Investigators have developed an assay to reliably detect acute promyelocytic leukemia (APL) RARA fusion gene transcripts, which are the hallmark of APL.

Cecilia Yeung MD, of the Fred Hutchinson Cancer Research Center, University of Washington, in Seattle, and colleagues published their findings in an abstract associated with the  National Comprehensive Cancer Network (NCCN) 2020 Annual Conference, which was postponed in response to the coronavirus disease 2019 (COVID-19) pandemic.

APL is difficult to definitively diagnose, and while it has become the most curable form of acute myelogenous leukemia (AML), explained the authors, it remains the deadliest form of the disease. Therefore, the team aimed to develop a rapid point of care diagnostic assay for APL. 

The newly developed assay is an isothermal polymerase chain reaction (iPCR) that amplifies the PML-RARA fusion gene transcripts with recombinase polymerase amplification (RPA). The researchers then used a lateral flow device to visualize the products from the RPA reaction and confirm positive PML-RARA fusions.

The test samples were both cell lines (NB4 with known PML-RARA fusion and K562 with no RARA fusion) and patient samples (16 patients in total; 12 patients with APL and 4 patients with CML). Patient samples were either fresh blood or bone marrow and were processed within 48 hours for RNA isolation. The target input RNA for the assay was 500 ng/µl; however, the authors noted that some samples yielded lower RNA concentrations, thus suboptimal input was used. 

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Using 59 samples from the known cell lines, the team demonstrated that the RPA reactions have a sensitivity of 100% (CI: 92.45%-100.00%), a specificity of 100% (CI: 73.54%-100%), a positive predictive value of 100%, a negative predictive value of 100%, and an accuracy of 100% (CI: 93.94%-100.00%).

They also designed the APL RPA reaction to be functional down to 30 degrees, which would be feasible in a point of care setting. The limit of detection (LOD) for the assay was revealed to be 5 ng of RNA from the NB4 cell line diluted with K562 RNA (for a total input of 500 ng).

From 12 patient samples, 7 were identified as positive; however, the 5 negative samples had inputs below the LOD.

“The potential for a point of care device is feasible, however, more optimization for improved sensitivity, lower input RNA requirements, and to achieve a lower limit of detection is needed,” the authors concluded.

Reference

Yeung C, Helton B, Radich J. YIA20-005: Rapid point of care assay by isothermal PCR for acute promyelocytic leukemia diagnosis. J Natl Compr Canc Netw. 2020;18(3.5):YIA20-005-YIA20-005.