Kristyna Wentz-Graff
Kevin Watanabe-Smith, PhD Photo courtesy of Oregon Health & Science University/
A newly discovered issue with the Ba/F3 transformation assay could “jeopardize attempts to characterize the signaling mechanisms and drug sensitivities of leukemic oncogenes,” researchers reported in Oncotarget.
The researchers said the assay “remains an invaluable tool” for validating activating mutations in primary leukemias.
However, the assay is prone to a previously unreported flaw, where Ba/F3 cells can acquire additional mutations.
This issue was discovered by Kevin Watanabe-Smith, PhD, of Oregon Health & Science University in Portland.
He identified the problem while studying a growth-activating mutation in a patient with T-cell leukemia. (The results of that research were published in Leukemia in April 2016).
“When I was sequencing the patient’s DNA to make sure the original, known mutation is there, I was finding additional, unexpected mutations in the gene that I didn’t put there,” Dr Watanabe-Smith said. “And I was getting different mutations every time.”
“After we saw this in several cases, we knew it was worth further study,” added Cristina Tognon, PhD, of Oregon Health & Science University.
So the researchers decided to study Ba/F3 cell lines with and without 4 mutations (found in 3 cytokine receptors) that have “known transformative capacity,” including:
- CSF2RB R461C, a germline mutation found in a patient with T-cell acute lymphoblastic leukemia (ALL)
- CSF3R T618I, a mutation found in chronic neutrophilic leukemia (CNL) and atypical chronic myelogenous leukemia (aCML)
- CSF3R W791X, also found in CNL and aCML
- IL7R 243InsPPCL, which was found in a patient with B-cell ALL.
The researchers said they observed acquired mutations in 1 of 3 CSF2RB wild-type cell lines that transformed and all 4 CSF3R wild-type cell lines, which all transformed.
Furthermore, most CSF2RB R461C lines (4/5) and CSF3R W791X lines (3/4) had additional acquired mutations. However, the CSF3R T618I lines and the IL7R 243InsPPCL lines did not contain acquired mutations.
The researchers said acquired mutations were observed only in weakly transforming oncogenes, which were defined as mutations with a weaker ability to transform cells (less than 1 in every 200 cells) or a slower time to outgrowth (5 days or longer to reach a 5-fold increase over the initial cell number).
The team also said their findings indicate that the majority of the acquired mutations likely exist before IL-3 withdrawal but only expand to levels detectable by Sanger sequencing in the absence of IL-3.
“The potential impact [of these acquired mutations] is that a non-functional mutation could appear functional, and a researcher could publish results that would not be reproducible,” Dr Watanabe-Smith said.
To overcome this problem, Dr Watanabe-Smith and his colleagues recommended taking an additional step when using the Ba/F3 assay—sequencing outgrown cell lines. They also said additional research is needed to devise methods that reduce the incidence of acquired mutations in such assays.
