Comparable b2a2/e13a2 and b3a2/e14a2 Reporting in BCR-ABL Assay When Calibrated By Who Is and By IVT-RNA Copy Number
Authors: Wei Huilin , Tran Tran , Shridhar Krupa , Wang Nick , Day Gwo-Jen 
Published: December 2023

Introduction:

Xpert® BCR-ABL Ultra, automated cartridge-based assay for monitoring BCR-ABL transcript levels, is calibrated by WHO IS to standardize the %reporting for both b2a2/e13a2 BCR-ABL(e13a2) and b3a2/e14a2 BCR-ABL(e14a2) relative to control ABL gene in peripheral blood of patients as a standard management of Chronic Myeloid Leukemia (CML) [1]. Studies have shown differences in patients carrying e13a2 or e14a2 in molecular response to Tyrosine Kinase Inhibitor (TKI) treatment [2-4]. Therefore, it is necessary to understand if there is %reporting differences between two transcripts calibrated by WHO IS[5] and explore the calibration method using breakpoint specific RNA input copy number (CN) for accurate TKI treatment monitoring.

Objectives:

To establish % reporting method calculated with known CN of e13a2, e14a2 and ABL IVT-RNA within a wide CN range and compare % e13a2 and % e14a2 to WHO IS quantification system.

Methods: Three IVT-RNAs, containing e13a2-ABL-BCR, e14a2-ABL-BCR and ABL-BCR, were synthesized as shown in Figure 1. Serial diluted e13a2 RNA, e14a2 RNA and ABL RNA panels were tested on Xpert BCR-ABL Ultra assay to generate individual standard curve of e13a2 Ct, e14a2 Ct and ABL Ct as a function of CN for % reporting calculation. Four levels of IVT-RNA panels with same CN of e13a2 and e14a2 were tested for breakpoint specific % reporting comparison. K562(e14a2), BV173 (e13a2) cell lysates and CML clinical samples carrying e13a2 or e14a2 transcripts were tested to evaluate the %CN reporting compared to WHO IS.

Result: Good linearity demonstrated in Ct vs CN input for e13a2, e14a2 and ABL IVT-RNA (Figure2) with comparable efficiency (E) between e13a2 (E=0.992) and e14a2 (E=0.986). %e13a2 reporting was ~1.50-fold (by WHO IS) and ~1.46-fold (by %CN) higher than %e14a2, by testing IVT-RNA panel (Table1). Minor differences in %reporting observed between % CN and WHO IS for e13a2 (84.5%~110.8%) vs e14a2 (82.5%~89.5%) from cell lysates (Table2) and e13a2 (92.6%~105.5%) vs e14a2 (88.1%~92.2%) from clinical samples (Table3).

Conclusions:

A %CN e13a2/ABL and e14a2/ABL reporting method with known CN of IVT-RNAs has been successfully established in Xpert BCR-ABL Ultra. Both WHO IS and %CN showed very minor differences between e13a2 and e14a2 for % reporting. %CN method demonstrated comparable %reporting to WHO IS in Xpert BCR-ABL Ultra.

Recent Publications

1. Dominy KM, Simon IM, Khorashad JS (2021) Evaluation of Xpert® BCR-ABL Ultra for the confirmation of BCR-ABL1 international scale conversion factors for the molecular monitoring of chronic myeloid leukaemia, Int J Lab Hematol, Feb;43(1):e31-e34.

2. Hanfstein B, Lauseker M, Hehlmann R, Saussele S, Erben P, Dietz C (2014) Distinct characteristics of e13a2 versus e14a2 BCR-ABL1 driven chronic myeloid leukemia under first-line therapy with imatinib. Haematologica.99:1441-7.

3. Jain P, Kantarjian H, Patel KP, Gonzalez GN, Luthra R, Shamanna RK (2016) Impact of BCR-ABL transcript type on outcome in patients with chronic-phase CML treated with tyrosine kinase inhibitors. Blood.127:1269-75.

4. Castagnetti F, Gugliotta G, Breccia M, Iurlo A, Levato L, Albano F (2017) The BCR-ABL1 transcript type influences response and outcome in Philadelphia chromosome-positive chronic myeloid leukemia patients treated frontline with imatinib. Am J Hematol. 92:797-805.

5. Matthew S, White HE, Hana Z, Nicholas C.P C (2022) Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy. Leukemia 36(7):1-8