Novel DNA Sequencing Technique Could Diagnose Liver Cancer Before Tumors Develop

A novel technique that involves the DNA sequencing of liver cells can determine whether the cells have been exposed to aflatoxin, and may help predict whether an individual has a higher risk of developing liver cancer before the tumors appear.

Investigators previously reported that exposure to aflatoxin usually results in a genetic mutation that converts guanine to thymine. This process can often lead to liver cancer, but in areas with high regulations, such as the United States and Europe, the risk of aflatoxin exposure is low.

In a study published in the Proceedings of the National Academy of Sciences, investigators sought to determine whether they could identify mutations derived from aflatoxin long before cancer develops.

“What we’re doing is creating a fingerprint,” said investigator John Essigmann. “It’s really a measure of prior exposure to something that causes cancer.”

For the study, mice were exposed to a single dose of aflatoxin just 4 days after birth. After exposure, all the mice eventually developed liver. The investigators sequenced DNA from the tumors, as well as from liver cells that were removed only 10 weeks after exposure, before the tumors developed.

The investigators used a powerful genome sequencing technique to identify rare mutations at 10 weeks. The novel DNA sequencing technique combines data from 2 complementary strands of DNA rather than sequencing each strand of double-stranded DNA alone. In most DNA techniques, each strand must be copied many times to get enough DNA to sequence, which results in the introduction of errors—–approximately 1 mistake for every 500 base pairs.

With the new technique, the 2 complementary strands are barcoded so that the sequence information can be recombined later. This approach allows the investigators to distinguish true mutations from copying errors. This technique is 1000 to 10,000 times more accurate than conventional DNA sequencing, according to the authors.

“Detecting rare events is something that this technology was designed to do,” said author Bogdan Fedeles.

The results of the study showed that at 10 weeks, a distinctive pattern of mutations that can serve as a “fingerprint” for aflatoxin exposure had already emerged. Approximately 25% of mutations occurred in CGC sequences. The authors noted that aflatoxin is more likely to produce mutations in guanine when flanked by cytosine on both sides.

“Even at 10 weeks, a very distinct mutational signature comes up,” Essignmann said. “It’s very early-onset, and you don’t see it with other carcinogens, to our knowledge.”

The investigators compared the mutational profile of the mice exposed to aflatoxin to genetic sequences found in liver tumors of more than 300 patients from around the globe. The results showed that the signature of the mouse cells closely matched the signatures of 13 patients believed to have been exposed to aflatoxin in their diet. Most of the 13 patients were from sub-Saharan Africa and Asia.

In the future, the investigators hope to devise a simple test—–such as a blood test––that could easily be examined for this mutational profile. For patients who tested positive, they would likely benefit from regular screening of their liver to determine if tumors had begun forming so that they could be surgically removed. Additionally, the test could be used to study new cancer-protective drugs or dietary regimens that might prevent these DNA mutations produced by aflatoxin.

The investigators also plan to look for mutational profiles produced by other liver carcinogens, such as dimethylnitrosamine, which can be found in local drinking water.

“The hypothesis that drives this field is that each agent that contributes to the genetic changes responsible for cancer has its own unique mutational signature, and those signatures can be used to identify the contributions of each of these agents to the tumor that ultimately develops,” said author Robert Croy.