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Analysis of HPV-16 viral load, integration status, and p16 expression in relation to Ebv co-infection and cervical…

Abstract

Cervical cancer, one of the most common cancers in women, is primarily driven by high-risk human papillomaviruses (HPV) infections, particularly HPV-16. Co-infection with Epstein-Barr virus (EBV) has been reported to exacerbate disease progression by influencing HPV genome integration. This study examines HPV-16 integration status, p16INK4a expression, and their relationship with EBV co-infection and viral load in cervical cancer cases. In this study, 134 HPV-16-positive formalin-fixed, paraffin-embedded cervical samples were collected and analyzed for HPV-16 viral load, genome integration and EBV co-infection, followed by p16INK4a immunohistochemistry. Statistical analysis was performed to examine the association between viral markers and cervical cancer progression. HPV-16 viral loads varied significantly by histological grade, with the highest loads observed in cervical intraepithelial neoplasia 2 (CIN 2) lesions. HPV integration status revealed episomal forms in 32.8% of samples, mixed forms in 56%, and fully integrated forms in 11.2%. p16INK4a expression correlated with disease progression, increasing with CIN grade and in squamous cell carcinoma (SCC). EBV was detected in 13.4% of samples, but no significant associations were found between EBV infection and HPV integration, viral load, or p16INK4a expression levels. HPV-16 viral load and integration status are strongly associated with cervical lesion severity, while p16INK4a expression increases with lesion grade, indicating its utility as a diagnostic marker. EBV co-infection did not significantly impact lesion progression, suggesting that its role in cervical cancer remains unclear.

Introduction

Cervical cancer is the fourth most common cancer in females around the world, with 85–90% cases occurring in low- and middle-income countries1. The incidence of cervical cancer is increasing in some regions, with approximately 500,000 women affected by this tumor each year. It is estimated that 260,000 people die from cervical cancer annually2. Recent studies indicate a slight decline in the age-standardized incidence rate (ASR) of cervical cancer in Asia, with South and Southeast Asia showing the most significant decreases from 1990 to 20193. In Iran, the ASR is approximately 1.90 per 100,000 population, with a median age of around 52 years4 Each year, approximately 1,056 women in Iran are diagnosed with cervical cancer5. The most common risk factor for cervical cancer is infection with high-risk human papillomaviruses (HR-HPVs), with HPV-16 responsible for 50–60% of cases depending on the geographic region6,7,8,9. In addition to HPV infection, the progression of cervical lesions also requires other host or environmental factors, including: tobacco smoking (TS), oral contraceptives, and immunosuppression, especially human immunodeficiency virus (HIV) infection10. The co-occurrence of Epstein-Barr virus (EBV) and HPV in cervical cancer has been reported in many studies11,12, although the role of EBV/HPV co-infection in its pathogenesis of cervical cancer remains unclear10.

EBV is a gamma herpesvirus that infects more than 90% of the population worldwide. EBV is associated with several B-cell-derived malignancies such as Burkitt’s lymphoma (BL) and Hodgkin’s disease (HD). Importantly, EBV is associated with several epithelial tumors, including a subset of anaplastic nasopharyngeal carcinoma (NPC) and gastric cancer (GC)10,13. Additionally, EBV has been reported to be associated with colorectal and brain cancer14,15. EBV is detected in less than 16.7% of normal and non-neoplastic cervical samples10, but several studies have reported an increased frequency of EBV detection in cervical cancer, ranging from 27.8–100%10,16. The co-infection of EBV and HR-HPV in squamous intraepithelial lesions (SIL) and cervical cancer ranges from 12.7–81.8%17,18. Moreover, EBV is often associated with HPV16 and HPV1819, potentially increasing the risk of HPV16 integrating into the host genome11,18. Additionally, HPV+/EBV + cervical cancers show increased methylation of the RB1 and E-cadherin (CDH1) gene promoters compared to HPV+/EBV − tumors20. In patients infected with HIV and HR-HPV, additional EBV infection significantly increases the risk of SIL compared to uninfected women21.

Papillomaviruses typically enter through basal epithelial cells and are usually episomal in low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL)22. Although integration of the HR-HPV genome into the host chromosomes is an important outcome, it is not a prerequisite for epithelial carcinogenesis23,24. This phenomenon is more commonly observed in HSIL and cervical squamous cell carcinoma (SCC) compared to normal or LSIL tissue25. Furthermore, integration of HR-HPVs (HPV-16 and HPV-18) into HSIL is often accompanied by chromosomal aberrations26, and the loss of the E2 region during integration leads to constitutive expression of the E6 and E7 proteins27. These proteins contribute to the immortalization of primary epithelial cells by inhibiting tumor suppressors P53 and Rb28. Overall, the E2/E6 ratio is the useful factors for predicting viral genome integration29. However, other factors, such as genetic changes and time since the initial infection, also play a role in cancer progression30. Premalignant lesions of the cervix, including LSIL and HSIL, have varying rates of progression to invasive cancer. It is estimated that 1–2% of women with HSIL or cervical intraepithelial neoplasia 2 (CIN 2) will progress to CIN 3 or invasive cancer, with progression rates of approximately 20% and 5%, respectively31.

The p16 protein (p16INK4a) of the CDKN2A gene is a tumor suppressor protein that inhibits cyclin-dependent kinases 4 and 6. Although p16INK4a acts as a down regulator of cell proliferation, this effect is ineffective in the context of proliferating cells infected with HR-HPV32. Therefore, p16INK4a may serve as a sensitive surrogate marker for such HR-HPV infections. Expression of p16INK4a causes negative feedback regulation of pRb protein, thereby increasing p16INK4a levels33. Inactivation of pRb by HPV E7 proteins can lead to increased expression of p16INK4a as a cellular response to cell cycle dysregulation34. Evidence suggests that p16INK4a expression is observed in HPV-transformed cell lines, cervical cancer, and HSIL. While studies have confirmed overexpression of p16INK4a in nearly all HSILs, p16INK4a expression is rarely observed in normal squamous epithelium. In metaplastic epithelium, lower levels of expression are typically seen. High expression of p16INK4a is closely associated with lesions infected with high-risk HPV types but not low-risk HPV types33.

Several studies have suggested that the integration status of the viral genome and expression levels of p16INK4A could serve as a diagnostic marker for cervical cancer progression35,36,37,38,39, but there are controversial data in this field40,41,42,43. Therefore, we conducted this study to investigate the role of HPV integration status and p16INK4A expression level as potential markers of disease progression and their relationship with EBV co-infection and viral loads in HPV-16 infected cervical specimens.

Materials and methods

Sample collection

In this retrospective study, formalin-fixed, paraffin-embedded (FFPE) cervical samples were collected from women referred to the pathology department of Yas Hospital in Tehran. These samples were submitted for analysis due to abnormal cytology or histopathology results, and a pathologist conducted the cytological examination. The pathologist utilized the World Health Organization (WHO) classification system for tumors of the female genital tract to categorize various histological subtypes of the cervix, encompassing CIN 1, 2, 3, and SCC44. Sections of the samples were used for DNA extraction, using the FavorPrep Tissue Genomic DNA Extraction Mini Kit (Favorgen, Biotech Corp, Taiwan) according to the manufacturer’s instructions. Additionally, the AMPLIQUALITY HPV-TYPE EXPRESS v3.0 method (Catalog No. 03–35 A-20 M AB, Analitica, Italy) was employed for HPV detection and typing. This method utilizes a Single-Step PCR to amplify the L1 viral region, followed by a Reverse Line Blot assay. It can identify 40 different HPV types, including 6, 11, 16, 18, and others. Samples with multiple infection with other HPV types were excluded, and only HPV-16 positive samples were included in the study. One hundred and thirty four HPV-16 positive samples were analyzed for integration evaluation. The histopathological diagnosis for these samples included 29 patients with CIN grade 1, 28 patients with CIN 2, 29 patients with CIN 3, 29 patients with cervical cancer and 19 healthy individuals in the control group. The reason for performing hysterectomies on 19 patients with normal tissue pathology was abnormal uterine bleeding caused by uterine fibroids. All methods were performed in accordance with the relevant guidelines, regulations of the institutional ethical committee of the ethics committee of Shiraz university of medical sciences (SUMS) and in accordance with the Declaration of Helsinki. Informed written consent was obtained from each participant, and the study was approved by the Ethics Committee of SUMS (IR. SUMS. REC. 1400.601).

Isolation of DNA from paraffin-embedded tissues

Before genome DNA extraction, deparaffinization was performed by incubating the samples in 1 ml of xylene for 30 min at room temperature. DNA was then extracted using the FavorPrep Tissue Genomic DNA Extraction Mini Kit according to the manufacturer’s instructions (Favorgen, Biotech Corp, Taiwan). The quality of extracted DNA was assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). Beta-globin was used as a control for genome DNA extraction and Real-time PCR assays. Eluted DNA was stored at − 70 °C until further use.

Real-time PCR assays

For the detection of HPV-16 E2 and E6 genes, TaqMan Real-time PCR amplification was performed. The reaction mixture contained 12.5 µl of 2× master mix (Pishgam, Tehran, Iran), 1 µl (10 pmol) of each forward and reverse primer, 0.5 µl (5 pmol) of probe, and 5 µl (50 ng) of each sample or control, making a total volume of 25 µl. Table 1 provides the sequences of all primers and probes used in this study. The temperature program for the HPV-16 E2 gene was as follows: 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s, 50 °C for 40 s, and 72 °C for 5 s, with data acquisition performed during the extension step. For the E6 gene, the temperature program was: 95 °C for 15 min, followed by 40 cycles of 95 °C for 20 s, 56 °C for 30 s, and 72 °C for 20 s, with data acquisition during the extension phase.

Table 1 Primer and probe sequences that were used in this study.

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The E2/E6 ratio was used to predict the viral genome integration status. An E2/E6 ratio of 0 indicates a “fully integrated state,” whereas a ratio between 0 and 1 indicates a “mixed state.” A ratio of 1 indicates an “episomal state.” The episomal state suggests that HPV has not integrated into the host genome and contains equal amounts of E2 and E6. In contrast, the “fully integrated state” leads to E2 loss, resulting in an E2/E6 ratio close to zero45.

For detecting the Epstein-Barr nuclear antigen-1 (EBNA-1) gene of EBV, the temperature program was as follows: 95 °C for 15 min, followed by 40 cycles of 95 °C for 20s, 57 °C for 30s, and 72 °C for 20s. Fluorescent signal acquisition was performed during the extension step.

For β-globin amplification and quantification, the reaction mixture consisted of Ampliqon RealQ Plus 2x Master Mix for Probe High Rox, with 0.16 µM of probe and 0.4 µM of each primer, in a final volume of 25 µL. The PCR cycling conditions were as follows: initial denaturation at 95 °C for 15 min, followed by 35 cycles of 95 °C for 20 s, 60 °C for 20 s for annealing, and 72 °C for 20 s for elongation.

Standard curves and detection limit of qPCR assays

Amplicons of the E2, E6, EBNA-1, and b-globin gene regions were cloned into the pTG19-T vector using the PCR TA Cloning Kit (SinaClon, Tehran, Iran) according to the manufacturer’s protocol. The quantification of the plasmids was performed using a NanoDropTM spectrophotometer (NanoDropTM 2000; Thermo Scientific). Based on the plasmid size and concentration, the absolute copy number of DNA in each sample was calculated. Standard curves were generated from 10-fold serial dilution of each plasmid. The detection limit for each test was determined by identifying the final dilution where the fluorescent signal showed exponential amplification. A PCR Ct value of ≤ 40 was used as the threshold for this determination.

For cell quantification, a standard curve of the β-globin gene plasmid, ranging from 3*10^5 to 30 copies per reaction, was used. Standard curves for E2, E6, and EBNA-1, created from tenfold serial dilutions (ranging from 3.9*10^7 to 30 copies for E2, 3.9*10^6 to 30 copies for E6, and 3.9*10^6 to 30 copies for EBNA-1), were employed to quantify the HPV and EBV genomes in each sample, with a background of 200 ng of human DNA per reaction. The genomic copies of HPV and EBV were normalized by calculating the ratio of viral copy numbers to half the β-globin gene copy numbers, thereby determining the absolute copy number of HPV and EBV DNA per cell.

p16INK4a immunohistochemistry

Immunohistochemical staining was performed on 5 μm thick FFPE tissue sections. Slides were deparaffinized twice for 30 min using xylene, and rehydration was achieved by gradually adding ethanol solution to Tris-buffered saline (TBS). Epitope recovery was performed by heat-induced microwave treatment in 10 mM citrate buffer (pH 6.0). Endogenous peroxidase activity was abolished by 10% H2O2. A protein blocker (Dako, Glostrup, Denmark) was used to block nonspecific binding sites. Slides were incubated with a monoclonal antibody against human p16 (1: 50; BD Pharmingen™; BD Biosciences, Franklin Lakes, NJ, USA) for 1 h at room temperature in a humidified chamber, followed by incubation in the EnVision + Dual Link System. HRP solution (Dako, Glostrup, Denmark) was used for 30 min in the presence of hydrogen peroxide, and 3,3′-Diaminobenzidine (DAB) was used as the chromogen. Nuclei were counterstained with Mayer’s hematoxylin. Sections without the primary antibody served as negative controls in each run. Formalin-fixed HeLa cell (national cell bank of Iran code: C115, Pasteur Institute, Tehran, Iran) block sections, known to react positively to p16 immunostaining, were used as positive controls. The interpretation of p16 immunostaining was carried out as described elsewhere32. For analysis, the 4-level scoring system was simplified into binary categories: scores of 0 and 1 + were classified as negative, while scores of 2 + and 3 + were classified as positive.

Statistical analysis

Statistical analysis was performed using SPSS software version 22 (SPSS Inc., USA). Odds ratios and 95% confidence intervals for cytogenetic abnormalities were calculated, and p values lower than 0.05 were considered statistically significant. Additionally, frequency counts and differences between continuous variables were calculated using the χ2 test and Anova test, respectively.

Result

Sample characterization

The age of women in the study ranged from 19 to 79 years, with a median age of 36 years. The age distribution was stratified according to pathological diagnoses, as illustrated in Fig. 1A. A total of 134 cervical tissue samples with HPV16 single infection were eligible for the study, consisting of 19 (14.17%) cases of normal cervical tissue, 29 (21.64%) cases of CIN1, 28 (20.89%) cases of CIN II, 29 (21.64%) cases of CIN III, and 29 (21.64%) cases of SCC. The clinicopathologic features of the study population are summarized in Table 2.

Fig. 1

figure 1

HPV viral load and Age distribution among different histological grades of cervical lesions. (A) Age distribution of patients in relation to the physical state of HPV-16. CIN, cervical intraepithelial neoplasia, SCC, squamous cell carcinoma. (B) Mean HPV-16 viral load in normal squamous epithelium and different histological grades of cervical lesions.

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Table 2 Age distribution, HPV-16 viral load, virus DNA integration status, P16INK4a expression and EBV infection according to pathological diagnosis.

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HPV-16 viral load

The viral load reported for HPV-16 was determined by measuring E6 DNA copies and was reported as copies per 1000 cells. The viral load for each group was reported as the mean ± standard deviation. The mean viral loads were as follow: normal (367 ± 540), CIN I (7085 ± 15229), CIN II (25655 ± 102624), CIN III (4824 ± 8803), and SCC (8497 ± 17416). Based on the results of the Kruskal-Wallis test, there was a statistically significant difference in HPV viral load between patients with different histological grades (P = 0.005). Pairwise comparisons revealed that the difference was significant only between patients with normal tissue and those with CIN II (P < 0.005), as shown in Table 2 (Fig. 1B).

E2/E6 ratio in normal squamous epithelium and different histological grades of cervical lesions

The E2/E6 ratio was used as a predictive factor for viral genome integration status. As shown in Fig. 2A, a significant difference in the mean E2/E6 ratios was observed across the five groups (P < 0.05). No significant difference was found between the normal tissue group and the CIN I and CIN II groups in terms of mean E2/E6 ratios (mean, 0.75 for both) (*P* > 0.05). However, the E2/E6 ratios in CIN III and SCC specimens were significantly lower than those in normal tissue (mean, 0.15) and CIN I and CIN II specimens (mean, 0.2) (P < 0.05) (Fig. 2A).

Fig. 2

figure 2

E2/E6 ration in HPV-16 positive cases and frequency of HPV-16 genome status in normal squamous epithelium and different histological grades of cervical lesions. (A) E2/E6 ration in HPV-16 positive normal squamous epithelium and different histological grades of cervical lesions. (B) Frequency of HPV-16 genome status in normal squamous epithelium and different histological grades of cervical lesions. CIN, cervical intraepithelial neoplasia, SCC, squamous cell carcinoma.

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Physical state of HPV integration in histological samples

Analysis of the physical state of HPV integration in 134 samples revealed the following distribution: 32.8% episomal, 56.0% mixed, and 11.2% fully integrated forms (Fig. 1A). Episomal forms were detected in 36.8% (7 of 19) of patients with normal pathology, 41.4% (12 of 29) of CIN I, 50% (14 of 28) of CIN II, 24.1% (7 of 29) of CIN III, and 13.8% (4 of 29) of SCC. Fully integrated HPV was found in 10.5% (2 of 19) of normal cases, 6.9% (2 of 29) of CIN I, 14.3% (4 of 28) of CIN II, 6.9% (2 of 29) of CIN III, and 17.2% (5 of 29) of SCC. Furthermore, the mixed form of HPV was observed in 52.6% (10 of 19) of normal histology, 51.7% (15 of 29) of CIN I, 35.7% (10 of 28) of CIN II, and 69% (20 of 29) in both CIN III and SCC (Fig. 2B). No statistically significant association was found between the grade of cervical lesion and HPV integration status (p = 0.1).

Association between viral load and integration status of HPV-16

The distribution of viral load across the various categories of integration status is shown in Table 3. The median viral load in the mixed DNA group was higher than in the episomal and fully integrated DNA groups. The difference between the three groups was statistically significant, as determined by the Independent-Samples Kruskal-Wallis test (P < 0.001).

Table 3 The association between HPV-16 DNA integration status with HPV-16 viral load, P16 expression level, and EBV infection.

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Analysis of P16INK4a expression in relation to CIN grade

Fifty-eight samples were analyzed for the expression of p16INK4a using IHC test. Eight samples were from normal squamous epithelium, 8 from CIN I, 11 from CIN II, 9 from CIN III, and 10 from SCC. p16INK4a expression levels were classified as zero (no expression), one (low expression), two (moderate expression), and three (high expression).

p16INK4a antigen expression was not observed in any of the normal squamous epithelium specimens. Low expression was observed in 12.5% (1 of 8) of CIN I and 72.7% (8 of 11) of CIN II. In CIN III, low expression was observed in 11.1% (1 of 9), moderate expression in 77.8% (7 of 9), and high expression in 11.1% (1 of 9). In SCC samples, low expression was observed in 10% (1 of 10), moderate expression in 20% (2 of 10), and high expression in 70% (7 of 10) (Fig. 3). The level of reactivity correlated with CIN grade (chi-square test for trend, p = 0.0001). The results showed that the expression of p16INK4a has a direct and strong relationship with the lesion progression; as the lesion progresses, the level of p16INK4a expression increases (Table 2).

Fig. 3

figure 3

Representative hematoxylin and eosin (H&E) staining and immunohistochemical analysis of P16INK4a expression in normal squamous epithelium and different histological grades of cervical lesions. (A) Normal epithelium, negative. (B) CIN 1 negative. (C) CIN 2, weak expression of pl6INK4a, (D) CIN 3 and (E) invasive cancer, strong nuclear and cytoplasmatic expression of p16INK4a. CIN, cervical intraepithelial neoplasia, SCC, squamous cell carcinoma (100X magnification).

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Association between P16 antigen expression levels and HPV-16 viral load

The analysis of the relationship between P16 antigen expression and viral load showed that in samples with moderate expression exhibited significantly higher viral loads compared to those with low or no expression (P < 0.05). However, the difference was not statistically significant when EBV infection was compared across all groups (*P* > 0.05), as shown in Table 4.

Table 4 Association between P16 expression levels with HPV-16 viral load and EBV infection.

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Association between P16 expression levels and HPV-16 integration status

The results regarding the relationship between P16 expression and HPV-16 integration status showed that the majority of samples with an episomal HPV-16 status exhibited low to no P16 expression, with a predominant presence of no expression (61.1%). In other words, the episomal form of HPV-16 tended to be associated with lower P16 expression levels. The integrated HPV-16 form, however, exhibited a more heterogeneous distribution of P16 expression, with a notable proportion showing high expression, suggesting that HPV integration alone did not consistently correlate with increased P16 expression. The mixed HPV-16 form was associated with higher P16 expression, with a significant proportion of samples exhibiting moderate to high levels of P16. This suggested that the combination of episomal and integrated HPV-16 forms might have been linked to higher P16 expression levels. The statistically significant difference (P < 0.007) in P16 expression levels between the various HPV-16 integration statuses indicated that integration status played a role in determining P16 expression levels. These findings were summarized in Table 3.

Prevalence of EBV infection in cervical samples and association with lesion severity

The results of real-time PCR showed that EBV EBNA1 gene sequences were detected in 18 (13.4%) of the cervical samples (Table 2). The EBV viral load for each group was reported as the mean ± standard deviation. The mean EBV viral loads were as follow: normal (205.33 ± 125.29), CIN I (36.75 ± 33.81), CIN II (1230 ± 1351), CIN III (2383.25 ± 2446.61), and SCC (1575.33 ± 2534.33) (Fig. 4). Statistical analysis indicated that there is no significant association between the prevalence of EBV infection and different pathological grades of the disease (P = 0.905) (Table 2).

Fig. 4

figure 4

Mean EBV viral load in normal squamous epithelium and different histological grades of cervical lesions. CIN, cervical intraepithelial neoplasia, SCC, squamous cell carcinoma.

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Association between EBV infection with HPV integration status, HPV viral load, and P16 expression levels

The results of the Pearson chi-square analysis showed no statistically significant relationship between EBV infection and the expression level of p16 (P = 0.54) or HPV viral load (P = 0.09). However, a significant association was observed between EBV infection and HPV integration status (P = 0.034), with a higher proportion of EBV-positive cases in the episomal HPV integration status group, as shown in Tables 3 and 4.

Discussion

This study investigates the relationship between HPV-16 viral load, integration status, and p16INK4a expression in cervical tissue samples of varying histological grades, along with the presence of EBV co-infection. A key strength of this study is its focused examination of HPV-16, eliminating the confounding effects of other high-risk and low-risk HPV types. To ensure this focus, all samples with concurrent infections of different HPV types were excluded, and only samples with a single HPV-16 infection were included in the analysis. The results contribute to understanding the complex interplay between these factors in the progression of cervical neoplasia and cancer.

We found that the HPV-16 viral load is significantly associated with the severity of cervical lesions. Research indicates that higher HPV-16 oncoprotein expression correlates with advanced cervical lesions, particularly in the context of E6 expression, which shows a significant association with histological grade46. Additionally, a study found that HPV-DNA load was markedly higher in patients with SCC compared to those with CIN and normal cervix, suggesting a direct relationship between viral load and lesion severity47. Furthermore, a retrospective analysis demonstrated that high-risk HPV viral load is an independent risk factor for developing high-grade cervical lesions, with odds ratios increasing significantly from CIN I to cervical cancer48. Notably, HPV-16 load serves as a potential biomarker for predicting high-grade lesions, with a specific cut-off indicating increased risk49.

The analysis of HPV-16 viral load across different histological grades of cervical lesions indicates a significant increase from normal tissue to CIN III and SCC, suggesting a correlation between higher viral loads and advanced disease stages. Specifically, the findings align with previous research that demonstrates elevated HPV-16 viral loads in CIN III compared to normal tissue, reinforcing the notion that higher viral loads may facilitate the progression of cervical neoplasia50. However, the lack of a significant difference in viral load between CIN III and SCC raises questions about the role of viral load in the transition from high-grade lesions to invasive cancer, indicating that other factors may also contribute to this progression. This complexity highlights the need for further investigation into the interplay between viral load and other biological mechanisms in cervical cancer development50.

HPV integration into the host genome is indeed a pivotal event in cervical carcinogenesis, leading to the disruption of the E2 gene and the unchecked expression of the oncogenes E6 and E7. Research indicates that the integration status of HPV-16 varies significantly with cervical lesion grades, showing a higher prevalence of integrated forms in CIN III and SCC51. The transition from episomal to integrated forms as lesions progress supports the notion that integration is a hallmark of cervical cancer development52. However, the presence of episomal forms in lower-grade lesions suggests that integration is not strictly necessary for disease progression, highlighting the complexity and heterogeneity of cervical cancer pathogenesis53. Furthermore, while integration often leads to the loss of E2, which is critical for regulating E6 and E7 expression, some tumors can still exhibit oncogenic activity without integration, primarily through episomal mechanisms54. This underscores the multifaceted nature of HPV-related carcinogenesis. In this study, the integration status of HPV-16 varied significantly across different cervical lesion grades, with a higher prevalence of mixed and integrated forms in CIN III and SCC. The observed shift from episomal to integrated and mixed forms as the disease progresses corroborates existing literature suggesting that integration is a hallmark of cervical carcinogenesis. The presence of episomal forms in lower-grade lesions and even in some high-grade lesions indicates that HPV integration is not a prerequisite for progression, highlighting the heterogeneity of cervical cancer pathogenesis.

The expression levels of p16INK4a correlate with the degree of cervical neoplasia, reinforcing its utility in identifying lesions that are likely to progress to invasive stages55. In studies evaluating various cervical pathology specimens, high p16INK4a expression was consistently associated with high-grade CIN and invasive carcinomas, demonstrating a strong predictive value for progression56. These findings support the argument that immunohistochemical analysis of p16INK4a could serve not only as a critical diagnostic tool but also as a potential screening test for high-risk HPV infections55. The current findings indicate that p16INK4a expression correlates with HPV-16 integration and is more prominent in higher-grade lesions, reinforcing its role as a surrogate marker for HPV-associated cervical neoplasia. The increased expression of p16INK4a in CIN III and SCC compared to lower-grade lesions and normal tissue supports its use as a diagnostic marker for identifying high-risk lesions that may progress to invasive cancer.

EBV appears to have a complex and somewhat ambiguous role in the context of HPV infections and cervical cancer. While HPV, particularly high-risk types like HPV-16 and HPV-18, is well-established as a primary cause of cervical cancer, the contribution of EBV remains less clear. Studies indicate that EBV co-infection with HR-HPV may increase the risk of cervical abnormalities, with one study reporting a 7.86-fold increased risk of ASCUS-type lesions in co-infected women57. Some studies report that EBV is frequently detected in cervical tissues, but its presence does not consistently correlate with increased cancer risk. For instance, one study found that higher EBV viral loads were present in control groups compared to those with CIN or SCC, suggesting a potential “hit and run” mechanism where EBV may initiate oncogenic processes but does not play a sustained role in cancer progression58. Moreover, a systematic review indicated a significant association between EBV infection and cervical carcinoma, with odds ratios suggesting a potential role in carcinogenesis59. However, other studies have shown that while EBV co-infection with HPV is common, it does not significantly impact the clinical features or prognosis of cervical cancer patients60,61. This inconsistency highlights the need for further research to clarify the interactions between EBV and HPV in cervical cancer development and to explore potential therapeutic implications62. Our study also explored the impact of EBV co-infection on cervical lesion progression. However, no statistically significant association between EBV presence and the histological grade of the lesions was observed, suggesting that EBV may not play a major role in the progression of HPV-related cervical lesions, at least within the context of this study population. Further research is needed to elucidate the potential synergistic effects of HPV and EBV co-infection in cervical carcinogenesis.

This study which focuses on HPV-16-positive samples limits the generalizability of the findings to other high-risk HPV types. While PCR is effective for detecting EBV and HPV, it does not provide information on their tissue localization. As a result, PCR can confirm co-infection but cannot reveal the specific interactions or mechanisms by which EBV may influence HPV-related carcinogenesis. Future studies should include larger, prospective cohorts and explore the role of other high-risk HPV types and co-infections with other high-risk HPV types or viruses, such as EBV, in cervical carcinogenesis. Moreover, investigating the molecular mechanisms underlying the interaction between HPV integration, viral load, p16INK4a expression, and host factors could provide deeper insights into cervical cancer pathogenesis.

Conclusion

This study highlights the complex interplay between HPV-16 viral load, integration status, p16INK4a expression, and EBV co-infection in the progression of cervical lesions. The findings suggest that while HPV-16 viral load and integration status are important markers of disease progression, the role of EBV co-infection remains unclear. The significant association between HPV-16 viral load, integration status, and p16INK4a expression with cervical lesion grade underscores the potential of these biomarkers in risk stratification and management of patients with cervical neoplasia. Monitoring HPV-16 viral load and integration status, along with p16INK4a expression, could aid in identifying patients at higher risk for progression to invasive cancer, thereby informing treatment decisions and follow-up strategies. The study reinforces the utility of p16INK4a as a biomarker for high-risk lesions and underscores the need for further research to unravel the molecular mechanisms driving cervical cancer development.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The current study was extracted from PhD thesis written by Azam Khamseh which was funded by Shiraz University of Medical Sciences (Grant No. 98-21986).

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Authors and Affiliations

Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Science, Shiraz, Iran

Azam Khamseh, Saied Ghorbani & Jamal Sarvari

Research Center for Clinical Virology, Tehran University of Medical Sciences, Tehran, Iran

Azam Khamseh & Seyed Mohammad Jazayeri

Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

Ali Farhadi

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

Ali Farhadi

Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Somayeh Jalilvand & Seyed Mohammad Jazayeri

Department of Obstetrics and Gynecology, Yas Hospital, Tehran, Iran

Fariba Yarandi

Department of Pathology, Yas Hospital, Tehran University of Medical Sciences, Tehran, Iran

Narges Izadi-Mood

Department of Epidemiology and Biostatistics, School of Health, North Khorasan University of Medical Sciences, Bojnurd, Iran

Hassan Saadati

Yas Hospital, Tehran University of Medical Sciences, Tehran, Iran

Elham Shirali

Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Jamal Sarvari

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Azam Khamseh

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