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Quantitative analysis of human papillomavirus type 16 in cervical neoplasm: A study in Chinese population

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Abstract

Background:

Human papillomavirus (HPV) infection was recognized as a major causal factor for the development and progression of squamous intraepithelial lesions (SIL). It is possible to use HPV test for the detection of cervical lesions as an adjunct to cervical cytology.

Objectives:

To evaluate the relation between HPV 16 viral load and the severity of cervical lesions in a Chinese population.

Methods:

Study population was recruited from the colposcopy and general outpatient clinic. The presence of HPV 16 E6 and E7 in cytological specimens was detected using HPV 16 specific polymerase chain reaction (PCR). The viral load in the specimens that were positive for HPV 16 specific PCR, was quantified by using real-time PCR assay.

Results:

The study recruited 394 women, in which 148 were high-grade SIL (HG-L), 121 were low-grade SIL (LG-L) and 125 were Normal. Sufficient DNA integrity was proven in 347 samples. Among 121 positive cases for HPV 16, 70 were HG-L, 34 were LG-L and 17 were Normal. Using quantitative real-time PCR, the percentages of samples with greater DNA copies were found to increase with the severity of diseases. There was also a significant difference in DNA copies among the three groups (HG-L versus Normal, p < 0.001; HG-L versus LG-L, p < 0.001). Area under receiver operating characteristic (ROC) curve of the HG-L versus LG-L and Normal was 0.836 indicating that quantitative PCR had a good diagnostic value in differentiating HG-L from the LG-L and Normal groups.

Conclusions:

Our data suggested HPV 16 viral load was significantly related to the severity of cervical lesions. Evaluation of viral burden could be a potential clinical tool in management of cervical lesions.

Introduction

Although cervical screening programmes have dramatically reduced the incidence of cervical cancer, 50% of invasive cervical cancers arise in women screened with existing cytological methodologies due to some inherent limitations (Cuzick et al., 1998). The sensitivity of cytology for high-grade squamous intraepithelial lesion (SIL) was only 40% possibly due to sampling or interpretation errors (Cuzick et al., 1995). On the other hand, over-reading of high-grade SIL leads to unnecessary colposcopic examinations and over-treatment. Various researchers have suggested for additional methods to improve the accuracy of smear, especially in distinction between low-grade and high-grade disease. In late 1970s, the hypothesis that human papillomavirus (HPV) were related to cervical cancers was proposed and throughout 1980s gained rapid support. Those positive for HPV DNA have a risk of developing cervical cancer 15–50 times higher than those without HPV DNA (Lo et al., 2002). Similar correlation is found between HPV and SIL in epidemiological studies (Kulasingam et al., 2002). Therefore, it is possible to use HPV test for the detection of cervical lesions as an adjunct to cervical cytology. The present study was conducted to establish a quantitative real-time polymerase chain reaction (PCR) assay for a precise determination of HPV 16 viral load, and to investigate the role of viral load in the development of cervical precancerous lesions and invasive cancers.

Section snippets

Specimens

Patients with normal and abnormal smears were recruited with informed consents and ethical approval from the local institute. All patients with abnormal smears were examined colposcopically with biopsies when indicated. According to WHO criteria, they were classified as high-grade SILs (HG-L), low-grade SILs (LG-L) and Normal. Smears collected by cervical brush were immersed into phosphate buffered saline and centrifuged. The pellet at the bottom of the tube was homogenized by TRIzol® Reagent

Grading of smears

The study design is summarized in Fig. 1. There were 269 cervical smears collected in colposcopic clinic. After colposcopic examination with biopsies, 148 cases with cervical cancer, CIN 2 or CIN 3 were classified as HG-L; 121 with CIN 1, HPV or inflammation were LG-L. Another 125 smears collected from women with normal cytology at out-patient clinic served as control.

Incidence of HPV 16

All the smears underwent DNA extraction and subsequently screened with PCR for β-actin and HPV 16 specific primers. There were

Discussion

The accumulated molecular and clinical evidences have left no doubt that HPV directly influences the pathogenesis of cervical neoplasia (Alani and Munger, 1998). Several markers have been proposed for the use in prediction of disease severity of SILs. The presence of HPV DNA, the integration state of HPV genome and the viral load were three major areas currently under investigations.

A study showed that 75.4% of patients with abnormal cytology were HPV-positive, a much higher proportion than

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