NEW YORK (GenomeWeb) – University of Delaware spinout Genome Profiling (GenPro) is developing technology to analyze genome-wide methylation profiles with the ultimate goal of developing diagnostic tests based on those profiles.
During a presentation last month at Cambridge Healthtech Institute's Clinical Genome Conference in San Francisco, GenPro Co-Founder and CSO Adam Marsh discussed the technology, which makes use of restriction enzymes and proprietary algorithms to evaluate methylation status of CpG islands and is compatible with next-generation sequencing workflows.
Marsh also highlighted current studies GenPro and academic collaborators are conducting in triple negative breast cancer and acute myeloid leukemia to identify epigenetic biomarkers that correlate with prognosis and treatment response.
Marsh had originally started studying environmental imprinting on organisms living in the extreme cold environment of Antarctica's sea ice, he told GenomeWeb after the conference. He developed methods and statistical platforms to study DNA methylation in these organisms, but realized he could also use these tools to study methylation profiles of any organism. Eventually, he turned his attention toward cancer.
Currently, the most common, gold-standard method for analyzing genomic methylation is to first bisulfite convert the genome, which can be done in either a targeted fashion or genome-wide. Bisulfite conversion converts unmethylated cytosines to uracil, leaving methylated cyotosine untouched, so that sequencing can distinguish between the two.
However, said Marsh, bisulfite sequencing has a number of drawbacks. For one, it requires a large amount of starting material because there is significant loss at each step during the chemical oxidation reactions.
In addition, he said, much of the actual sequence information is lost. Because all the unmethylated cytosines have been converted to uracil, they are read out as adenine and indistinguishable from thymines on the template strand, which are also read out as adenine. "In the end, it's a real challenge to map those reads back to the reference," he said.
By contrast, GenPro's DNA prep involves the use of restriction enzymes that do not chemically alter the DNA. The prep "doesn't impact other sequencing applications," such as normal variant calling, Marsh said. DNA is "prepped as it would be for a normal whole-genome analysis, aside form one fragmentation step up front." It can then be run through a standard variant calling pipeline and also GenPro's methylation profile analysis pipeline, which computationally reconstructs methylation status of the CpG islands.
The approach measures the probabilities that a particular cytosine within a CpG island is methylated or unmethylated. According to Marsh, incorporating both probabilities gives a more accurate picture and identifies cells within a "dynamic range" of between 20 percent and 80 percent methylated.
For instance, in an initial comparison of its technique with standard bisulfite sequencing of Parkinson's disease blood samples, GenPro demonstrated that bisulfite sequencing tended to give a bimodal distribution — with mean methylation scores either 0 percent or 100 percent. Nearly 40 percent of CpG sites were scored as being 0 percent methylated. By contrast, GenPro's approach classified less than 20 percent of those sites having 0 percent methylation, and found many more CpG sites with intermediate scores between 20 percent and 80 percent methylated.
In a clinical proof-of-principle study with Christiana Care Cancer Health System in Delaware, GenPro applied its technology to four 30- to 40-year-old women with triple negative breast cancer. Biopsy tissue was collected during mastectomy and both tumor and normal tissue was analyzed. The team identified 12 CpG sites that accounted for methylation differences between the tumor and normal samples. In the normal samples, those sites had mean methylation of nearly 60 percent, while in the tumor samples mean methylation was around 38 percent.
Looking at the genes and pathways of those 12 CpG sites, the researchers found that many were related to inflammation response pathways. GenPro and Christiana Care Cancer Health System are now expanding the study to 18 patients. The goal is to identify biomarkers of triple negative breast cancer that can distinguish between tumors that are likely to become invasive.
"The idea is that by picking up the early biomarkers of tumor aggressiveness, clinicians can decide at a very early stage whether a particular patient has an aggressive form and should immediately go to surgery, versus a patient with a non-aggressive form" for whom chemotherapy would be more appropriate, Marsh said.
The firm is also collaborating with an undisclosed cancer institution to study acute myeloid leukemia and epigenetic markers of patient response to Vidaza, a first-line hypomethylating agent. Some patients have a strong remission response, while others have no response to the agent..
GenPro CEO and Co-founder Jeb Connor told GenomeWeb that the firm's business model is one of collaboration and not developing and selling kits. It is looking to partner with academic institutions, pharmaceutical companies, and diagnostic firms interested in epigenetic profiling as a way of stratifying patients for a specific therapeutic, or predicting disease prognosis, he said.
The ultimate goal after identifying predictive biomarkers is to develop a clinical assay to test for such biomarkers. Such diagnostic tests would likely be based on quantitative PCR, Marsh said, and would "deliver very defined and clear therapeutic recommendations."
Epigenetic profiling is still a relatively new field in the clinical space, but researchers are increasingly realizing its importance, particularly in cancer. For instance, last year, researchers identified an association between methylated BRMS1 and reduced overall survival in non-small cell lung cancer patients.
Another group found that assessing methylation status in a panel of genes could help monitor breast cancer recurrence and treatment response.