments (e.g. , immunotherapy) that modulate the immune system’s response to the virus, altering such a balance.
Conclusion
This study highlighted the predictive role of the stromal CD25+/CD8+ ratio in patients with CIN2-3 treated with LEEP conization. A CD25+/CD8+ ratio >1.25 was significantly associated with a shorter DFS and HPV persistence at 18 months, underscoring its potential as a biomarker for recurrence risk. Consistently with previous studies, smoking, HPV-16/18 infection, and positive surgical margins emerged as critical recurrence factors, while adjuvant vaccination demonstrated a protective effect.
Our findings support the integration of immune profiling into cervical dysplasia management, thus allowing the identification of patients at higher risk of recurrence and the adoption of a tailored intensive surveillance program. Future studies with larger, multicenter cohorts are warranted to validate these results, refine cutoff values for immune markers, and further investigate their role in optimizing clinical outcomes for high-risk patients.
Footnotes
Authors’ Contributions
Conceptualization: ES, LD; writing-original draft: ES, AF; data curation, formal analysis, methodology: ES, SP, LD; figure development: ES, FDC; writing-review and editing: ES, SP, AF, MDS, FM, FT, SC, AB, AG, TS, PB, AGN, LD.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received February 13, 2025.
- Revision received March 30, 2025.
- Accepted March 31, 2025.
- Copyright © 2025 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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