A Fast and Accurate Cartridge-Based RT-qPCR Assay to Quantify PML-Rara mRNA Fusion Transcripts Near the Point of Care
Authors: Wu Natalie Chi-hwa , Malek Monisha , Ho Ken , Chen Julia , Komarla Anvita , Chu Victor , Wei Huilin , Day Gwo-Jen , Cayuela Jean-Michel , Ferrand Christophe , Cassinat Bruno 
Published: October 2019

Abstract
Background
Acute Promyelocytic Leukemia (APL) is a distinct subtype of Acute Myelogeneous Leukemia (AML), accounting for approximately 5-10% of AML cases. APL is caused by a chromosomal translocation t(15;17) which fuses the retinoic acid receptor alpha (RARA) and the promyelocytic leukemia gene (PML) in more than 95% of cases. Timely confirmation of PML-RARA fusion and prompt treatment with all-trans retinoic acid (ATRA)-based regimens reduces the risk of fatal hemorrhagic complications in APL patients, leading to complete remission rates of >90%. Monitoring PML-RARA transcript levels by RT-qPCR is also essential in the management of APL patients during and after ATRA based treatment to monitor for response to therapy and to identify early evidence of relapse.
In response to the unmet need for fast and reliable RT-qPCR PML-RARA assay results closer to the point of care, we sought to develop a sensitive and specific cartridge-based PML-RARA assay (RUO)* for use on the widely distributed GeneXpert ® Instrument System (GX). Our goals were two-fold: 1) to develop a simplified hands-on sample preparation procedure to process either whole blood or bone marrow samples into lysates for testing, and 2) to develop a single use PML-RARA cartridge prototype* preloaded with sample processing and RT-qPCR reagents to which a sample lysate could be added and then tested on the GX system, which automates the rest of the RT-qPCR process, with turn-around time to results of less than 3 hours, including hands on time.
Methods
Primers and probes capable of targeting and discriminating bcr1, bcr2, and bcr3 isoforms of PML-RARA fusion transcript were designed for the cartridge-based RT-qPCR assay prototype. Primers and probe were also developed for ABL1, which serves as a reference gene to normalize the total mRNA level across different samples. Bone marrow and whole blood samples from healthy individuals, as well as NB4 cells (bcr1) and UF1 cells (bcr3), were used to evaluate the specificity of the assay prototype. In vitro transcribed (IVT) RNAs coded with three different isoforms were generated and spiked into whole blood or bone marrow lysate to assess the dynamic range of the assay. Banked leukocytes from 20 APL patients with known PML-RARA fusion isoforms were lysed and tested with the PML-RARA assay cartridge prototype* on the GX system.
Results and Conclusion
A one-step off-board sample preparation procedure, requiring less than three minutes of hands-on time, was established to process EDTA whole blood and bone marrow samples into lysates for testing in the prototype PML-RARA assay*. When tested with IVT RNA spiked-in blood and bone marrow lysate, the assay prototype was shown to cover a 4 log dynamic range for each of the three PML-RARA fusion isoforms (bcr1, bcr2, bcr3). No false positive results were detected when using bone marrow or blood samples from healthy individuals. PML-RARA breakpoint (bcr) calls from clinical sample testing were 100% concordant with historical laboratory developed test data.
Here we demonstrate a new cartridge based RT-qPCR assay* to sensitively detect and quantify PML-RARA mRNA fusion transcripts using the widely distributed GeneXpert system. The assay prototype requires minimal hands-on time and results are available in less than three hours. Testing of prospectively collected APL clinical samples is in progress.
* For Research Use Only. Not for use in diagnostic procedures. Not reviewed by any regulatory body.

Disclosures
Wu: Cepheid: Employment, Equity Ownership. Malek:Cepheid: Employment. Ho:Cepheid: Employment. Chen:Cepheid: Employment. Komarla:Cepheid: Employment. Chu:Cepheid: Employment. Wei:Cepheid: Employment. Day:Cepheid: Employment. Cayuela:Cepheid: Other: financial sponsor to attend John Goldman Conference 2017. Ferrand:Cepheid: Other: financial sponsor to attend John Goldman Conference 2017.