High-throughput detection of human papillomavirus-18 L1 gene methylation, a candidate biomarker for the progression of cervical neoplasia

Abstract

The L1 gene of human papillomavirus-18 (HPV-18) is consistently hypermethylated in cervical carcinomas, but frequently hypo- or unmethylated in exfoliated cells from asymptomatic patients. In precancerous lesions, L1 is sporadically hypermethylated, correlating with the severity of the neoplasia. In order to explore the potential of using L1 methylation as a workable biomarker for carcinogenic progression of HPV-18 infections in routinely taken samples, our aim was to develop methylation-detection techniques that were sensitive and rapid without being overly complex technically. Therein, we developed a methylation-specific PCR (MSP) through the design of primer sets that specifically amplify either methylated or unmethylated HPV-18 L1 DNA within bisulfite-modified sample DNA. Amplification of unmethylated and in vitro methylated HPV-18 DNA by MSP resulted in 2500 copies of either of the two L1 DNA species being detected, a satisfactory sensitivity considering that bisulfite treatment leads to the fragmentation of about 99% of sample DNA. The primers proved specific and did not generate false positive results at concentrations exceeding the lowest limit of detection by a factor of 400. DNA from carcinomas yielded PCR signals only with the methylation-specific primers, and not with primers specific for unmethylated L1 genes. The inverse result was obtained with DNA from precursor lesions that contained only hypomethylated DNA. High-grade precursor lesions and carcinomas that contained hyper- as well as hypomethylated L1 DNA yielded PCR signals with both primers. By developing a fluorescence based real-time PCR, we quantitatively analyzed samples with in vitro methylated and unmethylated L1 DNA, and could distinguish clinical samples with hyper- and hypomethylated DNA or mixtures of both DNAs. The methylation-specific and real-time PCR techniques permitted efficient HPV-18 L1 methylation analyses and open the door for larger-scale clinical studies where the utility of methylation status to predict the progression of HPV-18 infection and HPV-18 associated lesions is assessed.

Human papillomavirus‐16 DNA methylation patterns support a causal association of the virus with oral squamous cell carcinomas

“…It has been pointed out by others 35 that promoter methylation may favor carcinogenesis, as it prohibits binding of the E2 protein, 38 which is a repressor of the E6 promoter, and assures continued oncogene expression even in the subpopulation of lesions that did not lose E2 as a consequence of genomic interruption. 39 The data of this study and our previous studies provide strong support that the measurement of DNA methylation is a useful biomarker to characterize malignant lesions. Ten of the 12 oral lesions that we examined showed hypermethylation typical for malignancies.…”

Viral epigenomes in human tumorigenesis

“…They also found differential methylation of L1 in carcinomas, premalignant lesions and asymptomatic carriers, supporting the results of Turan et al (2007), who proposed L1 methylation as a biomarker in neoplastic progression.…”

“…Previous studies of HPV16 DNA methylation have analyzed the entire LCR and L1 regions (Badal et al, 2003(Badal et al, , 2004Kim et al, 2003;Kalantari et al, 2004;Bhattacharjee and Sengupta, 2006;Turan et al, 2007;Hublarova et al, 2009). Even more recent studies have been able to map the DNA methylation of every CpG (DNA methylome) in a collection of human cervical samples corresponding to the different progressive stages of the disease, from asymptomatic carriers to primary cervical carcinomas and several established cervical cancer cell lines (Brandsma et al, 2009;Fernandez et al, 2009).…”

“…In HPV16 and HPV18 types, the LCR and E6 sequences were commonly found to be unmethylated independent of the stage of neoplastic progression, although the L1 region was densely methylated (Badal et al, 2003(Badal et al, , 2004Turan et al, 2006Turan et al, , 2007Hublarova et al, 2009).…”

Detection, management, and follow‐up of pre‐malignant cervical lesions and the role for human papillomavirus

“…Other mechanisms than integration and disruption of the E2/E1 genes must therefore be present to derepress E6/E7 expression as well. Such mechanisms could include promoter methylation, direct mutation of E2 [40][41][42].…”