Methylation of CpG sites in the upstream regulatory region, physical status and mRNA expression of HPV-6 in adult-onset laryngeal papilloma

Abstract

The methylation status of HPV-6 upstream regulatory region (URR) in adult-onset laryngeal papillomatosis (AO-LP) remains unclear. The purpose of this study was to investigate the methylation status of URR and the physical status of HPV-6, as well as the dynamic variations of viral load and mRNA expression in AO-LP. We examined 18 specimens from 11 patients with AO-LP by real-time polymerase chain reaction (PCR), bisulfite-sequencing PCR, and amplification of papilloma oncogene transcripts. HPV-6 was identified in 9 of 11 patients (81.8%), and all the 15 specimens derived from 9 HPV-6-positive cases contained only episomal HPV-6 transcripts with intact E2. Three HPV-6-positive patients developed recurrent lesions, and HPV-6 copy numbers and mRNA expression decreased after surgical treatment. Among the 96 CpG sites (16/case), 67 (69.8%) were unmethylated, while 23 (30.2%) were heterogeneous (≥ 1 methylated CpG clone). High viral loads and episomal status of HPV-6 were frequently observed in AO-LP; thus, persistent E6/E7 mRNA expression of LR-HPV-6 may be associated with AO-LP recurrences. Hypomethylation and scattered patterns of methylated CpGs at the URR of HPV-6 were identified in AO-LP.

Keywords: adult-onset laryngeal papilloma; human papillomavirus-6; mRNA expression; methylation; physical status.

Figure 1

(A) Dynamic variation of viral load and (B) E6 mRNA expression of HPV-6 in AO-LP before the first treatment, and after recurrences. Compared with the primary lesion, a gradual decline in the tendency of HPV-6 copy numbers and mRNA expression in the recurrent lesions was found in cases 5, 6, and 7 over time after surgical treatment. Pri.: primary tumor without treatment; 1st-R: first recurrence; 2nd-R: second recurrence; 3rd-R: third recurrence; 4th-R: fourth recurrence.

Figure 2. Amplification of papillomavirus oncogene transcripts (APOT) of HPV-6

In several typical cases with HPV-6-positive status, including cases 5, 6, and 7, amplimers of approximately 1200 bp in length were suspected to originate from the E7-E1^{^}E4 episomal transcript, and were confirmed by direct sequencing of amplification products. Case 2 was HPV negative.

Figure 3. Methylation of CpGs sites at the URR of HPV-6 in AO-LP

For every CpG site, 6 clones were sequenced to identify the presence and frequency of meCpG clones. The left vertical bar indicates the percentages of meCpG clones. The structural diagrams of the URR and the positions of the CpG sites are shown at the top and bottom of the figure, respectively. The CpGs in the URR are numbered according to the amended version of HPV6b.

Figure 4. Immunohistochemistry for p16, p53, and pRb in AO-LP

Positive expression was defined as p16INK4a staining in > 40%, or p53 and pRb staining in > 25% of 2000 tumor cells. The six micrographs show the typical p16, p53, and pRb immunoreactivity patterns corresponding to positive and negative expression (×100; bar, 100 μm).