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Experiments in Mice Demonstrate Possibility of Inducing Biomarker Release to Detect Early Cancers

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NEW YORK (GenomeWeb) – Researchers from Stanford University have demonstrated in mice a technique for inducing tumors to release a detectable biomarker that they hope can be developed into a method for early cancer detection in humans.

The study, published this week in Proceedings of the National Academy of Sciences, was a proof of principle that the team's approach could distinguish mice implanted with human melanoma cells from those without.

The method involves administering a type of nonviral vector, a synthetic ring-shaped DNA molecule called a DNA minicircle, which contains a promoter that activates expression of a detectable reporter protein only in cancer cells.

In their initial study, the team used minicircles containing the reporter gene SEAP and the tumor-specific promoter pSurv to drive the expression of SEAP. PSurv normally promotes the expression of the protein survivin and is active in cancer cells and some fetal tissues but not in healthy adults.

According to the study authors, the approach could first be tested in populations who have had cancer and are being monitored for recurrence, and then could be explored for screening high-risk populations and eventually the general population.

Sanjiv Gambhir, the study's senior author, told GenomeWeb that the group sees another few years ahead of fine tuning the approach in mice before it can begin to take the first steps of advancing it for human use.

The team's minicircle approach borrows from the world of gene therapy, in which the goal is to induce expression of a therapeutic transgene within a tumor but not in any other tissues in the body. But instead of causing the expression of a therapeutic target, the Stanford group hoped to be able to induce expression of a detectable biomarker only in cancer cells and not in healthy cells.

In their study they tested their pSurv-driven, SEAP-expression inducing DNA minicircles in a group of immunocompromised mice — seven mice that had been injected with cancer cells from human melanoma cell lines and six which had not. They also injected a control solution with no minicircles into another five mice.

The researchers then measured SEAP levels in the mice's blood after one, three, seven, 11 and 14 days.

According to the authors, within 48 hours, SEAP was present in the blood of mice with tumors, at levels clearly distinguishable from the tumor-free animals. Their ROC analysis yielded an AUC of 0.92, a very robust delineation between the tumor-bearing and cancer-free mice.

The team did also observe a slight SEAP signal from tumor-free mice who were administered minicircles. Gambhir said that this background signal is due to the inherent background of the team's SEAP detection assay.

Moving forward, the group plans to work on developing better assays for SEAP, and also to experiment with other reporter genes.

Interestingly, the researchers found that the maximum strength of the SEAP signal in the melanoma mice varied with the tumor volume in their lungs, suggesting that the method could potentially detect not only the presence of cancer but also its extent or progression.

The study did not investigate the limit of detection for the minicircles in terms of the lowest detectable tumor burden in cancer-carrying mice. If the method is to be useful as an early cancer detection tool in humans, it will have to be established that it can detect the presence of cancer cells at very low prevalence in the body or blood.

According to the authors, the team is currently testing the strategy using animal models in which tumor volume can be measured as a way of calculating the detection sensitivity.

Future work will also have to establish whether the minimal detectable cancer volume in mice can translate to a sufficiently sensitive test in humans, who possess a much larger blood volume.

Gambhir said that prior to publishing this paper, his team also experimented with delivering molecules that cause cancer cells to express a target that makes them imageable, instead of a biomarker to be detected in the blood.

Potentially, these two approaches could be combined, allowing for blood-based early cancer detection and also pinpointing of that cancer's location in the body.

This combination is something the team is now planning to tackle. The group is also looking into the possibility of integrating a gene therapeutic component, so that cancer could be detected, imaged, and treated using a single minicircle. 

"If you are detecting the cell, why not also go ahead and kill it?" Gambhir said.

There is also the potential to use alternative promoters in order to create minicircles that are specific to a particular cancer type. "The survivin promoter is switched on in many cancers, but we are looking at other promoters that are only active in specific cancers — only in breast or only in breast and ovarian cancer, for example … We have not perfected that in mice yet, but that is one of the next steps," he said.

In terms of advancing the technique for clinical use, there are several hurdles to overcome, Gambhir said. One is the development of an oral delivery system. In the study the team injected the minicircles into the mice's tails, but for human use, developing a swallowable pill is a primary goal for the group.

Gambhir estimated that the team will need about three more years of animal studies before it can then move on to begin to establish the safety of the minicircle approach in humans.

Encouragingly, minicircles have some benefits over other transfection agents, he explained. "One of the reasons we chose minicircles is because they don't integrate with the host genome. That makes them much more clinically translatable."

"They get into the body and into the cells and they ramp up the expression of the gene, but then over two weeks or so, they disintegrate," he said. The synthetic molecules also do not contain pathogen DNA that could potentially elicit a strong immune response as do viruses and plasmids, the study authors wrote.

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