The boy was quickly running out of options. His legs and belly were swelling like sponges, his lungs kept filling with fluid so badly that he needed an oxygen tank, and the extreme measures his physicians had tried worked only temporarily.
“He got worse and worse and worse,” said Dr. Hakon Hakonarson, a pediatric lung specialist and director of the Center for Applied Genomics at Children’s Hospital of Philadelphia (CHOP). “He was going to die.”
But the boy lived. Born with a rare, complex genetic condition, he owes his life to zebrafish, the nearly transparent creatures that have become the go-to lab animal in countless studies of genetics, development, and disease.
Hakonarson and his colleagues identified the mutation causing the boy’s illness, in which lymphatic vessels proliferate out of control and leak fluid into the lungs and other organs; engineered the mutation into zebrafish; waited for the fish to develop a piscine version of the boy’s disease; tested multiple drugs on the fish; found one that stopped the vessels’ kudzu-like growth; and got permission from federal health officials to try it on their young patient.
It worked, they reported in Nature Medicine on Monday, a success that shows “precision medicine” can be expanded well beyond cancers, where it has shown the greatest promise. Much like, say, lung cancer, the child’s illness, central conducting lymphatic anomaly, can be caused by any of several mutations. Each leads down a different biological road to the disease. Only a drug that targets the right road can stop the disease, just as blockading only the roads taken by an invading army can stop it from laying siege to a city.
Researchers not involved in the boy’s case said it made a compelling case for applying genetic techniques to lymphatic disorders, which strike about 1 in 4,000 newborns. “I think this approach definitely could be used clinically,” said medical geneticist Dr. V. Reid Sutton of Texas Children’s Hospital, which treats about 50 children with lymphatic anomalies every year. “It suggests that we should do this sort of genetic testing to identify where in the biological pathway the problem is and try to find a drug that targets it.”
Daniel was 12 when his family took him to CHOP. As a little kid he had played competitive soccer and run a 5K in 25 minutes. But when he was 10 his legs suddenly began swelling, he had trouble breathing, and he couldn’t tolerate exercise. The Virginia hospital near the family’s home diagnosed a buildup of lymphatic fluid around his heart and drained it, but with lymphatic fluid continuing to leak in, it was like bailing out a boat without repairing the leak.
Transferred to CHOP, Daniel received palliative treatment, including the cauterization of lymphatic vessels and sirolimus, an immune-suppressing drug (also called rapamycin) that helps some patients with one type of lymphatic disease but not others. But nothing helped for long, and sirolimus didn’t help at all.
Daniel “was getting measured for a wheelchair and had to be tutored at home,” his mother said in a statement to CHOP.
Hakonarson and his colleagues suspected that getting to the genetic root of the problem was Daniel’s only hope. They sequenced his exome, the part of the genome that produces proteins. A dozen genes that had previously been linked to lymphatic anomaly looked normal.
But a gene on the X chromosome, called ARAF, didn’t. ARAF makes an enzyme called a kinase that adds dangly molecules to a cell’s proteins. Too much of that can cause a cell to proliferate so wildly it becomes cancerous or, in Daniel’s case, to sprout lymphatic vessels like creepers from a neglected patch of ivy. (Neither of his parents has the mutation. It arose by random bad luck when he was only a days-old embryo.) But the exact mechanism by which the lymphatic vessels proliferated was one that sirolimus doesn’t affect.
ARAF mutations had never been linked to lymphatic disorders, however. Maybe it was an innocent bystander unrelated to Daniel’s now-life-threatening illness. To determine that, the scientists genetically engineered zebrafish embryos to carry the mutation.
Within five days, the fish had developed a lymphatic system. “That’s one of the nice features of zebrafish,” said CHOP’s Christoph Seiler, who led this part of the study: “They develop lightning-fast.” With the ARAF mutation, what the quarter-inch-long fish developed were lymphatic vessels that grew and grew, just like Daniel’s.
It was “proof that this mutation causes overgrowth” of lymphatic vessels, Hakonarson said.
That was scientifically interesting, but the researchers had a dying patient. They gave groups of fish with the ARAF mutation one of 10 different drugs. “We just put it in the water and they take up the drug,” Seiler said. “You don’t need to inject it like you do with a mouse.”
All the drugs are “MEK inhibitors,” which hit exactly the biological pathway that Daniel’s ARAF mutation takes the brakes off. The scientists anxiously examined the fish for evidence that the chaotic lymphatic vessel proliferation had stopped but that nothing else had been harmed.
They had a winner: trametinib, a melanoma drug that Novartis sells as Mekinist.
Although physicians can prescribe a drug for conditions other than those the Food and Drug Administration approved it for, Hakonarson was reluctant to do that. “We did not want to take the chance of giving a toxic, adult drug to a child” without outside experts deciding that the potential benefits outweighed the risks, he said.
With FDA approval, the CHOP team gave Daniel trametinib. Within two months, his breathing improved. At three months, the fluid in his lungs had receded enough that he no longer needed supplemental oxygen. The swelling in his legs disappeared. An MRI showed that his lymphatic vessels reshaped themselves into something close to normal. It was the first time a drug had remodeled an entire organ system.
“You try a lot of things in research and only a few work,” Seiler said. “It’s so great to go back into the clinic and treat” on the basis of what the zebrafish revealed.
Now 14, Daniel plays basketball, rides his bicycle, and helps coach soccer.
Experts not involved in the study cautioned that one success is suggestive, but not proof, that exome sequencing and targeted therapy will help patients with rare lymphatic anomalies. Still, “this is the first paper to implicate ARAF in vascular malformation and to demonstrate MEK inhibition may be effective,” said Dr. Matt Warman, a medical geneticist at Boston Children’s Hospital. “This is important since several clinicians are currently using MEK inhibitors in other patients whose vascular malformations are due to mutations.”
The CHOP results would be more persuasive with more patients followed for longer, he said, but the researchers “have performed a service to patients affected by these distressing disorders.”
Daniel’s case argues for clinical trials in which existing drugs, especially for cancer, are matched to the precise genetics of patients with lymphatic anomalies, said Sutton of Texas Children’s. Scientists in France reported last year that the Novartis breast cancer drug alpelisib (Piqray) saved 19 patients with vascular malformations similar to Daniel’s.
“As we identify additional causes [of lymphatic anomalies], we should be able to do more mutation-specific therapies,” Warman said. Especially if zebrafish point the way.