Analysis of human papillomavirus type‐16 variants in Italian women with cervical intraepithelial neoplasia and cervical cancer

Abstract

Human papillomavirus type 16 (HPV-16) classes (E, AA, As, Af1, Af2) and their variants have different geographic distribution and different degrees of association with cervical lesions. This study was designed to examine HPV-16 variants among Italian women and their prevalence in case patients (affected by invasive cervical carcinoma or cervical intraepithelial neoplasia grade 2-3 and cervical intraepithelial neoplasia grade 1), versus control subjects with normal cervical epithelium (controls). A total of 90 HPV-16 positive cervical samples from women of Italian Caucasian descent have been tested, including 36 invasive cervical carcinomas, 21 with cervical intraepithelial neoplasias grade 2-3, 17 with cervical intraepithelial neoplasia grade 1 and 16 controls. HPV-16 was detected with an E6/E7 gene-specific polymerase chain reaction, and variant HPV-16 classes and subclasses were identified by direct nucleotide sequencing of the region coding for the E6 and the E7 oncoproteins, the MY09/11-amplified highly conserved L1 region, and the long control region (LCR). Among the 90 HPV-16 samples, nine viral variants have been identified belonging to the European (Ep-T350 and E-G350) and non-European (AA and Af-1) branches. The E-G350 is the prevalent variant in all analyzed different disease stages being present in 55.5% of ICC, 52.4% of cervical intraepithelial neoplasias 2-3, 47.1% of cervical intraepithelial neoplasia grade 1, and 50.0% of control samples. The non-European variants AA and Af1, rarely detected in control samples, represent 33.3% of all HPV-16 infections in invasive cervical carcinoma (with a peak of 19.4% and 13.9%, respectively), showing a statistically significant increase in frequency in more advanced lesions (chi(2) trend = 7.2; P < 0.05). The prevalence of HPV-16 Ep-T350, however, is higher in controls (43.7%) and in of cervical intraepithelial neoplasia grade 1 (41.2%) than in cervical intraepithelial neoplasia grade 2-3 (28.6%) and in invasive cervical carcinoma (11.1%) cases strongly suggesting lack of progression for pre-neoplastic lesions associated with such variant. The increased frequency of non-European variants in invasive lesions suggests that they are more oncogenic than European variants. This could have implications for future diagnostic and therapeutic strategies.

Human papillomavirus genotypes and HPV16 variants in penile carcinoma

“…The percentage of penile carcinoma testing positive for non-European variants (55.6% of HPV16-positive samples) is much higher than that reported for cervical carcinoma in the same geographical area (33.3%). 42 In particular AA variants have a prevalence of 44.4% in penile cancer compared to the 19.4% in cervical carcinoma. Furthermore, it is relevant to note that Af1 variants, that account for 60.9% of all HPV16-associated cervical carcinoma in the African continent and have not been previously reported in samples collected in other European countries, represent the 11.1% and 13.9% of HPV-16 identified in Italian penile carcinoma and cervical carcinoma, respectively.…”

“…HPV-16 E6/E7 whole region was amplified by nested 47 The HPV-16 LCR sequences were amplified by seminested PCR using the primer pair 16LCR-1N (5 0 -GCTTGTGTAACTATTG TGTCA, nt 7289-7309) with 16LCR-E6-5C (5 0 -ATCCC GAAA AGCAAAGTCAT-3 0 , nt 231-250) and the primer pair 16LCR-1N with 16LCR-2C (5 0 -GTCCTGAAACATTGCAG TTCT-3 0 , nt 95-115) as previously described. 42 Both outer and inner PCR amplification reactions were performed in 50 ll reaction mixture containing 20 pmoles of each primer, 50 mM KCl, 2.5 mM MgCl2, 100 mM Tris-HCl pH 8.3, 0.1% Triton X-100, 50 mM of each dNTP and 1.8 units of thermostable AmpliTaq DNA polymerase (Applied Biosystems, Foster City CA ). In the first-step PCR, 5 ll of DNA (10-100 ng) was used as target DNA (outer reaction); in the nested PCR, 5 ll of the first-step PCR was used as input of amplified DNA (inner reaction).…”

“…[1][2][3][4][5] The more than 40 HPV genotypes infecting the genital mucosa can be divided into 3 groups: 'high-riskÕ or 'carcinogenicÕ types (16,18,31,33,35,39,45,51,52,56,58,59) associated with a high relative risk of cervical cancer; 'low riskÕ viruses (6,11,40,42,43,44,54,61,70,72,81,89) associated with benign epithelial proliferations in the genital area, but not associated with invasive cervical cancer; and a group of 6 types (26,53,66,68,73,82) that is classified as 'probably carcinogenicÕ since there is limited data associating these HPV types with cervical cancer. 6,7 Additionally, carcinogenic HPVs are involved in the development of a subset of other carcinomas arising from mucosal squamous epithelial cells including penile carcinoma.…”

“…[30][31][32][33][34][35][36][37][38][39] Several epidemiological studies, focused on the association of HPV16 variants and risk progression of cervical carcinoma, have observed an increased risk of disease associated with either European variants 34,40 or with non-European variants of HPV16. 37,38,[41][42][43] Much less is known about the involvement of HPV variants in the development of HPVrelated tumors other than cervical neoplasia. In one study that included 5 HPV16-positive penile carcinoma and 5 cervical neoplasia biopsies from Uganda the presence of Af1 HPV16 variants was observed in 100% of the cases.…”

Human papillomavirus (HPV) genotypes and HPV16 variants in human immunodeficiency virus-positive Italian women

“…Thus, genomic variants can be considered markers of specific HPV genomes and accordingly can be used in epidemiological and aetiological studies to investigate transmission of HPV within and among populations (Xi et al, 2006;Tornesello et al, 2007). The most studied HPV16 isolates, classified as European and non-European (Asian, Asian-American, African 1 and 2) variants, differ in their biological properties and in their oncogenic potential (Tornesello et al, 2000(Tornesello et al, , 2004Xi et al, 1997Xi et al, , 2002Xi et al, , 2007Hiller et al, 2006). Only a few studies, all performed in the USA, have evaluated the prevalence and natural history of HPV16 variants in HIV-positive women and compared them with HIV-negative women (Chaturvedi et al, 2004;Schlecht et al, 2005).…”