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Researchers Find Plasma Tumor Marker in Canine Indicating Metastasis

NEW YORK (GenomeWeb News) – Similar to human breast cancers, canine mammary tumors are beset by chromosome instability, researchers from Chronix Biomedical and the Institute of Veterinary Medicine at the University of Göttingen in Germany reported in PLOS One yesterday. While the dogs' tumors exhibited variations in chromosomal and copy-number changes, about half of the cancers analyzed contained a deletion on chromosome 27.

The researchers also were able to detect specific chromosomal breakpoints in cell-free DNA collected from plasma. For one animal, they found such tumor-specific DNA more than a year after the dog had been treated surgically, indicating the presence of metastases.

"We were able to detect the persistence of a plasma tumor marker after surgery in one of the canine patients, directing veterinarians to schedule a tomographic examination that revealed metastatic lesions in the lung," Julia Beck, a senior scientist at molecular diagnostics firm Chronix Biomedical and first author of the study, said in a statement. "We're confident that the same approach could be reliably used to analyze human blood samples."

Chronix has developed a droplet digital PCR-based assay to measure circulating cell-free DNA indicative of organ transplant rejection, and said it is now extending its cell-free DNA studies to monitor tumor status and residual disease in people. Clinicians, the company added, could use such 'liquid biopsies' early on as well as after treatment or surgery.

For this study, Beck and her colleagues collected tumor and normal samples from five dogs with mammary carcinomas belonging to three histological subtypes. Those samples were then subjected to mate-pair sequencing using Life Technologies' SOLiD4+ system, which generated an average 37 million mappable reads.

Using CNV-seq, the researchers generated copy-number profiles of the five tumors. From this, they found that one dog, referred to as T49, appeared to have a tumor with shattered chromosomes and with a number of copies of chromosomes CFA27 and CFA35. Meanwhile, another dog's tumor seemed to have lost a number of chromosomes and yet another appeared to have nearly normal copy-number levels.

Further, by using SVDetect the researchers unearthed other structural aberrations, including some 54 somatic rearrangements. As before, T49 contained a number of aberrations. That tumor genome also housed a number of breakpoints that clustered on chromosomes CFA27 and CFA35.

"The five tumor genomes are heterogeneous, with most of the detected aberrations not shared by more than two tumors," the researchers noted.

One region, though, was deleted in a number of the tumors. Two tumors lacked chromosome CFA27 in its entirety while another two had segmental deletions in that region.

Using the QX100 Droplet Digital PCR System from Bio-Rad, Beck and her team confirmed the deletion in those four tumors and found it in six of a further 15 canine tumors that they analyzed.

The deletion, the researchers noted, likely affects the nearby tumor suppressor gene PFDN5, which usually represses the expression of the MYC oncogene product.

"This is the first report showing that the gene is recurrently deleted in canine tumors," Beck and her colleagues added. "Future studies are needed to determine whether the recurrent PFDN5 deletion can be confirmed in a larger group and whether this deletion is specific to mammary tumors."

For four of the samples, Beck and her colleagues determined tumor-specific signatures based on their breakpoint patterns and developed PCR primers specific to those patterns.

Using droplet digital PCR and those specific primers, they were able to detect cell-free DNA from the tumors floating in the dogs' plasma. For the dog T49, before surgery, they noted that its fraction of tumor-specific DNA was 11 percent, and those levels decreased to 5 percent 83 weeks later, though not all the way down to zero. A tomographic examination of the dog found metastatic lesions in the lung.

"With the advance of more cost-effective digital PCR instrumentation with a high inherent sensitivity and precision, it appears possible to detect such subtle quantitative differences in cfDNA samples directly," Beck and her colleagues wrote, noting that similar approaches have been used to detect fetal aneuploidies in maternal blood.

However, they cautioned that detecting individual breakpoints to create the signatures is an "elaborate and costly" process and noted that tumor monitoring is not commonly done for dogs.

Such an approach, though, could be applied to people, and Chronix, the company for which most of the study authors work, said that it is developing such a tumor-tracking blood test for human cancers.