Human papillomavirus 16 L1 gene methylation as a potential biomarker for predicting anal intraepithelial neoplasia in men who have sex with men (MSM)

The human papillomavirus (HPV) 16 early promoter and L1 gene methylation were quantitatively measured using pyrosequencing assay in anal cells collected from men who have sex with men (MSM) to determine potential biomarkers for HPV-related anal cancer. The methylation patterns of HPV16 genes, including the early promoter (CpG 31, 37, 43, 52, and 58) and L1 genes (CpG 5600, 5606, 5609, 5615, 7136, and 7145), were analyzed in 178 anal samples. The samples were diagnosed as normal, anal intraepithelial neoplasia (AIN) 1, AIN2, and AIN3. Low methylation levels of the early promoter (< 10%) and L1 genes (< 20%) were found in all detected normal anal cells. In comparison, medium to high methylation (≥ 20–60%) in the early promoter was found in 1.5% (1/67) and 5% (2/40) of AIN1 and AIN2-3 samples, respectively. Interestingly, slightly increased L1 gene methylation levels (≥ 20–60%), especially at the HPV16 5’L1 regions CpGs 5600 and 5609, were demonstrated in AIN2-3 specimen. Moreover, a negative correlation between high HPV16 L1 gene methylation at CpGs 5600, 5609, 5615, and 7145 and a percentual CD4 count was found in AIN3 HIV positive cases. When comparing the methylation status of AIN2-3 to that of normal/AIN1 lesions, the results indicated the potential of using HPV16 L1 gene methylation as a biomarker for HPV-related cancer screening.


Introduction
Anal carcinoma is a rare disease found globally in men and women, with an incidence of < 1-2 cases per 100,000 [1]. However, a high incidence of anal cancer was present in HIVinfected women and HIV-infected men who have sex with men (MSM), accounting for 30/ 100,000 and 131/100,000, respectively [2]. There is an association between human papillomavirus (HPV) infection and anal carcinoma; HPV DNA was found in men (68. [51,52]) were highly methylated in cervical cancers [49, [51][52][53][54]. A recent study reported that a high methylation at CpG 5611 and 7145 respectively predicted the presence of CIN2 + and CIN3+ with high accuracy [55]. Our group has previously reported an association between high methylation of the HPV16 L1 gene, especially at the CpG sites 5600 and 5609, and high-grade cervical lesions and cervical carcinoma [49]. However, there is a limited number of studies on the HPV16 genome methylation in anal cells [56][57][58], and to the best of our knowledge, there has been no HPV methylation study in anal cells collected from Asian countries. Therefore, the investigation of the HPV16 methylation status of these CpG positions in anal cells is of scientific interest. We aimed to detect the methylation pattern of the HPV16 genome in the CpG positions within the early promoter and L1 regions in anal cells obtained from the Thai men who have sex with men (MSM), analyzed by a quantitative pyrosequencing assay.

Clinical samples and cell lines
This study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (COA No. 053/2016). The present study is a retrospective study of 178 archived HPV16-positive DNA samples extracted from anal cells; therefore, no informed consent was required from the patients. These samples were collected from MSM at the Thai Red Cross AIDS Research Centre (TRC-ARC) Bangkok, Thailand, between May 2013 and December 2013. All samples were anonymized. There were 134 HIV-infected cases and 44 HIV-uninfected cases. The percentages of CD4+ results were only obtained from HIV-infected men. The DNA was extracted from human cervical cancer cell lines that contained integrated HPV 16. CaSki (CRL-1550 Lot No.3794357) and SiHa (HTB-35 Lot No.4031219) were used as positive controls for the amplification and pyrosequencing. They respectively contained approximately 500-600 copies or 1-2 copies per cell.

Specimen collection and DNA extraction
A moistened, non-lubricated flocked swab (Rovers EndoCervex-Brush, Rover Medical Devices B.V., Netherlands or FLOQSwabs, Copan Italia S.p.A., Italy) was used to collect anal cells from the anal canal surfaces. After sample collection, the anal swab was placed and kept in a liquidbased cytology (LBC) fluid (Liqui-PREPTM LGM International, Inc., Florida, USA) at 2-8˚C until the DNA extraction within 7 days. DNA was extracted from anal cells according to the manufacturer's protocol using the AmpliLute Liquid Media Extraction kit (Roche Molecular Diagnostics, California, and USA) and DNA was collected in 120 μL of elution buffer.

HIV detection and CD4 cell count
At an anonymous clinic, the Architect HIV Ag/Ab combo kit (Abbott Laboratories, GmBH, Wiesbaden, Germany) was used as the screening test, followed by Alere DetermineTM HIV ½ (Abbott Laboratories) and Serodia HIV ½ (Fujirebio, Tokyo, Japan) as the confirmatory test. BD FACSCount™ CD4 reagents (BD Biosciences, San Jose, CA, USA) were used to enumerate the absolute counts and percentages of CD4 T lymphocytes in unlysed whole blood (CD4 counts and CD4 percentages). The reagents were intended for in vitro diagnostic on a BD FACSCount™ instrument.

Methylation analysis by a pyrosequencing assay
The extracted DNA (100-1000 ng) from anal cells was subjected to bisulfite conversion by using the EZ kit Gold Bisulfite Conversion Kit (Zymo Research) according to the manufacturer's instructions. The sequences of forward, reverse, and sequencing primers for the early promoter CpG positions 31, 37, 43, 52, and 58, the 3'L1 CpG positions 7136, and 7145, and the 5'L1 CpG positions 5600, 5606, 5609, and 5615 are shown in Table 1.
The PCR amplification protocol was as follows: 1x PCR buffer, 2.5 mM MgCl 2 , 250 μM dNTP, 12.5 pM of each forward and reverse primer, 1 Unit DNA polymerase (HotStart HiFidelity Polymerase, Affymetrix, USA), 2.5 μl of bisulfite-treated DNA and DNase/RNase-free water were added to the final volume of 25 μL. The PCR amplification was started with an initial denaturing at 95˚C for 10 minutes, followed by 50 cycles of 95˚C for 30 seconds, 55˚C for 1 minute, and 72˚C for 1 minute, and a cycle for the final extension at 72˚C for 10 minutes. The PCR products were detected by 1.5% agarose gel electrophoresis. Prior to pyrosequencing, all reagents including 70% ethanol, denaturation buffer and washing buffer were prepared in Milli-Q water and placed on the PyroMark Q96 Vacuum Workstation. For sample preparation, 20 μL of biotin-labeled amplification products were mixed with 2 μL of streptavidin sepharose beads (GE Healthcare Bio-science AB, Uppsala, Sweden) in 40 μL of PyroMark binding buffer (QIAGEN, Hilden, Germany), added Milli-Q water to a total volume of 80 μLand then agitated at 1400 rpm for at least 10 minutes. After switch on the vaccum pump, The filter probes were placed into the tube containing the beads, after all liquid was aspirated and beads were captured onto the filter probed. Next, the filter probes were washed by flushing with 70% ethanol for 5 seconds, denatured in the PyroMark denaturation solution (QIAGEN, Hilden, Germany) for 5 seconds. The filter probes were then flushed again in PyroMark wash buffer (QIAGEN, Hilden, Germany) for 10 seconds.Switch off the vacuum pump, mixed the beads captured PCR products with 0.4 μM of the sequencing primers in 40 μL of PyroMark annealing buffer (QIAGEN, Hilden, Germany) prepared in pyromark plate low, after that, placed the plate on a heating block at 80˚C for 2 min and cooled down to room temperature for at least 5 min. Then, the PyroMark nucleotides, substrate and enzyme mixture (QIAGEN, Hilden, Germany) were loaded into cartridge according to the calculated volume after setting up the run in the PyroMark Q96 Software. Finally, the pyromark plate low and filled reagent cartridge were loaded onto the PyroMark™ Q96 machine (Qiagen, Hilden, Germany). After the run, the software will measure the percent methylation value of each CpG site shown in the analyzed pyrogram. The bisulfite conversion control (Single cytosine was completely converted to uracil) was highlighted in yellow bar must be lacking an intensity signal, thus, no peak was found within the analyzed sequence. The gray bar represents the analyzed CpG sites within the sequence. The percent methylation value of each CpG site that perfectly pass quality control are indicated in blue on the top of the gray bar (S1-S3 Figs).

Statistical analysis
The Kruskal-Wallis test was used to analyze the differences in the mean methylation values among the groups of specimens. The Fisher exact test was used to examine the significant differences of the sample proportions with a methylation � 20% between normal/AIN1 and AIN2-3. Pearson's correlation coefficient (r) was used to analyze the association between the percentage of methylation and the CD4 count. A P value less than 0.05 was considered a statistically significant difference.

Correlation between the CD4+ percentage and HPV16 gene methylation
There were no statistically significant differences in the mean HPV16 methylation percentage between HIV-uninfected and HIV-infected cases (Table 3). There was no correlation between the HPV16 L1 gene methylation and a low CD4 count in normal, AIN1, and AIN2 cases. Interestingly, high gene methylation of HPV16 L1 was moderately correlated with a low percentual CD4 count in AIN3 HIV-infected cases, especially at CpGs 5600, 5609, 5615, and 7145 (R = 0.4692-0.5412) (Fig 4).

Discussion
In the present study, the methylation patterns of the HPV16 early promoter and the L1 region in anal cells were studied using a pyrosequencing assay. The quantitative methylation analysis   [63][64][65][66][67][68]. Hypermethylation in the L1 gene was found to be correlated with an integration form of HPV16 [65,69]. The present study found a high methylation of the HPV16 L1 gene in some of the AIN2/3 samples compared to normal anal cells. We also found that the CpG sites 5600 and 5609 showed a higher methylation (� 20%) compared to the other CpG sites in the L1 region (5606, 5615, 7136, and 7145) (Fig 1 and Table 2). Previous reports in cervical cells showed that the CpG sites 5600 and 5609 were the best sites for separation of normal cervical cells and high-grade dysplasia [47,49,54]. The methylation patterns of the HPV16 L1 gene in anal cells were similar to cervical cells and might therefore be used to distinguish normal cells from HPV-related abnormal cells. Anal samples with intermediate to high methylation levels (� 20%) in the present study may indicate an increased risk of more rapid progression compared to those with low methylation levels (< 20%). Methylation patterns of the HPV16 early promoter have been widely studied in cervical cells. Nevertheless, controversial results were found. Some studies reported hypomethylation of HPV16 early promoter in cervical carcinoma or so-called progressive hypomethylation [40,41]. Other studies reported hypermethylation or progressive hypermethylation of the early promoter in cervical carcinoma [38,43,44,48,50]. The physical state and copy number of integrated HPV16 genomes were the main reason for these methylation differences within the early promoter, as shown in CaSki and SiHa cells. There was evidence for closed chromatin within the viral oncogene promoter region due to hypermethylation of multiple copies of the integrated HPV, leading to controlling the expression level of viral oncogenes to be optimal that facilitates the survival of cancer cells [70,71]. An episomal form of the HPV16 genome was found in high-grade cervical lesions, and cervical carcinoma displayed high methylation levels at E2BS in the early promoter compared to a single integrated HPV16 genome [45]. One study showed a high methylation of the early promoter in high-grade anal cells [58]. The E2 protein plays a role in controlling viral oncogene expression by either activating or suppressing viral oncogene promoter depending on the E2 protein concentration [72][73][74]. It was hypothesized that the E2BS methylation of episomal HPV16 with an intact E2 gene could prevent the binding of the E2 protein at the proximal E6/E7 oncogene promoter, thus preventing the suppressive activity of E2 protein leading to the overexpression of viral oncogenes [75].
An anal cytology assay has been used for screening of abnormal anal cells [76,77]; however, there was a wide range of assay sensitivity, varying from 19-89%, for detecting high-grade AIN [78][79][80][81]. The pooled specificity of the HR-HPV for detecting anal cells diagnosed as AIN2 + was only 33.1% [82]. The combination of the established methods with a more specific assay, such as HPV16 L1 methylation, would improve the specificity to detect abnormal anal cells as reported in high-grade cervical lesions and cervical carcinoma [45, 55,83].   It has been reported that HIV-infected patients were susceptible to HPV infection. One study reported that HIV-infected women with a CD4 count of fewer than 200 cells/mm3 have 59.3% of high-risk HPV infections, correlating with increasing severity of cervical lesions [84]. The study of oral samples reported that a low CD4 count (< 200 cells/mm3) increased the risk for oral HPV infections in HIV-infected patients [85]. There was an association between a low CD4 count (< 200 cells/mm3) and rapid anal cancer progression in HIV-infected MSM [86]. In the present study, a combined HPV16 L1 gene methylation (�20%) and a low percentage of a CD4 count might be beneficial to differentiate HIV-infected MSM who are at risk to rapidly progress to high-grade AIN and carcinoma. The limitation of the present study was that anal carcinoma samples could not be included due to the very low incidence of anal cancer. The low normal sample size may limit the significance of the statistical comparison between normal and high-grade AIN.

Conclusions
The methylation patterns of the HPV16 genome in anal intraepithelial neoplasia were similar to those of abnormal cervical cells. Hypermethylation of the HPV16 L1 gene, especially at CpG 5600 and 5609, found in AIN2/3, could be a biomarker for predicting HPV-related abnormal anal cells. Moreover, the combination of HPV16 L1 gene hypermethylation together with a low CD4 count in HIV-infected patients might be used as a biomarker for rapid progression to more severe lesions and anal cancer than those with a low methylation and high CD4 count. Thus, in order to employ methylation of specific CpG sites for screening of HPV-related abnormal lesions and cancer, a large sample size including normal and anal carcinoma samples should be further studied and evaluated.