The physical state of HPV16 infection and its clinical significance in cancer precursor lesion and cervical carcinoma

Abstract

Purpose: Integration of high-risk human papillomavirus (HR-HPVs) into the host DNA has been proposed as a risk for cervical carcinogenesis. HPV-16 is the predominant high-risk type and its integration ration varied largely in different cervical cancer (CC) samples. The aim of this study was to evaluate the correlation between physical state of HPV16 infection and extent of cervical lesion, as well as the clinical significance of virus existing state.

Methods: A total of 252 cases of paraffin-embedded blocks derived from cancer precursor lesion and cervical carcinoma samples were detected by HC-II for HR-HPV infection. HPV16 infection was confirmed by PCR and immunohistochemistry for HPV16 E7 simultaneously. The physical state of HPV16 infection were assessed by PCR for 3 overlapping fragments in E2 gene and multiple PCR for E2 gene and E7 gene.

Results: The infection ratio of HR-HPV in normal cervical tissue, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III and cervical cancer were 15.0, 32.8, 54.3, 69.7, 93.8%, respectively. HR-HPV positive samples of 62.8% were infected with HPV16. The integration ratio of HPV16 in CIN III and cervical carcinoma were 35.7 and 58.1% respectively, both of which were significantly higher than that of CIN I and normal cervical tissues. The discrepancy was statistically significant (P < 0.05). Furthermore, it was observed that persistent virus infection and progression of cervical lesion were more common in CIN I with integrated HPV16 than that with episomal HPV16.

Conclusion: The integration ratio of HPV16 was accompanied by an increase in the grade of cervical lesion. The integrated state of HPV16 infection was strongly associated with persistent HPV infection and progression of cervical lesions.

Fig. 1

Characterization of HPV-16 infection. a Gel photograph of PCR performed on DNA extracted from paraffin-embedded cervical lesion tissue samples with primers for house-keeping gene actin and gapdh. b Gel photograph of PCR performed on DNA extracted from paraffin-embedded cervical lesion tissue samples with primers located within the E7 region (315-bp product) of the HPV-16 genome. c Expression of HPV16E7 in different cervical tissues detected immunohistochemically. 1 A normal cervical squamous epithelium stained with HPV16E7 antibody showing no positive staining (20×). 2 A low-grade cervical intraepithelial neoplasia (CIN I) lesion was stained with HPV16E7 antibody showing weak positive staining localized in the lower one-third cells and scattered in some of the intermediate layer cells (20×). 3 A high-grade cervical intraepithelial neoplasia lesion (CIN III) showing strong immunohistochemical staining in the dysplastic cells (40×). 4 A poorly differentiated invasive squamous cell carcinoma. Cancer cells expressing intense immunohistochemical staining, distributing equally throughout the lesion (40×)

Fig. 2

Detection for physical status of HPV16 infection. a Characterization of HPV-16 integration. Gel photograph of PCR performed on DNA extracted from different tissues, demonstrating failure of amplification of those different fragments (amplimer a, b, c, shown as 475-bp, 477-bp, 276-bp products, respectively) of the E2 gene in CIN and CC lesions. M marker (100-bp DNA ladder), 1 Caski cell, 2 SiHa cell, 3 a CIN I lesion, 4 a CIN III lesion, 5–6 CC lesions (integration), 7 water blank negative control. b Discrimination between episomal infection and mixed infection. Gel photograph of multiplex PCR performed on DNA extracted from paraffin-embedded cervical lesion tissue samples with primers located within the E7 region (315-bp product) and E2 region (amplimers B, 477-bp product) of the HPV-16 genome. M marker, 1 integrated infection (CC), 2 episomal infection (normal cervix), 3 episomal infection (CIN III), 4 mixed infection (CC)