A study of anal tissue samples from HIV-positive men has shown that high-grade pre-cancerous lesions are associated with similar patterns of DNA methylation to cervical lesions. The study’s findings were published in the June 1st edition of The Journal of Acquired Immune Deficiency Syndromes.
Since these samples also had high levels of human papillomavirus (HPV) DNA, measuring the extent of methylation along with HPV viral loads could be used to predict the chances of a patient developing anal cancer.
DNA methylation is a mechanism used by human cells to turn off the genes of invading organisms, such as viruses. By chemically modifying some of the DNA’s nucleotide building blocks, methylation prevents the cell’s machinery from attaching to the DNA and co-ordinating the expression of the virus’s genes.
To investigate whether methylation is involved in controlling the severity of pre-cancerous lesions in the anus or ‘anal intraepithelial neoplasia’ (AIN), researchers from the University of California Los Angeles and the University of Virginia Health System examined anal tissue samples from 53 HIV-positive gay men. They looked for the presence of human papilloma virus (HPV) in the samples and examined the extent of methylation of the virus’s DNA.
The investigators found that all of the men had HPV-infection in the anus, with 16 of them carrying the high-risk type 16. All but one of these 16 men had HPV-16 in combination with up to twelve other high- or low-risk HPV types.
Although there was considerable variability between samples from different men and between multiple samples taken from each man, the investigators found that 12% of the HPV-16 samples had evidence of DNA methylation. They note that this is similar to the frequency seen in samples taken from the cervix of HIV-negative women with pre-cancerous lesions, highlighting the similarities between cervical and anal disease.
“HPV-16 DNA methylation occurs in anal lesions of HIV-infected men at a frequency similar to that observed in cervical samples,” write the investigators. “It thus has to be considered as a factor that influences aetiologic outcome of this infection at this site and in this patient population."
“Overall, methylation of HPV-16 genomes occurs qualitatively and quantitatively in a similar manner in cervical and anal tissue,” they conclude.
When they investigated which parts of the viral DNA were methylated, the research team saw that samples from patients with low-grade AIN had low levels of methylation in the ‘promoter’ regions (one [2%] of 50 samples) and high levels in the ‘enhancer’ regions (13 [30%] of samples).
The promoter region of a gene is the site of attachment for the ‘RNA polymerase’ enzyme that turns a gene on. In contrast, the enhancer regions are short stretches of DNA that turn a gene’s activity up or down in response to factors release in the cell.
In contrast, samples from men with high-grade AIN had high levels of methylation in the promoter regions (36 [32%] of 110 samples; p < 0.001) and low levels in the enhancer regions (8 [1%] of samples; p = 0.007).
In agreement with previous findings, the investigators also found that high-grade AIN was more common in men infected with more different types of HPV (median 9 vs. 1; p < 0.001). These men also had higher HPV viral loads (median 30,700 vs. 24,400 copies per 10,000 cells; p = 0.002).
“These data suggest a correlation of anal pathologic findings, multiplicity of HPV types, viral DNA load and preferential methylation of promoter sequences,” conclude the researchers.
Although it may be expected that methylation of the promoter regions would prevent the virus’s cancer-causing genes being turned on, the investigators suggest that the risk of progression towards cancer could be determined by a combination of methylation and viral load.
They suggest that, early in infection, HPV remains unincorporated into the human cell’s DNA. During this ‘latent’ phase, the virus is able to replicate but cannot express its cancer-causing genes.
Later in infection, the virus’s genetic material becomes incorporated into the human cell’s DNA, which stimulates the cell to methylate the promoter region in an attempt to prevent the production of the virus’s cancer-causing genes.
However, the investigators argue that high HPV viral loads mean that not all of the HPV DNA can be inhibited by methylation. In cells with high viral loads, the virus’s cancer-causing genes overcome the methylation process. “These cells would combine methylation with the high load of HPV genomes typical of many tumour cells, as reflected in our data,” explain the researchers, ”whereas latently infected cells would often combine methylation with a low viral load.
“Based on this report, we plan to follow the leads of our data and suggest that HPV-16 promoter methylation combined with a high viral genome load may identify clinical samples with clones that signal the emergence of tumour cells,” they conclude.
Wiley DJ et al. Methylation of human papillomavirus genomes in cells of anal epithelia of HIV-infected men. J Acquir Immune Defic Syndr 39: 143-151, 2005.