YIA20-005: Rapid Point of Care assay by isothermal PCR for Acute Promyelocytic Leukemia Diagnosis

Authors: Cecilia Yeung MDa,b, Bret Helton BSa, and Jerald Radich MDa,b
View More View Less
  • a Fred Hutchinson Cancer Research Center, Seattle WA
  • | b University of Washington, Seattle WA

Background: Acute promyelocytic leukemia (APL) accounts for ∼10% of acute myelogenous leukemia (AML) and may present as a medical emergency with risk of early death. Since the introduction of induction therapy with all-trans retinoic acid and arsenic, APL has become both the most curable yet deadliest form of AML. Definitive diagnoses are often not possible during a STAT evaluation because of APL’s unique morphological features that lack immature blasts and instead are either promyelocytes, or in another variant (the microgranular variant) appear like monocytes. Further complicating the picture is the inability of definitive identification by flow cytometry, which relies on the lack of CD34 and other typical blast markers seen in AML. Therefore, we propose a rapid point of care diagnostic assay is needed. Methods: We developed an iPCR reaction targeting PML-RARA with Recombinase Polymerase Amplification (RPA). We were able to use the products from the RPA reaction and apply to a lateral flow device for visualization of a positive PML-RARA fusion. Samples included cell lines, NB4 with known PML-RARA fusion, K562 without any RARA fusion and 16 patient samples including 12 APL, 4 CML. Patient samples were processed within 24-48 hours of draw from fresh blood or bone marrow and RNA isolated via trizol per manufacture protocol. Target input RNA was 500ng/ul, however some patient samples had low RNA yield and suboptimal input volumes were used. Results: We tested 59 samples on known cell lines and determined that our RPA reactions have a sensitivity of 100% (CI: 92.45% to 100.00%), a specificity of 100% (CI: 73.54% to 100%) of100ng/uL, a PPV/NPV of 100% and 100% respectively, and accuracy is 100% (CI: 93.94% to 100.00%). Our APL RPA reaction could be functional down to 30 degrees reaction which is optimal for a POC design. Limit of detection (LOD) can now detect down to 5ng of RNA from NB4 cell lines diluted with K562 RNA (for a total input of 500ng). On patient samples 7/12 were shown to be positive, the 5 negative samples had low input volumes (optimal = 500ng) below the LOD. Conclusions: Our initial design for this RPA assay has been able to reliably detect the PML-RARA fusion RNA transcript in a 30° reaction without the need of any additional electronic equipment. 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.

Corresponding Author: Cecilia CS Yeung, MD
All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 404 140 15
PDF Downloads 320 77 7
EPUB Downloads 0 0 0