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Keywords:

  • Cervical cancer;
  • human papillomavirus 18;
  • molecular marker;
  • physical status;
  • risk assessment

Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References

The aim of this work was to assess the value of the physical status of human papillomavirus (HPV) DNA as a disease marker for cervical cancer development in a set of 248 DNA samples previously genotyped as HPV 16 or 18, by calculating the E2/E6 ratio through real-time PCR. There was a significant difference in integration status according to disease grade for both genotypes (p <0.001). Furthermore, especially for HPV 18, determining the DNA physical status could be a useful biomarker in predicting cervical cancer risk development, with a lower E2/E6 ratio clinically associated with the development of a precancerous lesion.

Almost all cervical cancers are related to human papillomavirus (HPV) infection, and HPV 16 and 18 genotypes are responsible for more than 70% of the cases worldwide [1-3]. HPV 18 is more frequently detected in adenocarcinoma and adenosquamous carcinoma, whereas HPV 16 is more associated to squamous cell carcinoma [4, 5]. In Portugal, HPV 16 is the most frequently detected genotype by cytological grade (19.7%); HPV 18 has a low prevalence (4.4%) [6].

Integration of HPV into the host genome is a prerequisite for the development of malignant lesions, resulting from a disruption in the HPV genome through partial loss of the E2 gene [7, 8]. In addition, recent data suggest that integration frequency in premalignant and malignant lesions varies with HPV genotype [5, 9]. Physical status of HPV DNA (episomal, linear or concomitant/mixed forms) has also been considered as a marker of disease progression [10-12]. Absolute quantification of E2 and E6 genes by real-time PCR, followed by calculation of the E2/E6 ratio has been proposed [13], allowing identification of the different forms of the viral DNA. The aim of this study was to investigate these associations regarding HPV 18 in comparison with HPV 16 cases.

The study group comprised 248 frozen DNA aliquots of cervical samples, from women aged 18–65 years (mean age 33.1 ± 9.3 years; median 31 years), genotyped previously as HPV 16 (n = 132) or HPV 18 (n = 116) by CLART HPV2 (Genomica, Madrid, Spain). The cervical samples were collected in ThinPrep PreservCyt medium (CyticUK, Crawley, UK) for cytological analyses. Histological evaluation made on colposcopically directed biopsies was also available (Table 1). The histological result was applied as the reference standard in assessing the predictive value of the physical status in cervical cancer development using the cytological result as baseline.

Table 1. Characteristics of the studied samples
 Cases (%) (n = 248)HPV 16-positive (%) (n = 132)HPV 18-positive (%) (n = 116)
  1. NILM, normal cytology; ASC-US, atypical squamous cells of undetermined significance; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ICC, cervical carcinoma, including adenocarcinoma; CIN, cervical intraepithelial neoplasia grade 1–3.

Cytology
NILM79 (31.8)42 (31.8)37 (31.9)
ASC-US56 (22.5)26 (19.7)30 (25.9)
LSIL66 (26.6)35 (26.5)31 (26.7)
HSIL41 (16.5)27 (20.5)14 (12.1)
ICC6 (2.4)2 (1.5)4 (3.4)
Histology
Normal30 (12.1)30 (22.7)0 (0.0)
CIN197 (39.1)47 (35.6)51 (44.0)
CIN299 (39.9)44 (33.3)54 (46.6)
CIN316 (6.5)9 (6.8)7 (6.0)
ICC6 (2.4)2 (1.5)4 (3.4)

The physical status of HPV 16 and 18 DNA was assessed by real-time PCR amplification of E2 and E6 genes, as described previously [14, 15]. Real-time PCR was carried out on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). For each sample, absolute quantification of E2 and E6 genes was performed in duplicate, simultaneously in the same plate.

Mann–Whitney and Kruskal–Wallis non-parametric methods were used to discriminate differences in DNA physical status among the different cytological or histological categories. Student's t-test analysis was performed to measure the difference between the mean E2/E6 ratios for both genotypes through the clinical diagnosis categories. A p value ≤0.05 (two-sided) was considered statistically significant. All analyses were performed using the IBM SPSS Statistics version 20.0 (IBM Corporation Inc., Armonk, NY, USA).

Among the HPV 16-positive samples, 40.2% (53/132) were episomal, 58.3% (77/132) presented concomitant forms, and 1.5% (2/132) were in the linear form. The episomal forms were more frequent in normal cytologies, whereas the concomitant forms increased according to disease grade (p 0.011; Fig. 1a). An increasing proportion of concomitant forms was identified from normal histology through to cervical intraepithelial neoplasia (CIN) grade 3 lesions, ranging from 33.3% (10/30) to 88.9% (8/9), respectively (p <0.001; Fig. 1b). Concerning the E2/E6 ratio, no significant difference was found between normal histologies and CIN2 cases (0.959 ± 0.112) (p <0.001; Fig. 2a).

image

Figure 1. Physical status of viral DNA from (a) human papillomavirus (HPV) type 16 according to cytology; (b) HPV 16 according to histology; (c) HPV 18 according to cytology; and (d) HPV 18 according to histology. Data are presented with standard error bars. NILM, normal cytology; ASC-US, atypical squamous cells of undetermined significance; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ICC, cervical carcinoma, including adenocarcinoma; CIN, cervical intraepithelial neoplasia grade 1–3.

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image

Figure 2. Comparison of human papillomavirus (HPV) type 16 (a) and HPV 18 (b) E2/E6 ratio according to the different histological categories. Data are presented as T-plots, representing the 5th and 95th centiles. Normal, normal histology; CIN, cervical intraepithelial neoplasia grade 1–3; ICC, cervical carcinoma, including adenocarcinoma.

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Among the HPV 18-positive cases, 13.8% (16/116) were episomal, 65.5% (76/116) presented concomitant forms, and 20.7% (24/116) were in the linear state. Cytologically, the episomal forms were detected in 15% of the cases, whereas the concomitant form was the most frequent in every lesion group (>60.0%). An increasing proportion of linear forms was related to the severity of the lesion grade (p 0.029; Fig. 1c). The proportion of concomitant forms decreased from 72.5% (37/51) to 28.6% (2/7) in CIN1 and CIN3 lesions, respectively, whereas the proportion of linear forms increased from 24.1% (13/54) in CIN2 lesions, to 71.4% (5/7) in CIN3 lesions, and 100% (4/4) in adenocarcinomas (ICC) (p <0.001; Fig. 1d). Considering the E2/E6 ratio, there was a significant linear decrease in the value of the ratio associated with the severity of lesion grade (0.054 ± 0.112; p <0.001) (Fig. 2b).

The percentage of concomitant and linear forms (72.2%; 179/248) is consistent with other studies [8, 13, 14]. As expected, especially for HPV 16, our data showed that integration may be an early event during carcinogenesis, as concomitant forms were the most frequently detected [8, 12, 13, 16-18]. Yet, the fact that even in CIN2 cases episomal forms were detected could indicate that apart from HPV 16 integration, other mechanisms might lead to carcinogenesis. Considering the mean E2/E6 ratio in the histological diagnosis, values were similar from normal histology to CIN2 lesions, and only in CIN3 lesions the mean E2/E6 ratio was considerably lower.

Previous studies determined that the frequency rate of exclusively episomal forms of HPV 18 ranged from 9.8% to 36.7% in ICC [18, 19]. However, in our study no episomal form was present in adenocarcinomas, suggesting a more aggressive biological potential of this genotype, as previously stated [5]. In fact, we observed a relation between HPV 18 E2/E6 ratio and disease grade, as shown by a shift from the exclusively episomal form to the exclusively linear form according to the severity of the lesion.

Regarding the natural history of HPV, it is important to manage HPV 16-positive or HPV 18-positive women by improving diagnosis and taking into account type-specific characteristics associated with malignant transformation [5, 12]. Considering the systematic analysis proposed by Saunier et al. [8], in which a cut-off value for E2/E6 ratio lower than 0.520 represents a higher risk for the development of high-grade lesions, in this study, 16.7% (22/132) of HPV 16-positive and 55.2% (64/116) of HPV 18-positive women were at risk.

This study has some limitations. First, it is a cross-sectional study, precluding the determination of the longitudinal clinical relevance of this biomarker. Also, the low number of high-grade lesions included in this study shows the need for further studies to achieve significance in women with cervical cancer. Further evaluation of the determination of HPV 18 physical status as a predictive biomarker must be addressed. Nonetheless, HPV physical status, especially in women infected with HPV 18, seems to be important in patient management and cervical cancer prevention, helping to distinguish the clinical relevance of HPV infection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References

AGO is grateful to Fundação para a Ciência e Tecnologia for a PhD studentship. The authors are very grateful to Rogério Tenreiro, PhD, at the Faculty of Sciences in the University of Lisbon for providing reagents to study the physical status.

Transparency Declaration

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References

The authors declare that there are no conflicts of interest.

References

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References