Diagnostic performance of the molecular BCR-ABL1 monitoring system may impact on inclusion of CML patients in stopping trials

Abstract

In chronic myeloid leukemia (CML), the duration of deep molecular response (MR) before treatment cessation (MR4 or deeper, corresponding to BCR-ABL1 ≤ 0.01% on the International Scale (IS)) is considered as a prognostic factor for treatment free remission in stopping trials. MR level determination is dependent on the sensitivity of the monitoring technique. Here, we compared a newly established TaqMan (TM) and our so far routinely used LightCycler (LC) quantitative reverse transcription (qRT)-PCR systems for their ability to achieve the best possible sensitivity in BCR-ABL1 monitoring. We have comparatively analyzed RNA samples from peripheral blood mononuclear cells of 92 randomly chosen patients with CML resembling major molecular remission (MMR) or better and of 128 CML patients after treatment cessation (EURO-SKI stopping trial). While our LC system utilized ABL1, the TM system is based on GUSB as reference gene. We observed 99% concordance with respect to achievement of MMR. However, we found that 34 of the 92 patients monitored by TM/GUSB were re-classified to the next inferior MR log level, especially when LC/ABL1-based results were borderline to thresholds. Thirteen patients BCR-ABL1 negative in LC/ABL1 became positive after TM/GUSB analysis. In the 128 patients included in the EURO-SKI trial identical molecular findings were achieved for 114 patients. However, 14 patients were re-classified to the next inferior log-level by the TM/GUSB combination. Eight of these patients relapsed after treatment cessation; two of them were re-classified from MR4 to MMR and therefore did not meet inclusion criteria anymore. In conclusion, we consider both methods as comparable and interchangeable in terms of achievement of MMR and of longitudinal evaluation of clinical courses. However, in LC/ABL1 negative samples, slightly enhanced TM/GUSB sensitivity may lead to inferior classification of clinical samples in the context of TFR.

DNA Damage and DNA Damage Response in Chronic Myeloid Leukemia

“…The molecular response (MR) of CML was assessed by international scale (IS)-standardized polymerase chain reaction (PCR) of BCR-ABL1 in all CML samples [35]. For the quantitative real-time PCR reaction (qRT-PCR) of BCR-ABL1 fusion gene and GUSB control gene, a TaqMan detection system (TaqMan 7500 Fast Real-Time PCR System, Thermo Fisher Scientific, Waltham, US) was used for quantification of e13a2/e14a2 (b2a2/b3a2) transcripts and a LightCycler detection system (Roche Applied Science, Penzberg, Germany) for quantification of e1a2 transcripts as previously described [36,37].…”

Molecular Testing in CML between Old and New Methods: Are We at a Turning Point?

“…These data are constantly confirmed by new evidence and altogether stress the utility to move to dPCR for the monitoring of MRD in CML patients, particularly in subjects presenting a low level of BCR-ABL1 transcript and potentially eligible for stopping TKI therapy. In fact, the biased diagnostic performance of the BCR-ABL1 molecular detection and quantification may impact the inclusion of CML patients in therapy-stopping trials [49,50], which is currently one of the pivotal goals of CML management [4,51]. A comparison between the main features of RT-qPCR and dPCR is presented in Table 1.…”

Chronic Myeloid Leukemia Prognosis and Therapy: Criticisms and Perspectives

“…Assessing DMR through the molecular measurement of BCR-ABL1 transcript levels in the peripheral blood by RT-qPCR is necessary for addressing the patient to TKI discontinuation but, unexpectedly, a tight correlation between the depth and duration of DMR, and the rate of TFR maintenance does not exist. The reason might be sought in the intrinsic limitations of RT-qPCR, mainly consisting of lack of precision in the quantification of the low levels of the target (BCR-ABL1 transcript) [ 31 , 32 ]. Thus, the great majority of patients who undergo the treatment discontinuation are preferably selected among those with undetectable levels of BCR-ABL1 transcript by RT-qPCR [ 33 , 34 ].…”

“…Having a precise and accurate methods for MR measurement is mandatory to better design the future strategies, to optimize the CML therapy and to refine patient management, but it is difficult to properly manage CML therapy when RT-qPCR provides results of MRD expressed as “undetectable transcript” [ 32 ]. Digital PCR (dPCR) may overcome this intrinsic limit of RT-qPCR and is the only feasible tool in the event of stopping TKIs for a better selection of CML patients eligible to treatment discontinuation.…”