Type distribution of human papillomaviruses in ThinPrep cytology samples and HPV16/18 E6 gene variations in FFPE cervical cancer specimens in Fars province, Iran

Abstract

Background: There exists strong evidence that human papillomavirus (HPV) is associated with cervical cancer (CC). HPV E6 is a major oncogene whose sequence variations may be associated with the development of CC. There is not sufficient data on the distribution of HPV types in ThinPrep cytology specimens and HPV 16/18 E6 gene variations among CC patients in the southwest of Iran. This study was conducted to contribute to HPV screening and vaccination in Iran.

Methods: A total of 648 women screened for cervicitis, intraepithelial neoplasia or CC were included in the study. All participants underwent ThinPrep cytology testing, single-step HPV DNA detection and allele-specific reverse hybridization assays. Moreover, a total of 96 specimens previously tested positive for single infection with HPV16 or 18 were included for variant analysis. HPV16/18 lineages and sublineages were determined by PCR assays followed by sequencing the E6 gene and the construction of neighbor-joining phylogenetic trees.

Results: Overall, HPV DNA was detected in 62.19% of all the screened subjects. The detection rates of HPV DNA among individuals with normal, ASC-US, ASC-H, LSIL, and HSIL cervical cytology were 48.9%, 93.6%, 100%, 100%, and 100%, respectively. Low-risk HPVs were detected more frequently (46.9%) than high-risk (38.9%) and possible high-risk types (11.1%). Of 403 HPV-positive subjects, 172 (42.7%) had single HPV infections while the remaining 231 (57.3%) were infected with multiple types of HPV. Our results indicated a remarkable growth of high-risk HPV66 and 68 and low-risk HPV81 which have rarely been reported in Iran and HPV90 and 87 that are reported for the first time in the country. In addition, 3 lineages (A, D, and C) and 6 sublineages (A1, A2, A4, C1, D1, and D2) of HPV16, and one lineage and 4 sublineages (A1, A3, A4, and A5) of HPV18 were identified. The studied HPV16 and 18 variants mainly belonged to the D1 and A4 sublineages, respectively.

Conclusion: The present study suggests that the prevalence of HPV infection in women of all age groups with or without premalignant lesions in the southwestern Iran is high and the predominant HPV types in the southwest of Iran may differ from those detected in other parts of the country. This study also highlights the necessity of not only initiating HPV vaccination for the general population but also developing new vaccines that confer immunity against the prevalent HPV types in the area and national cervical screening programs using a combination of thinPrep cytology test and HPV detection assays in order to improve the accuracy of the screening.

Keywords: Cervical cancer; HPV; HPV16; HPV18; Lineage; Phylogenetic analysis; ThinPrep.

Fig. 1

Prevalence of HPV types among HPV-positive individuals

Fig. 2

Distribution of high-risk HPV types (a), possible high-risk HPV types (b), and low-risk HPV types (c) in HPV-positive groups of normal, ASC-US, ASC-H, LSIL, and HSIL cervical cytology

Fig. 3

Phylogenetic analysis of HPV16 E6 (a) and HPV18 E6 (b) gene regions was conducted using the maximum-likelihood method based on the Kimura 2-parameter model with bootstrap resampling (1000 replicates) by the MEGA 6 package [37]. Numbers above the branches indicate the bootstrap values. Ninety-six different nucleotide patterns of studied sequences were indicated by black triangles (GenBank accession numbers OP572427 through OP572474 for HPV16 and OP572475 through OP572522 for HPV18). The accession number of reference sequences of each sublineage used for phylogenetic analysis in this study was indicated by white triangles

Fig. 3

Phylogenetic analysis of HPV16 E6 (a) and HPV18 E6 (b) gene regions was conducted using the maximum-likelihood method based on the Kimura 2-parameter model with bootstrap resampling (1000 replicates) by the MEGA 6 package [37]. Numbers above the branches indicate the bootstrap values. Ninety-six different nucleotide patterns of studied sequences were indicated by black triangles (GenBank accession numbers OP572427 through OP572474 for HPV16 and OP572475 through OP572522 for HPV18). The accession number of reference sequences of each sublineage used for phylogenetic analysis in this study was indicated by white triangles