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

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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.
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RESEARCH ARTICLE
Human papillomavirus 16 L1 gene
methylation as a potential biomarker for
predicting anal intraepithelial neoplasia in
men who have sex with men (MSM)
Arkom ChaiwongkotID
1,2
*, Nittaya Phanuphak
3
, Tippawan Pankam
4
,
Parvapan Bhattarakosol
1,2
1Faculty of Medicine, Applied Medical Virology Research Unit, Chulalongkorn University, Bangkok,
Thailand, 2Faculty of Medicine, Department of Microbiology, Chulalongkorn University, Bangkok, Thailand,
3Institute of HIV Research and Innovation, Bangkok, Thailand, 4The Thai Red Cross AIDS Research
Centre, Bangkok, Thailand
*arkomchaiwongkot@gmail.com
Abstract
The human papillomavirus (HPV) 16 early promoter and L1 gene methylation were quantita-
tively 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 meth-
ylation 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 methyla-
tion (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 demon-
strated 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 nor-
mal/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 HIV-
infected 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 papilloma-
virus (HPV) infection and anal carcinoma; HPV DNA was found in men (68.7–91.2%) and
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OPEN ACCESS
Citation: Chaiwongkot A, Phanuphak N, Pankam T,
Bhattarakosol P (2021) Human papillomavirus 16
L1 gene methylation as a potential biomarker for
predicting anal intraepithelial neoplasia in men who
have sex with men (MSM). PLoS ONE 16(9):
e0256852. https://doi.org/10.1371/journal.
pone.0256852
Editor: Kalimuthusamy Natarajaseenivasan,
Bharathidasan University, INDIA
Received: October 5, 2020
Accepted: August 17, 2021
Published: September 1, 2021
Copyright: ©2021 Chaiwongkot et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was fully supported by
Research Grant for New Scholar
Ratchadaphiseksomphot Endowment Fund
Chulalongkorn University, Grants for Development
of New Faculty Staff, Ratchadaphiseksomphot
Endowment Fund, Chulalongkorn University and
was partly funded by TSRI Fund
(CU_FRB640001_01_30_9).
women (90.4–90.9%) with anal carcinoma worldwide [3]. Studies showed that HPV16 was the
most prevalent type found in anal carcinoma that detected in 70–71.6% of men and 74–83.4%
of women [2,4,5].
A high prevalence of HPV infection was reported in anal cells collected particularly from
HIV-infected MSM [611]. The worldwide HPV prevalence in anal cells of HIV-infected and
HIV-uninfected MSM was 92.6% and 63.9%, respectively, and HPV16 was found in 35.4% and
12.5% of HIV-infected and HIV-uninfected MSM, respectively [12]. Recent studies in Asian
countries, for example, in China reported a high prevalence of HPV infection among HIV-
infected MSM (82.69%) compared to HIV-uninfected MSM (62.81%) [13], in Korea and
Japan reported HPV infection rate were 82.7% and 75.9% of HIV-infected MSM, respectively
[14,15]. In Bangkok, Thailand, anal HPV infections were found in 85% of HIV-infected MSM
compared to 58.5% in HIV-uninfected MSM; HPV16 was detected in 22.5% and 9.8% of HIV-
infected and HIV-uninfected MSM, respectively [16]. The prevalence of anal HPV infections
in northern Thailand was 80% among MSM, in which 100% and 70% were found in HIV-
infected MSM and HIV-uninfected MSM, respectively, HPV16 was the most common high-
risk type, accounting for 40% in HIV-infected MSM and 22% in HIV-uninfected MSM [17].
One study reported a high prevalence of HPV16 infection in both HIV-infected (54.9%) and
HIV-uninfected (61.1%) MSM with a histological diagnosis of AIN3 [18]. It was reported that
HPV16 was the most persistent high-risk HPV type [19,20] and less likely to spontaneously
regress from cervical intraepithelial neoplasia (CIN)2-3 to normal when compared to other
HPV types [21,22]. The study in HIV-uninfected MSM [23] and HIV-infected MSM [6,24]
revealed that HPV16 showed the longest duration of infection with the lowest rate of viral
clearance compared to low-risk and other high-risk HPV types.
High-risk HPV is considered to be the causative agent of cervical cancer and other HPV-
related cancers such as vulva, anal, head, and neck cancer. The viral oncoproteins E6 and E7
disrupt the normal function of host proteins involved in cell cycle regulation, where E6 causes
p53 degradation and E7 inactivates retinoblastoma proteins [2527]. However, HPV-related
cancer development takes more than 10–20 years, while most HPV-infected populations spon-
taneously regress [28,29]. The up-regulation of the viral oncogenes E6 and E7 [3032] and the
down-regulation of viral proteins involved in viral particle assembly, such as the L1/L2 pro-
teins [3335], are correlated with cancer progression. Epigenetic modification such as methyl-
ation of the HPV genome is considered to be one factor that controls the expression of viral
genes during productive and transforming infections [36].
Differential methylation of the HPV16 genome has been reported in cervical samples dur-
ing productive infections [36,37], the HPV16 early promoter was unmethylated in basal and
intermediate cells at the proximal E2 binding sites 2–4 (E2BS) but became highly methylated
in superficial cells at the upper part of the epithelium [36]. In latent HPV16 infection, the viral
long control region (LCR), which encompasses the early promoter, was highly methylated
throughout the epithelium. In transforming HPV16-infected cells, the distal E2BS (E2BS1)
and enhancer regions were found to be methylated, while the early promoter was unmethy-
lated [36]. One study showed that the HPV16 p670 late promoter was highly methylated in
cervical carcinoma cases [38]. The low expression of the early viral gene and lack of the capsid
L1/L2 proteins expression in undifferentiated basal cells prevented the activation of an
immune response to viral infection [39].
The methylation pattern in early promoters, especially HPV16 E2BS (CpG positions 31, 37,
43, 52, and 58), has been widely studied in cervical cells, and the observed methylation patterns
were either progressive hypomethylation [4042] or progressive hypermethylation [38,43,44].
One study showed that a high methylation of E2BS was correlated with the episomal form and
multiple copies of integrated HPV genome in high-grade cervical lesions and cervical
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Competing interests: The authors have declared
that no competing interests exist.
carcinoma [45]. HPV 16 L1 gene hypermethylation was correlated with severe cervical lesions
and cervical carcinoma [4649]. It was reported that the HPV16 3’L1 CpG positions 7136 and
7145 [50] and the 5’L1 CpG positions 5600, 5606, 5609, and 5615 (some published paper men-
tioned 5602, 5608, 5611, and 5617, respectively, according to reference sequence used in the
studies [51,52]) were highly methylated in cervical cancers [49,5154]. 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 [5658], and to the best of our knowl-
edge, 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.
Materials and methods
Clinical samples and cell lines
This study was approved by the Institutional Review Board of the Faculty of Medicine, Chula-
longkorn 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 con-
sent 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 Decem-
ber 2013. All samples were anonymized. There were 134 HIV-infected cases and 44 HIV-unin-
fected 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 posi-
tive controls for the amplification and pyrosequencing. They respectively contained approxi-
mately 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 liquid-
based 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.
HPV DNA detection
Extracted DNA samples from LBC were subjected to HPV genotyping using the Linear Array
HPV genotype test (Roche Molecular System, Inc., Mannheim, Germany). Extracted DNA
samples were amplified for HPV genotypes and the beta-globin gene. HPV and beta-globin
amplicons were hybridized with oligonucleotide probes for specific HPV and beta-globin and
detected by colorimetric determination. The test kit could detect the following 37 HPV geno-
types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67,
68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and CP6108.
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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 FACSCountCD4 reagents (BD Biosciences, San Jose, CA, USA) were used to enumer-
ate 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
FACSCountinstrument.
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 manufactur-
er’s instructions. The sequences of forward, reverse, and sequencing primers for the early pro-
moter 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 HiFi-
delity 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 ini-
tial 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 prepara-
tion, 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
Table 1. The sequences of primers used in the present study.
Target gene Nucleotide sequences Size (bp) Reference
Early promoter: CpG 31,37,43,52,
and 58
FW: 5’-TTGTAAAATTGTATATGGGTGTG-3’
RV: Biotin-5´-AAATCCTAAAACATTA CAATTCTC-3’
Sequencing primer:
400S1: 5’-AATTTATGTATAAAATTAAGGG-3’
Sequence to analyze:
YGTAATYGAAATYGGTTGAATYGAAATYGGTTAGTA
192 [59]
3’L1: CpG 7136, and 7145 FW: Biotin-5’-GGTTAAATTAAAATTTATATTAGGAAAA-3’
RV: 5’-AAACATATACACAACAAACAACACTAATTC-3’
Sequencing primer:
800: 5’- TACATACAATACTTACAACT-3’
Sequence to analyze:
TACRTTTTTTACRTTTAACAATTATAAAA
140 [49]
5’L1: CpG 5600, 5606, 5609, and 5615 FW Biotin 5’-TAATATATAATTATTGTTGATGTAGGTGAT -3’ RV
5’-AACAATAACCTCACTAAACAACCAAAA-3’
Sequencing primer:
5600: 5’-CCAAAAAAACATCTAAAAAAAAATATAATA-3’
Sequence to analyze:
AACRTTTACRTCRTTTTCRTAACAT
130 [49]
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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 PyroMarkQ96 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 con-
verted 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 (S1S3 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 dif-
ferences 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 statis-
tically significant difference.
Results
Methylation levels of the HPV16 early promoter and the L1 gene in cervical
cancer cell lines
Of 178 HPV16 positive samples, including 134 HIV-infected and 44 HIV-uninfected samples,
the mean patient age was 31.23 years. Histology results were obtained from 123 samples classi-
fied as normal (n = 16), AIN1 (n = 67), AIN2 (n = 12), and AIN3 (n = 28). The methylation
patterns in the HPV16 early promoter and the two regions within the HPV16 L1 gene were
analyzed. The methylation levels of the early promoter comprising the 5 CpGs (31, 37, 43, 52,
and 58), including the proximal E2 binding sites (E2BSs) and the Sp1 binding site of CaSki,
were 70%, 60.5%, 73.5%, 66.5%, 77%, respectively. For SiHa cells, the methylation levels were
0–1% for all 5 CpGs. The methylation levels of the L1 gene (CpG 5600, 5606, 5609, 5615, 7136,
and 7145) were 84%, 59%, 76%, 65%, 69%, and 67% for CaSki and 95%, 96%, 80%, 80%, 69%,
and 76% for SiHa, respectively, as shown in Fig 1. A high methylation of the L1 gene was
detected in both cervical cell lines regardless of the copy number of integrated HPV16. Pyro-
gram of hyper-methylated and hypo-methylated HPV16 genome was shown in S1S3 Figs.
Methylation levels of the HPV16 early promoter and the L1 gene in anal
cells
The methylation levels of the early promoter in normal, AIN1, AIN2, and AIN3 are shown in Fig
2. The methylation level was <10% in all normal anal samples, while an intermediate to high
methylation level (20–60%) was found in 1.5% (1/67) and 5% (2/40) in AIN1 and AIN2-3 sam-
ples. The majority of AIN1, AIN2, and AIN3 showed a low methylation level in the early
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promoter (<10%). For the L1 gene, a low methylation level was found in all detected normal anal
samples (<20%). There was a slight increase in L1 gene methylation from normal to AIN3, espe-
cially at CpG 5600 and 5609, for which the methylation levels were higher than for other CpGs
(5606, 5615, 7136, and 7145) (Fig 3). Intermediate to high methylation levels (20–60%) of CpG
5600 were found in 0% (0/16) of normal, 10.5% (7/67) of AIN1, 25% (3/12) of AIN2, and 28.6%
(8/28) of AIN3 (P<0.05) cases, while CpG 5609 methylation was found in 0% (0/16) of normal,
3% (2/67) of AIN1, 16.7% (2/12) of AIN2 and 7.1% (2/28) of AIN3 cases (P >0.05) (Table 2).
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. Inter-
estingly, 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
Fig 1. A scatter plot of the methylation percentage of the 11 CpGs. The methylation level of CpGs located within the early
promoter (positions 31, 37, 43, 52, and 58), 3’L1 (positions 7136, and 7145), and 5’L1 regions (positions 5600, 5606, 5609, and
5615) of the Caski and SiHa cell lines. A and B were methylation levels obtained from SiHa and CaSki, respectively. C was the
methylation level of the L1 gene obtained from both SiHa and Caski.
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was first analyzed in CaSki and SiHa control cell lines, where the methylation levels were con-
sistent with previous reports [47,5962]. HPV16 and 18 L1 gene hypermethylation have been
reported previously in cervical carcinoma, vulva intraepithelial neoplasia (VIN), oral carci-
noma, and penile carcinoma [47,48,6368]. Hypermethylation in the L1 gene was found to be
correlated with an integration form of HPV16 [65,69]. The present study found a high methyl-
ation 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). Previ-
ous 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 pat-
terns 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 interme-
diate 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 inte-
grated 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
Fig 2. Methylation percentage of the early promoter in anal samples stratified by histology as normal, AIN1,
AIN2 and AIN3. Error bars in the bar graph represent the mean with standard deviation (SD). The Kruskal–Wallis
test was used to compare the differences among the groups. There were no statistically significant differences among
the groups (P >0.05).
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protein plays a role in controlling viral oncogene expression by either activating or suppressing
viral oncogene promoter depending on the E2 protein concentration [7274]. It was hypothe-
sized 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 sup-
pressive 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
[7881]. 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].
Fig 3. Methylation percentage of the L1 gene in anal samples stratified by histology as normal, AIN1, AIN2 and AIN3. The error bars in the bar graph represent
the mean with standard deviation (SD). The Kruskal–Wallis test was used to compare the differences among the groups. denotes statistically significant differences
among the different lesion severities of CpG 5600 (P <0.05).
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Table 2. Patient age and methylation status of the HPV16 early promoter and the L1 genes in anal cells with various grades of lesions.
Group Normal AIN1 AIN2 AIN3 P value
No. (123) 16 67 12 28
Age (years):
Mean 27.93 31.06 31.3 32.52 >0.05
SD 6.82 6.66 5.66 6.27
Range 22–47 19–50 25–42 22–52
% Methylation level:
CpG 31
Mean 0.300 1.597 0.667 3.00 >0.05
SD 0.483 4.568 0.888 7.299
Range 0–1 0–34 0–2 0–28
% samples 20% 0% 1.5% 0% 7.1% >0.05
% samples <20% 100% 98.5% 100% 92.9%
CpG 37
Mean 0.400 1.016 0.583 2.269 >0.05
SD 0.516 3.011 0.900 5.625
Range 0–1 0–23 0–3 0–23
% samples 20% 0% 1.5% 0% 7.1% >0.05
% samples <20% 100% 98.5% 100% 92.9%
CpG 43
Mean 0.600 1.065 0.250 2.538 >0.05
SD 0.699 4.745 0.452 7.824
Range 0–2 0–37 0–1 0–31
% samples 20% 0% 1.5% 0% 7.1% >0.05
% samples <20% 100% 98.5% 100% 92.9%
CpG 52
Mean 0.700 1.532 1.083 3.115 >0.05
SD 0.823 3.745 1.084 6.364
Range 0–2 0–28 0–3 0–26
% samples 20% 0% 1.5% 0% 7.1% >0.05
% samples <20% 100% 98.5% 100% 92.9%
CpG 58
Mean 0.400 1.500 0.333 3.231 >0.05
SD 0.516 5.331 0.492 8.878
Range 0–1 0–41 0–1 0–35
% samples 20% 0% 1.5% 0% 7% >0.05
% samples <20% 100% 98.5% 100% 93%
CpG 5600
Mean 8.111 9.00 11.500 14.346 >0.05
SD 3.887 8.976 12.494 13.069
Range 0–13 0–50 0–40 0–57
% samples 20% 0% 10.5% 25% 28.6% <0.05
% samples <20% 100% 89.5% 75% 71.4%
CpG 5606
Mean 6.889 3.593 4.417 4.000 >0.05
SD 5.183 3.616 5.017 4.699
Range 0–15 0–16 0–14 0–23
% samples 20% 22% 0% 0% 3.6% >0.05
(Continued )
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Table 2. (Continued)
Group Normal AIN1 AIN2 AIN3 P value
% samples <20% 78% 100% 100% 96.4%
CpG 5609
Mean 3.667 5.833 6.833 8.577 >0.05
SD 3.841 6.279 8.537 8.732
Range 0–10 0–37 0–24 0–35
% samples 20% 0% 3% 16.7% 7.1% >0.05
% samples <20% 100% 97% 83.3% 92.9%
CpG 5615
Mean 6.00 2.648 3.250 3.577 >0.05
SD 3.12 2.789 3.251 4.032
Range 2–12 0–13 0–9 0–20
% samples 20% 0% 0% 0% 3.6% >0.05
% samples <20% 100% 100% 100% 96.4%
CpG 7136
Mean 1.583 2.063 1.917 3.680 >0.05
SD 2.193 2.945 1.621 3.945
Range 0–8 0–17 0–5 0–16
% samples 20% 0% 0% 0% 8% >0.05
% samples <20% 100% 100% 100% 92%
CpG 7145
Mean 2.167 1.365 1.333 2.040 >0.05
SD 3.460 2.180 1.557 3.089
Range 0–10 0–12 0–5 0–12
% samples 20% 0% 0% 0% 8% >0.05
% samples <20% 100% 100% 100% 92%
The P- values were calculated using the Kruskal-Wallis and Fisher Exact tests. indicated a significant difference (P <0.05).
https://doi.org/10.1371/journal.pone.0256852.t002
Table 3. Mean HPV16 gene methylation level of each CpG and the percentual CD4 count between HIV-infected and HIV-uninfected patients.
Group Normal (16) AIN1(67) AIN2(12) AIN3(28) P value
HIV uninfected infected uninfected infected uninfected infected uninfected infected
No. (123) 4 12 18 49 2 10 1 27
Mean %CD4+, Mean cell count NA 19.5%, 389 NA 16.83%, 315 NA 19.2%, 359 NA 16.93%, 326 >0.05
Mean % Methylation
CpG 31 0% 0.43% 2.88% 1.11% 1.5% 0.5% 0% 3.12% >0.05
CpG 37 0% 0.57% 2.0% 0.64% 2.0% 0.3% 0% 2.36% >0.05
CpG 43 0% 0.86% 2.65% 0.47% 0% 0.3% 0% 2.64% >0.05
CpG 52 0% 1% 2.47% 1.18% 2.0% 0.9% 0% 3.24% >0.05
CpG 58 0% 0.57% 3.1% 0.91% 0.5% 0.3% 0% 3.36% >0.05
CpG 5600 10.3% 7% 12.33% 7.72% 5.5% 12.7% 14% 14.36% >0.05
CpG 5606 6.67% 7% 3.73% 3.54% 0% 5.3% 6% 3.92% >0.05
CpG 5609 7% 2% 8.07% 4.98% 1.0% 8.0% 10% 8.52% >0.05
CpG 5615 4.67% 6.67% 3.13% 2.46% 0% 3.9% 5% 3.52% >0.05
CpG 7136 3% 1.11% 2.5% 1.92% 0.5% 2.2% 0% 3.83% >0.05
CpG 7145 3.67% 1.67% 1.69% 1.26% 1.0% 1.4% 0% 2.13% >0.05
NA: Not applicable.
https://doi.org/10.1371/journal.pone.0256852.t003
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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
Fig 4. Correlation between the percentage of HPV16 L1 gene methylation andthe percentage of CD4 counts in AIN3 cases. There
was an inverse correlation between CpGs 5600, 5606, 5609, 5615, 7136, and 7145 methylation and a low percentual CD4 count. A
moderate correlation (R = 0.4692–0.5412) was found at CpGs 5600, 5609, 5615, and 7145.
https://doi.org/10.1371/journal.pone.0256852.g004
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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 nor-
mal 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 sam-
ples should be further studied and evaluated.
Supporting information
S1 Fig. Pyrogram from quantification of methylation by pyrosequencing of HPV16 early
promoter. The highlighted yellow bar represents the internal control (No intensity signal was
found when cytosine was completely converted to uracil) within the analyzed sequence. The
percent methylation value of each CpG site that perfectly pass quality control were indicated
in blue box on the top of the gray bar.
(TIF)
S2 Fig. Pyrogram from quantification of methylation by pyrosequencing of HPV16 5’L1
regions. The highlighted yellow bar represents the internal control (No intensity signal was
found when cytosine was completely converted to uracil) within the analyzed sequence. The
percent methylation value of each CpG site that perfectly pass quality control were indicated
in blue box on the top of the gray bar.
(TIF)
S3 Fig. Pyrogram from quantification of methylation by pyrosequencing of HPV16 3’L1
regions. The highlighted yellow bar represents the internal control (No intensity signal was
found when cytosine was completely converted to uracil) within the analyzed sequence. The
percent methylation value of each CpG site that perfectly pass quality control were indicated
in blue box on the top of the gray bar.
(TIF)
Acknowledgments
We would like to thank Mrs. Vanida Mungmee for her kindly help in CaSki and SiHa cell
cultures.
Author Contributions
Conceptualization: Arkom Chaiwongkot.
Formal analysis: Arkom Chaiwongkot, Parvapan Bhattarakosol.
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HPV16 L1 methylation for predicting anal intraepithelial neoplasia
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Investigation: Arkom Chaiwongkot, Tippawan Pankam.
Methodology: Arkom Chaiwongkot, Tippawan Pankam.
Project administration: Arkom Chaiwongkot.
Resources: Nittaya Phanuphak, Tippawan Pankam.
Supervision: Parvapan Bhattarakosol.
Writing – original draft: Arkom Chaiwongkot.
Writing – review & editing: Arkom Chaiwongkot, Parvapan Bhattarakosol.
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Article
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The human papillomavirus (HPV) can cause different cancers in both men and women. The virus interferes with functions of the cervix, vulva, vagina, anus in the anogenital area, breast, and head and neck cancer due to the local lesions. The tumors lead to death if not treated as a result of distant metastasis to internal organs and brain. Moreover, HPV attenuates the immune system during chronic infection and releases viral antigens into the tumor microenvironment. The tumors know how difficult is to win the battle with a strong united army of immune cells that are equipped with cytokines and enzymes. They confuse the immune cells with secreting viral antigens. The immune system is equipped with cytokines, a complement system, antibodies, and other secretory proteins to overcome the foreign invaders and viral antigens. However, the majority of the time, tumors win the battle without having all the equipment of the immune cells. Thus, in this review, we describe the recent progression in cellular and humoral immunity studies during the progression of HPV-related cancers. First of all, we describe the role of B, plasmoid cells, and B regulatory cells (Breg) in their functions in the tumor microenvironment. Then, different subtypes of T cells such as T CD8, CD4, T regulatory (Treg) cells were studied in recently published papers. Furthermore, NK cells and their role in tumor progression and prevention were studied. Finally, we indicate the breakthroughs in immunotherapy techniques for HPV-related cancers.
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Background: How to best triage human papillomavirus (HPV) positive women remains controversial in an era of HPV primary screening of cervical cancer. Here, we assessed the long-term risk stratification for triaging HPV 16 positive women by standalone HPV 16 methylation and combined with E6 oncoprotein. Methods: A total of 1742 women underwent screening with HPV DNA testing, cytology, and visual inspection with acetic acid (VIA) in 2005 and were followed for 10 years. Seventy-seven women with HPV 16 positivity determined by HPV genotyping test were examined via E6 oncoprotein detection and bisulfite pyrosequencing for quantitative methylation of L1 and LCR genes of HPV 16. Results: The 10-year cumulative incidence rate (CIR) of cervical intraepithelial neoplasia grade 3 or severe (CIN3+) for HPV 16 positive women was 25.3% (95% CI 14.7-37.3%), which significantly increased in women with high methylation at six sites (CpG 5602, 6650, 7034, 7461, 31, and 37) and in women with positive E6 oncoprotein. A methylation panel based on the above six sites showed a competitive risk stratification compared to cytology (HR 11.5 vs. 8.1), with a higher 10-year CIR of CIN3+ in panel positives (57.2% vs 36.8%) and comparable low risk in panel negatives (5.7% vs 4.8%).The sensitivity and specificity for accumulative CIN3+ was 85.7% (95%CI 60.1-96.0%) and 78.4% (95%CI 62.8-88.6%) for a methylation panel and 57.1% (95%CI 32.6-78.6%) and 86.5% (95%CI 72.0-94.1%) for E6 oncoprotein. The AUC values of methylation standalone and the co-testing of methylation panel and E6 oncoprotein were around 0.80, comparable to 0.68 for cytology, 0.65 for viral load, and superior to 0.52 for VIA (p < 0.05). Conclusions: Our findings indicated the promising use of HPV 16 methylation alone or combined with E6 oncoprotein for triaging HPV 16 positive women based on the long-term risk stratification ability.
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Human papillomaviruses (HPV) are a large family of viruses which contain a circular, double-stranded DNA genome of approximately 8000 base pairs. The viral DNA is chromatinized by the recruitment of cellular histones which are subject to host cell-mediated post-translational epigenetic modification recognized as an important mechanism of virus transcription regulation. The HPV life cycle is dependent on the terminal differentiation of the target cell within epithelia-the keratinocyte. The virus life cycle begins in the undifferentiated basal compartment of epithelia where the viral chromatin is maintained in an epigenetically repressed state, stabilized by distal chromatin interactions between the viral enhancer and early gene region. Migration of the infected keratinocyte towards the surface of the epithelium induces cellular differentiation which disrupts chromatin looping and stimulates epigenetic remodelling of the viral chromatin. These epigenetic changes result in enhanced virus transcription and activation of the virus late promoter facilitating transcription of the viral capsid proteins. In this review article, we discuss the complexity of virus- and host-cell-mediated epigenetic regulation of virus transcription with a specific focus on differentiation-dependent remodelling of viral chromatin during the HPV life cycle.
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Background: About 23% of patients develop CIN2+ after LEEP treatment due to residual or recurrent lesions. The majority of patients with HPV infection were HPV negative before treatment, but 16,4% were still HPV 16 positive after treatment, indicating that conization do not necessarily clear HPV infection rapidly. The aim of this retrospective study was to evaluate the possible correlation existing between the appearance of recurring high-grade lesions and the viral genotype 16, and other risk factors such as residual disease. Methods: One hundred eighty-two HPV positive patients underwent LEEP for CIN2+. The follow-up post treatment was carried out every 6 months. Abnormal results during follow-up were confirmed histologically and considered recurrent high-grade intraepithelial cervical lesions (CIN2/CIN3 or CIS). Statistical analysis was performed by using the SPSS software package for Windows (version 15.0, SPSS, Chicago, IL, USA). Descriptive statistics are expressed as frequency, arithmetic mean, standard deviation (S.D.) and percentages. We calculated significance (P < 0.5) with the Easy Fischer Test. We calculated the Odds Ratio (OR) of women with peristent HPV 16 infection and positive margin, to have a recurrence. Results: In our study, the rate of persistent infection from HPV 16, after LEEP, was 15.9% (29/182) with 94% (17/18) of the recurring disease occurring within 18 months of follow up. From this study it was found that the persistence of genotype 16 is associated with a greater rate of relapse post-conization of CIN 2+ lesions, with respect to other genotypes. Our study further supports those studies that demonstrate that the risk for residual disease or relapse is not to be overlooked, also when the margins are negative, but persistent HPV infection is present. In our case study, 40% of relapses were in women with negative margin, but with persistent HPV 16 infection. Even more so, the margins involved in HPV16 positive subjects is another prediction factor for relapse. Conclusions: Our results show the importance of genotyping and that persistent HPV 16 infection should be considered a risk factor for the development of residual/recurrent CIN 2/3.
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Objective: Efficient and highly predictive biomarkers reflecting the prognosis of persistent atypical squamous cells of unknown significance(ASCUS) and low grade squamous intraepithelial lesion(LSIL)s are unavailable and need to be developed urgently. We aimed to develop a predictive model for diagnosis of cervical intraepithelial neoplasia(CIN)2+ by analyzing the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS and LSIL with a high risk of HPV infection. Methods: Cervical cytology samples comprising (70 ASCUS and 215 LSIL Pap smears) were analyzed. Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed. Expression levels of HPV L1 capsid protein in cervical cytology samples were measured, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. The risk for CIN2+ was calculated using the results of immunocytochemistry and the HPV DNA test. Results: Negative results for HPV L1 immunochemistry test were more frequently observed in CIN2+, and expression of the HPV L1 capsid protein was higher in CIN1 or cervicitis (Fisher's exact test, p<0.05). Diagnosis rates for CIN2+ were highest for the combination of HPV L1 capsid protein immunocytochemistry, cytology and HPV test when compared with other combinations (Akaike information criterion (AIC): 191.7, Schwarz criterion(SC): 206.3, p<0.001). Conclusion: Absence of HPV L1 capsid expression and presence of HPV type 16 or 18 infection are reliable predictors of progression to CIN2+ in patients showing persistent ASCUS and LSIL.
Article
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Objectives Cervical cancer is the most common malignant tumors in women leading to serious morbidity and mortality worldwide, especially among developing countries. A main cause of the disease is the high-risk human papillomavirus (HR-HPV) infection. HSIL usually progress to cervical cancer, and low-grade lesions, including LSIL and ASCUS, mostly turn to normal or benign lesions, but there are still a small number of patients who will progress to HSIL. Up to now there is no efficient biomarker clinically available to predict people with high risk to progress into HSIL. This study was conducted to evaluate the value of human papillomavirus (HPV) DNA, p16INK4a protein, and HPV L1 capsid protein in predicting HSIL and minimizing unnecessary colposcopy treatments. Methods 1222 patients with HR-HPV infection or with abnormal Thinprep cytologic test (TCT) were chosen to conduct colposcopy in the cervical out-patient clinic of Shanghai First Maternity and Infant Hospital affiliated to Shanghai Tongji University from June 2014 to January 2017. TCT, cervical biopsy, HPV DNA and HPVL1 were performed on all patients. 110 patients were selected to detect p16INK4a protein. Hybrid capture 2 (HC-2) was used to detect HPV DNA, and their subgroups using gene typing system. Immunohistochemical technology was used to detect HPV L1 and p16. Results HPV DNA was positive in 1097 cases, with the positive rate of 89.7% (1097/1222). In particular, the positive expression rates of HPV DNA were 82.3, 95.7, 96.6 and 100% in Normal/CC, LSIL, HSIL and cervical cancer groups, respectively (p < 0.001). HPV L1 was negative in 781 cases with HR-HPV infection, and the overall negative rate is 71.1%. In patients with Normal/CC, LSIL and HSIL, the negative expression rates of HPV L1 were 91.3, 40 and 81.2%, respectively (p value < 0.001). In the 110 patients, HPV L1 was negative in 98.1% (53/54) of Normal/CC, 42.9% (12/28) of LSIL and 85.1% (23/27) of HSIL (p value = 0.0043). P16-positive rates in patients with Normal/CC, LSIL and HSIL were 33.3% (18/54), 75% (21/28) and 96.2% (26/27), respectively (p value < 0.001). 18 out of 28 cases express low positive (+) in LSIL, 25 out of 27 cases express strong positive (3+) in HSIL. Patients with L1(−) p16(+) including 18.5% (10/54) of normal/cervicitis, 60.7% (17/28) of LSIL and 85.1% (23/27) of HSIL (p value < 0.005). Furthermore, patients with L1(−) p16(1+) included 37% (10/27) of normal/cervicitis 59.3% (16/27) of LSIL and 3.7% (1/27) of HSIL; patients with L1(−) p16(2+) consisted of 0% of normal/cervicitis/LSIL and 100% (1/1) of HSIL; patients with L1(−) p16(3+) were composed of 0% of normal/cervicitis, 4.5% (1/22) of LSIL and 95.5% (21/22) of HSIL (p value < 0.005) (Table 6). Conclusion With the increase in the degree of the cervical lesions, the expression of HPV DNA and p16 is up-regulated while HPV L1 protein is down-regulated. HPV DNA, HPV L1 and p16 are useful markers for the prediction of HSIL. Combined detection of these three markers has important potential to predicting HSIL and minimizing unnecessary colposcope examination.
Article
Background Prospective data on the natural history of anal human papillomavirus (HPV) infection are scarce in human immunodeficiency virus (HIV)-infected men who have sex with men (MSM). Methods We analyzed incidence and clearance of HPV-16 and HPV-18 in a French cohort of HIV-infected MSM, aged ≥35 years, followed-up annually (n = 438, 2014–2018). Results Human papillomavirus-16 and HPV-18 incidence were similar (~10% incident infections at 24 months). Human papillomavirus-16 incidence was higher among high-grade versus no lesion at baseline (adjusted incidence rate ratio = 3.0; 95% confidence interval, 1.07–8.18). Human papillomavirus-16 cleared significantly slower than HPV-18 (32% versus 54% by 24 months). Conclusions In conclusion, anal HPV-16 is more persistent than HPV-18, and its incidence correlates with a prior detection of high-grade lesions.
Article
Background: People living with HIV (PLWH) have a markedly elevated anal cancer risk largely due to loss of immunoregulatory control of oncogenic human papillomavirus infection. To better understand anal cancer development and prevention, we determined whether recent, past, cumulative, or nadir/peak CD4+ T-cell count (CD4) and/or HIV-1 RNA level (HIV RNA) best predict anal cancer risk. Methods: We studied 102,777 PLWH during 1996-2014 from 21 cohorts participating in the North American AIDS Cohort Collaboration on Research and Design. Using demographics-adjusted, cohort-stratified Cox models, we assessed associations between anal cancer risk and various time-updated CD4 and HIV RNA measures, including cumulative and nadir/peak measures calculated within pre-specified moving time windows. We compared models using the Akaike's information criterion. Results: Cumulative and nadir/peak CD4 or HIV RNA measures from ~8.5 to ~4.5 years in the past were generally better predictors for anal cancer risk than their corresponding measures from ~4.5 years to ~6 months in the past. However, the best model included CD4 nadir (i.e., the lowest CD4) from ~8.5 years to ~6 months in the past (hazard ratio for <50 vs. ≥500 cells/µL: 13.4; 95% confidence interval: 3.5-51.0) and proportion of time CD4 <200 cells/µL from ~8.5 to ~4.5 years in the past (a cumulative measure; hazard ratio for 100% vs. 0% of time: 3.1; 95% confidence interval: 1.5-6.6). Conclusions: Our results are consistent with anal cancer promotion by severe, prolonged HIV-induced immunosuppression. Nadir and cumulative CD4 may represent useful markers for identifying PLWH at higher anal cancer risk.
Article
The incidence and mortality of anal squamous cell carcinoma (SCC) are expected to continue to increase in the next 20 years. High-risk groups for anal SCC, i.e., human immunodeficiency virus (HIV)-positive patients, men who have sex with men (MSM), women with previous genital neoplasia, and solid-organ transplant recipients, have been identified. HIV-positive MSM have the highest risk, and some societies have advocated for anal cancer screening to be done in this population. Screening for anal SCC follows the same principles as that for cervical cancer since there are similarities between the two types of cancers. Anal cytology has been recommended as an initial screening method for high-risk groups, e.g., HIV-positive MSM. Normally, the cytology is liquid based and collected blindly by a clinician using a Dacron swab and it is especially used for internal lesions detection. The sensitivity to predict anal high-grade squamous intraepithelial lesions is higher in immunosuppressed patients with a high burden of the disease. The report should include the classification, normally according to the Bethesda terminology and the sample adequacy, in a manner similar to that for cervical cytology. In cases involving unsatisfactory samples, it is important to repeat the procedure given the prevalence of anal squamous cytological abnormalities in follow-up cytology procedures. The absence of transformation zone cells in anal cytology seems to increase the risk of false-negative results.
Article
Background: Anal canal carcinoma is relevant because it commonly occurs in high-risk groups, and its incidence has been increasing. Objective: This study evaluated the accuracy of anal cytology in the screening of precursor lesions of anal cancer, compared with histopathologic examination as the reference, in all subjects and in men who have sex with men, HIV-infected men and women, and men who have sex with men and HIV-infected subgroups. Data sources: The data included studies identified in the MEDLINE, Latin American and Caribbean Health Sciences, Cochrane Library, and Embase electronic databases, as well as in the grey literature. The search terms included anal cancer, anal dysplasia, anal intraepithelial neoplasia, screening, and anal cytology. Study selection: After excluding studies with no histopathological data and those with duplicate and missing data, 34 primary studies were included. Intervention: Cytology of anal smears was studied. Main outcome measures: Sensitivity, specificity, diagnostic OR, and area under the curve were measured. Results: A total of 5093 patients were included. The pooled sensitivity of anal cytology was 85.0% (95% CI, 82.0%-87.0%) and pooled specificity was 43.2% (95% CI, 41.4%-45.1%) for the detection of anal intraepithelial neoplasia grade 2 or worse versus anal intraepithelial neoplasia grade 1 and normal when measuring all subjects. The accuracy of anal cytology was higher in the men who have sex with men and HIV-infected and men who have sex with men only subgroups. Limitations: This study was limited by its specificity. Conclusions: The study results support the hypothesis that cytology is a good test for the screening of anal cancer.
Article
Anal cancer incidence and mortality have been increasing over the past decade. Although the incidence in the general population remains low, it is much higher in certain subgroups, including those living with human immunodeficiency virus and men who have sex with men. Approximately 90% of anal squamous cell cancers are caused by infection with carcinogenic human papillomavirus (HPV). Given the common etiology between anal and cervical carcinogenesis, screening for anal cancer has been proposed in certain high‐risk populations using strategies adapted from cervical cancer prevention. In this review, the authors discuss important differences in anal and cervical cancer regarding the populations at risk, disease natural history, and clinical procedures and outcomes that need to be considered when evaluating strategies for anal cancer screening. They also performed a systematic review and meta‐analysis of the performance of anal cytology, anal HPV testing, and various biomarkers for the detection of anal precancers and cancers. The implications of these performance estimates are summarized in the context of risk‐based screening and management of anal precancers, and important research gaps are highlighted that need to be addressed to fully understand the benefits and harms of anal cancer screening. Cancer Cytopathol 2018. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.