ZNF154 hypermethylation signature
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Pan-Cancer Epigenetic Signature Readable in Circulating Tumor DNA
If cancer has a signature, the dotted line may be a gene called ZNF154, say scientists at the National Institutes of Health. Although the scientists don’t know exactly what the gene does, they do know that it may carry distinctive methylation marks, and that these marks have been associated with multiple types of cancer. In their most recent work, the scientists have evaluated whether these marks might serve as a universal cancer biomarker.
The team, led by Laura Elnitski, Ph.D., a computational biologist at the NIH’s National Human Genome Research Institute, previously identified hypermethylation around the ZNF154 gene in 15 solid epithelial tumor types from 13 different organs. In their current work, which was described February 5 in the Journal of Molecular Diagnostics, these scientists are going further. They are testing whether ZNF154 hypermethylation can distinguish tumor samples from normal tissue samples. What’s more, the scientists are running computer simulations to evaluate whether the tiny amounts of tumor DNA that end up circulating in blood can be detected on the basis of ZNF hypermethylation.
The results of this work led the scientists to conclude that ZNF hypermethylation is a relevant biomarker for identifying solid tumor DNA. Moreover, the scientists say that it “may have utility as a generalizable biomarker for circulating tumor DNA.”
Details appeared in the Journal of Molecular Diagnostics article (“Robust Detection of DNA Hypermethylation of ZNF154 as a Pan-Cancer Locus with in Silico Modeling for Blood-Based Diagnostic Development”), which described how the magnitude and pattern of ZNF hypermethylation across colon, lung, breast, stomach, and endometrial tumor samples were measured using next-generation bisulfite amplicon sequencing.
“To evaluate this site as a possible pan-cancer marker, we compare the ability of several sequence analysis methods to distinguish the five tumor types (184 tumor samples) from normal tissue samples (n = 34),” wrote the authors. “The classification performance for the strongest method, measured by the area under (the receiver operating characteristic) curve (AUC), is 0.96, close to a perfect value of 1. Furthermore, in a computational simulation of circulating tumor DNA, we were able to detect limited amounts of tumor DNA diluted with normal DNA.”
Even when the scientists reduced the amount of methylated molecules by 99%, the computer could still detect the cancer-related methylation marks in the mixture. Knowing that tumors often shed DNA into the bloodstream, the scientists calculated the proportions of circulating tumor DNA that could be found in the blood.
Going forward, Dr. Elnitski’s group will begin screening blood samples from patients with bladder, breast, colon, pancreatic, and prostate cancers to determine the accuracy of detection at low levels of circulating DNA. Tumor DNA in a person with cancer typically constitutes 1–10% of all DNA circulating in the bloodstream. The group noted that when 10% of the circulating DNA contains the tumor signature, their detection rate is quite good. Because the methylation could be detected at such low levels, it should be adequate to detect advanced cancer as well as some intermediate and early tumors, depending on the type.
Dr. Elnitski’s group will also collaborate with Christina Annunziata, M.D., Ph.D., an investigator at the National Cancer Institute (NCI). The scientists will test blood samples from women with ovarian cancer to validate the process over the course of treatment and to determine if this type of analysis leads to improved detection of a recurrence and, ultimately, improved outcomes.
Current blood tests are specific to a known tumor type. In other words, clinicians must first find the tumor, remove a sample of it and determine its genome sequence. Once the tumor-specific mutations are known, they can be tracked for appearance in the blood. The potential of the new approach is that no prior knowledge of cancer is required, it would be less intrusive than other screening approaches like colonoscopies and mammograms and it could be used to follow individuals at high risk for cancer or to monitor the activity of a tumor during treatment.
“Finding a distinctive methylation-based signature is…a technical challenge, but we found an elevated methylation signature around the gene known as ZNF154 that is unique to tumors,” declared Dr. Elnitski. “We have laid the groundwork for developing a diagnostic test, which offers the hope of catching cancer earlier and dramatically improving the survival rate of people with many types of cancer.”
NIH Team IDs Recurrent Methylation Site in Five Cancer Types
NEW YORK (GenomeWeb) – Multiple tumor types are recurrently methylated at a specific CpG island, according to researchers from the National Institutes of Health. In addition, the biomarker could potentially be analyzed noninvasively to detect late stage and potentially intermediate and early stage cancers.
In a study published today in the Journal of Molecular Diagnostics, the researchers described a specific site, ZNF154, that is methylated across five different tumor types — lung, stomach, colon, breast, and gynecologic — and can be identified through a targeted bisulfite sequencing strategy. In addition, the researchers performed a computational simulation of circulating tumor DNA, demonstrating that they could detect the methylation biomarker on tumor DNA in a solution composed of 99 percent normal DNA.
The work builds on a previous study in which the team discovered that the epigenetic marker was found across a wide variety of samples in the Cancer Genome Atlas. In the more recent study, the researchers sought to test the reproducibility of the marker as well as whether it could be identified from blood.
The researchers looked at 184 tumor samples across the five different cancer types as well as genomic DNA extracted from 34 normal tissue samples. First, they validated the idea that the targeted bisulfite sequencing protocol would be robust even on small amounts of DNA, since bisulfite conversion can fragment and damage DNA. After verifying that the methylation signal was “robust” with “minimal variation” they moved on to testing the 184 tumor samples.
The DNA from the samples was bisulfite converted and the researchers designed a 302-base PCR product covering the ZNF154 CpG island, including the transcriptional start site, which they had previously found to be hypermethylated in the TCGA data. They then sequenced the amplicons on the Illumina MiSeq instrument.
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“The large magnitude of this hypermethylation bodes well for a strong discriminant in each tissue type,” the authors wrote.
Nonetheless, they noted that there were still a few tumor samples that had lower levels of methylation than the average methylation in the normal samples, including one sample each from lung, stomach, and colon, and three breast samples.
Within each tumor type, the researchers noted a couple of additional interesting findings. For instance, within the lung cancer samples, small cell carcinomas and squamous cell carcinomas had average methylation levels around 15 percent higher than adenocarcinomas and bronchioalveolar carcinomas.
The group next wanted to assess whether ZNF154 methylation could be used as a pan-cancer marker in blood-based screening. The researchers randomly matched one of the 34 normal samples to each of the 184 tumor samples and created mixtures of various dilutions ranging from 10 percent normal to 99 percent normal.
They were able to reliably identify tumor DNA in mixtures containing up to 90 percent normal tissue. Using the area under a curve measurement (AUC), which plots the false positive rate and true positive rate, the team achieved an AUC of .89. When the percentage of normal data was increased to 99 percent, AUC dropped to .74.
Recurrent patterns of DNA methylation in the ZNF154, CASP8, and VHL promoters across a wide spectrum of human solid epithelial tumors and cancer cell lines.
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