NEW YORK — Researchers in the US and China said on Tuesday that they are collaborating to develop a liquid biopsy test that detects life-threatening complications related to damaged blood vessels in patients with type 2 diabetes.
They reported details of the test, which uses next-generation sequencing to detect changing levels of 5-hydroxymethylcytosine (5hmC) in circulating cell-free DNA, recently in Clinical Chemistry. The test enabled them to distinguish patients with type 2 diabetes who had vascular complications from patents without complications.
The researchers are using the technology to develop a commercial diagnostic test for early detection of diabetes complications and several cancers, said Wei Zhang, one of the study authors and an associate professor of cancer epidemiology and prevention at the Chicago-based Northwestern University Feinberg School of Medicine.
Cesar Castro, director of the cancer program at Massachusetts General Hospital Center for Systems Biology, who is not affiliated with the current study, said that "the opportunity to detect emerging disease or its complications through a blood test has galvanized a cadre of multidisciplinary investigators." Castro along with colleagues at MGH and Harvard Medical School are developing a liquid biopsy assay that targets early detection of pancreatic cancer.
He said that the morbidity and mortality conferred by diabetes makes it an ideal disease process to test. "The promise of the [5hmC biomarker] work centers around the potential to intervene earlier and hopefully redirect disease trajectories," he said.
In the Clinical Chemistry study, the collaborators developed a selective chemical labeling strategy for 5hmCs and deployed it to search for the markers in cfDNA samples from patients with type 2 diabetes. They evaluated whether the biomarkers could identify patients who had developed complications but were not manifesting symptoms.
The researchers investigated how well the test performed using blood samples from 62 diabetic patients. Twelve patients had no vascular complications, 34 had a singular vascular complication, and 16 had multiple vascular complications.
The researchers reported finding "statistically significant changes" in 5hmC markers associated with type 2 diabetes-related with both macrovascular and microvascular complications.
They said a 16-gene 5hmC panel for vascular complications performed well in distinguishing patients with or without complications and proved itself better than frequently used clinical variables including the duration of diabetes, body mass index, and estimated glomerular filtration rate (eGFR), a routine test for evaluation of kidney function.
In measurements of clinical accuracy for detecting eGFR, the investigators saw an overall area under the curve of .53, while the 16-gene panel for vascular complications showed an area under the curve of. 85.
The group reported that a separate 13-gene 5hmC panel for vascular complications outperformed clinical variables in distinguishing patients with single from multiple complications. The researchers discovered that while a creatinine biomarker showed an area under the curve of .75 and was the best performer among the clinical variables that they tested, the 13-gene panel showed an area under the curve of .84.
About two-thirds of the 424 million diabetic patients worldwide die from vascular complications, according to Northwestern. Patients attend clinics so their physicians can measure blood sugar, glucose, and A1C levels associated with diabetes. Measurements of blood pressure are indicative of cardiovascular health, and the level of albumin in urine is a clinical indicator of kidney disease for diabetes patients.
However, none of these or other routine tests predict with much certainty that a diabetes patient is developing vascular complications, Zhang said, adding, "In the study, our 5hmC markers performed significantly better than existing clinical indicators."
Zhang said he anticipates that the test will focus on monitoring the progress of patients with diabetes. When patients attend clinics for routine tests, clinicians could order the biomarker test to detect whether their patients are developing specific life-threatening complications, he said.
Along with Chuan He, a developer of the technology and professor in the department of chemistry at the University of Chicago, Zhang anticipates commercializing a diagnostic test based on the findings from this and earlier studies. But, the marker also has applications beyond evaluating complications associated with diabetes.
In July, Zhang, He, and their colleagues had published the results of a prospective study in the journal Gut that involved the application of the platform to find 5hmC in cfDNA for early detection of hepatocellular carcinoma, or liver cancer, in 2,554 patients.
"In our cancer studies we have shown that the technology is effective when a couple of hundred cells are available in the blood," Zhang said. To operate effectively, a 2 milliliter plasma sample needs less than 1 nanogram of cell-free DNA, he added.
In some instances, when a medical condition requires only a few genes for diagnosis, a lab could use PCR instead of next-generation sequencing, he noted. For its sequencing work, the group is using the Illumina NextSeq 500, but any Illumina sequencer works with the test, Zhang said.
To commercialize its test, the group is considering several options, including getting US Food and Drug Administration clearance and establishing or engaging with a laboratory to conduct lab-developed testing.
The group is exploring its platform's potential as an alternative to tissue biopsy-based testing in detecting lymphoma, multiple myeloma, and colon cancer, among other medical conditions.
The University of Chicago holds a patent for the 5hmC technology that the researchers have developed, while a company under the control of Zhang, He, and colleagues have licensed the technology and established a commercialization plan. Zhang said that the firm anticipates making the test available at an "affordable" price that would be less expensive than tests that require whole-genome sequencing because it sequences only part of the genome.
"As for the relevance of 5hmC to detect early-stage cancers, there remains significant work to pursue to ensure it does not meet the fate of various others’ prior attempts to identify [a universal marker or markers] for all cancers," Castro said. "The interplay between inflammation and tumor cannot be emphasized enough, so clarity as to whether signals are actually arising from non-tumor protagonists would be needed."
Castro noted that 5hmC is an epigenetic marker found in every cell that is differentially expressed from cell to cell. The main value proposition offered by the recently reported biomarker test is a selective chemical labeling strategy for 5hmC to enable more sensitive mapping in limited clinical materials, he said. "Whether the labeling strategy can extend to other biomarkers is unclear, but it could be an important advance given the increasing multiplexing needs of the field," Castro added.
He said that if the 5hmC biomarker platform is vetted in a larger number of patient samples, it "would derive more near-term benefits with the more defined vascular complication approach" than its application to "the higher hanging fruit of developing universal cancer screening which is being alluded to."
If Zhang, He, and their colleagues are successful in developing a test for liver cancer, they would face a highly competitive and rapidly evolving market, as several firms and research groups are exploring the potential of liquid biopsy-based DNA tests for cancer detection.
For example, while its Cologuard colorectal cancer diagnostic test has been steadily gaining market share, Exact Sciences has been collaborating with the Mayo Clinic to develop a liquid biopsy test for liver cancer. Geisinger Health and Johns Hopkins University have also teamed up to study whether a liquid biopsy test that combines targeted circulating tumor DNA analysis with a number of protein markers can detect early cancers, including liver cancer, in healthy people.
Meanwhile, there are many diagnostic players, such as Foundation Medicine, Guardant, Personal Genome Diagnostics, OncoCyte, and Grail pursuing, or have already commercialized, liquid biopsy tests for a variety of cancers.