Sequential Quantitative Minimal Residual Disease in BCR-ABL Positive Acute Lymphoblastic Leukaemia: Differential Observations between Major and Minor Fusion Gene Rearrangements and Variation Due to Source Material.
Authors: Gerrard Gareth , Mitchell Wayne , Goldstone Anthony , Chopra Raj , Buck Georgina , Richards Sue , Foroni Letizia 
Published: October 2019

Abstract
Background: Minimal Residual Disease (MRD) monitoring in BCR-ABL+ acute lymphoblastic leukaemia (ALL) is a valuable tool in the management of Philadelphia positive adult ALL due to the markedly poor prognosis engendered by this leukemia subtype. As part of the UKALLXII trial we received samples from 104 de novo adult ALL patients, 26 of which (25%) were found to be BCR-ABL+. BM and PB received at presentation and during chemotherapy or post SCT were tested for MRD as part of a modified UKALLXII trial protocol which aimed at assessing the efficacy of combined chemotherapy and Imatinib treatment and SCT.
Aims: 1) To establish which sample source offers the highest level of sensitivity; 2) To determine if differences in disease level is associated with the BCR-Abl rearrangement exhibited.
Methods: BM and/or PB samples were received at presentation and at monthly follow-up points and were tested by quantitative real-time PCR (QRT-PCR) using a Roche LightCycler 1.3 with the SYBR-green fluorescent detection system. The BCR-ABL transcript levels were normalised as a ratio against Abl levels. Operating procedures were adapted from the recommendations of the European Study Group for MRD in ALL.
Results: 138 samples (65 PB, 73 BM) were analysed from 26 BCR-ABL+ patients: 15M, 11F; median age 43y (range 17y to 58y). 17 (65%) were found to be minor and nine (35%) were major. Ten patients have received Imatinib and nine have undergone transplant (7 allo-SCT, 2 autograft). 2 patients have relapsed and 2 have died (1 following relapse). Paired t-tests between PB and BM sample BCR-Abl transcript levels show that BM offers a significantly higher level of sensitivity with a median BCR-ABL level of 2.93 x 10−4 against 1.8 x 10−4 for PB (p= 0.0056, n=46). Baseline ABL levels show the converse (1.49 x 104 BM against 1.77 x 104 PB, p= 0.016, n=46) suggesting that the generally greater quantity of material associated with PB samples may result in higher quality RNA. BCR-Abl levels between major and minor patients show that patients exhibiting the major form have significantly higher levels of disease in both PB (median 3.1 x 10−2 major (n= 24) against 3.3x10−4 in patients exhibiting minor BCR-Abl (n=41), (p< 0.0001) and BM samples (median 2.0 x 10−2 major (n= 28) against 3.3 x 10−4 minor (n=45), (p= 0.0009). Paired sequential analysis for 11 patients with 2 or more follow-up samples confirms this observation (median 3.5 x 10−2 major (n=4) against 3.5 x 10−4 minor (n=7), (p= 0.008).
Conclusions: In the QRT-PCR MRD monitoring of adult ALL BM samples offer a small but significantly higher level of sensitivity than PB samples. Patients exhibiting the major BCR-ABL rearrangement have significantly higher levels of disease than patients with the minor form. These data describing differential disease status within the BCR-ABL+ subgroup and variance due to sample type may be important in providing guidelines for patient management.