Human papillomavirus type 16 integration in cervical carcinoma in situ and in invasive cervical cancer

Abstract

Integration of human papillomavirus type 16 (HPV-16) into the host DNA has been proposed as a potential marker of cervical neoplastic progression. In this study, a quantitative real-time PCR (qRT-PCR) was used to examine the physical status of HPV-16 in 126 cervical carcinoma in situ and 92 invasive cervical cancers. Based on criteria applied to results from this qRT-PCR assay, HPV-16 was characterized in carcinoma in situ cases as episomal (61.9%), mixed (i.e., episomal and integrated; 29.4%), and integrated (8.7%) forms. In invasive cervical cancer samples, HPV-16 was similarly characterized as episomal (39.1%), mixed (45.7%), and integrated (15.2%) forms. The difference in the frequency of integrated or episomal status estimated for carcinoma in situ and invasive cervical cancer cases was statistically significant (P = 0.003). Extensive mapping analysis of HPV-16 E1 and E2 genes in 37 selected tumors demonstrated deletions in both E1 and E2 genes with the maximum number of losses (78.4%) observed within the HPV-16 E2 hinge region. Specifically, deletions within the E2 hinge region were detected most often between nucleotides (nt) 3243 and 3539. The capacity to detect low-frequency HPV-16 integration events was highly limited due to the common presence and abundance of HPV episomal forms. HPV-16 E2 expressed from intact episomes may act in trans to regulate integrated genome expression of E6 and E7.

FIG. 1.

Detection threshold of the HPV-16 qRT-PCR assay. Values in fluorescence units for the E6 and E2 gene targets are shown on the y axis. The ratio of integrated HPV (SiHa) to episomal HPV-16 (W12) DNA in each reaction is shown on the x axis. Below the x axis scale in parentheses is the mean ratio derived from E6:E2 qRT-PCR values at each defined combination of integrated and episomal forms. SiHa (1 × 10⁴ to 1 × 10¹ HPV-16 genome equivalents per reaction) was combined with 10² genome equivalents of W12. Bars indicate standard errors; open and filled circles indicate individual quantitative PCR values obtained for E6 and E2, respectively (see the text for details).

FIG. 2.

Deletion mapping of HPV-16 E1 and E2 genes in CIS and CC samples. Selected samples demonstrating E1 and E2 losses (n = 35) and two tumors containing rearrangements with no losses (C29 and C57) are included. ID column indicates individual specimen identifiers; fresh frozen cervical tumors are designated by C followed by numeric assignments; paraffin-embedded specimens are denoted by numeric assignments with no alpha modifier for CIS and the addition of an i preceding the number for invasive CC tumors; qPCR column designates integrated, episomal, and mixed forms based on E2:E6 ratio determinations. Various columns indicated by F represent the upstream primer for each pair specified in Table 1. R, rearrangements detected by gel analysis; ND, not determined.

FIG. 3.

Rearrangements in HPV-16 E2 sequences. A. Nucleotide positions per HPV-16R sequence are noted at the top of the sequence information and correspond to nucleotide positions 2690 to 2870 and 3469 to 3871. The 17 nucleotides that serve as the recombination site are shown in italics. B. Illustration of the proposed end-joining recombination event shown here as an example in tumor C28 that leads to reconstruction of the 583-bp fragment obtained.