A new method for more quickly diagnosing a rare type of childhood leukemia has broader implications in the testing of other disorders in which cell-signaling pathways are disrupted.
Juvenile myelomonocytic leukemia (JMML) accounts for 2% of all childhood leukemias, affecting the youngest children — typically just a year old — and the prognosis is grim, with survival ranging from 10 months to four years. So it's vital to start treatment, typically bone marrow transplants, quickly.
Current diagnostic methods take weeks. But researchers at Stanford University School of Medicine (Stanford, California) and the University of California, San Francisco, have put a new spin on a well-known cell-sorting technique that may provide physicians with a diagnosis in just a few hours.
"JMML poses a couple of challenges," Nikesh Kotecha, PhD, told Diagnostics & Imaging Week. "One is that the clinical presentation of JMML is not easy to detect. It looks like a lot of other common infections like HPV and even the common cold. So from a clinical standpoint, before you diagnose JMML, you do a rule-out diagnosis; then you want to confirm it with blood chemistry tests."
When a child has JMML, the body tells too many blood stem cells to develop into two types of white blood cells called myelocytes and monocytes. Some of the blood stem cells never mature and are called blasts. Eventually the myelocytes, monocytes and blasts crowd out the red blood cells and platelets in the bone marrow and the child will start to have symptoms, such as infections and anemia.
Kotecha said that immunology, signaling biology, medicine, statistics and informatics are combined in a multidisciplinary technique to produce the diagnosis. Flow cytometry, in which fluorescently labeled antibodies are used to classify and sort cells based on proteins displayed on their outer surface, is used with a new twist.
Small holes are made in the cell membrane prior to sorting. The holes allow other antibodies to enter the cell and bind to signaling molecules involved in the cell's internal monologue — in this case, a protein called STAT5.
Kotecha and his collaborators used an antibody that binds only to the activated, or phosphorylated, version of the protein to determine the signaling status of the pathway in individual cells exposed to a variety of conditions.
"In our technique, we use flow cytometry," Kotecha said. "The new thing is that we're not only looking at the types of cells. We have a new protocol to look at not only cell surface level but also at cell-signaling level. Signaling is really important because it gives you a way to understand the mechanisms."
A reliable indicator of JMML cells is their tendency to proliferate in response to very low levels of a growth-stimulating factor called GM-CSF; normal cells respond only at higher levels. But it can take two to three weeks to grow enough cells in the lab to get a definitive answer.
Kotecha knew that GM-CSF activates a particular cellular signaling cascade called the JAK-STAT pathway. Although that pathway had not previously been directly implicated in JMML, Kotecha used an antibody that binds only to activated STAT5 to determine whether the cells of 12 patients with JMML displayed abnormally high levels of the protein in response to low doses of GM-CSF. Eleven of the 12 did so, confirming the involvement of the STAT pathway in the disorder.
This technique shows promise in diagnosing other types of leukemia, follicular lymphoma, and could also be used as a tool in drug discovery.
Identifying populations of cells by their response to specific stimuli may help in the ability to assess the efficacy of specific therapies.
"It's a really good way of trying to get at diseases that incorporate cell type as a dimension," he said. "With traditional biochemistry methods, it isn't easy to do."