Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy.

Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.

Salmon M ，White HE ，Zizkova H ，Gottschalk A ，Motlova E ，Cerveira N ，Colomer D ，Coriu D ，Franke GN ，Gottardi E ，Izzo B ，Jurcek T ，Lion T ，Schäfer V ，Venturi C ，Vigneri P ，Zawada M ，Zuna J ，Hovorkova L ，Koblihova J ，Klamova H ，Markova MS ，Srbova D ，Benesova A ，Polivkova V ，Zackova D ，Mayer J ，Roeder I ，Glauche I ，Ernst T ，Hochhaus A ，Polakova KM ，Cross NCP ... -  《-》

Variant-specific discrepancy when quantitating BCR-ABL1 e13a2 and e14a2 transcripts using the Europe Against Cancer qPCR assay.

Molecular monitoring of treatment response in patients with chronic myelogenous leukemia is performed using the Europe Against Cancer (EAC) qPCR assay using the International Scale (IS). The assay amplifies both e13a2 and e14a2 BCR-ABL1 transcript variants. Observing distinct variant-dependent amplification curves during qPCR, we aimed to determine if this affected quantitation of BCR-ABL1. We investigated the qPCR efficiency at three Danish diagnostic centers (Zealand University Hospital [ZUH], Aarhus University Hospital [AU], and Rigshospitalet [RH]) on cell lines expressing either the e13a2 or e14a2 BCR-ABL1 transcript variants and compared %IS values from 219 chronic myeloid leukemia patients from the centers with either the e13a2 (n = 113) or e14a2 (n = 106) transcript variants obtained by qPCR with absolute quantitation by droplet digital PCR (ddPCR). Although no significant differences were found in amplification efficiencies of the transcript variants, Bland-Altman analysis of qPCR vs ddPCR values for patient samples revealed a significant average difference in the bias between variants (e3a2/e14a2) of 4.6-, 6.5-, and 1.8-fold for ZUH, AU, and RH, respectively. Furthermore, qPCR %IS values of diagnostic patient samples revealed a significant 4.7-fold difference between the e13a2 and e14a2 variants. Our findings suggest that the EAC qPCR assay may underestimate the e14a2 variant compared to the e13a2 variant.

Kjaer L ，Skov V ，Andersen MT ，Aggerholm A ，Clair P ，Gniot M ，Soeby K ，Udby L ，Dorff MH ，Hasselbalch H ，Pallisgaard N ... -  《-》

Impact of the BCR-ABL1 fusion transcripts on different responses to Imatinib and disease recurrence in Iranian patients with Chronic Myeloid Leukemia.

One genomic breakpoint can result in variable BCR-ABL1 transcript types due to alternative splicing. The influence of different BCR-ABL1 transcript types on clinical outcome is still controversial. The objective of this analysis was to determine the impact of transcript type on response, clinical outcome, recurrence risk after treatment with Imatinib mesylate in Chronic Myeloid Leukemia (CML) patients. Sixty CML patients in chronic phase were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and banding standard protocols. There was a significant difference in collective incidence of complete cytogenetic response (CCR) between the e14a2 and e13a2 groups (P=0.04). The median time to achieve CCR was shorter in e14a2 patients than to e13a2 (P=0.01). This finding is paralleled by the molecular response where the median of the BCR-ABL1/ABL1 expression levels were significantly lower in e14a2 transcript compared to e13a2 type at 3, 6, 9 and 12months from the start of therapy (P<0.01). The probability of recurrence after treatment discontinuation was 9.33 fold higher in e13a2 transcript, that is reported here for the first time (χ2=5.49; P=0.01; OR: 9.33; 95% CI: 1.59, 54.67). No significant difference was observed regarding overall survival (OS), although Patients with e14a2 transcript displayed a significant tendency toward a higher event free survival (EFS) ratio (P=0.03). We found that patients with the e14a2 transcript achieved better and faster responses to Imatinib mesylate. In this study, parallel data regarding molecular and cytogenetic responses, impact of transcript type on the probability of recurrence might suggest a general outcome that the type of transcript can be used as a prognostic marker at diagnosis.

Rostami G ，Hamid M ，Jalaeikhoo H 《-》

RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia.

Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region-c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1IS (%BCR-ABL1IS) by reverse transcription-quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification. BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay. Measurements on all instruments correlated well when the %BCR-ABL1IS was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range. RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.

Alikian M ，Whale AS ，Akiki S ，Piechocki K ，Torrado C ，Myint T ，Cowen S ，Griffiths M ，Reid AG ，Apperley J ，White H ，Huggett JF ，Foroni L ... -  《-》

Comparison of Real-Time Quantitative PCR and Digital Droplet PCR for BCR-ABL1 Monitoring in Patients with Chronic Myeloid Leukemia.

Real-time quantitative PCR (qPCR) is routinely used to detect minimal residual disease in chronic myeloid leukemia patients. The absolute quantification with droplet digital PCR (ddPCR) could reduce the inherent variability of qPCR. We established a duplex ddPCR assay using the Europe against Cancer (EAC) primer/probe system for breakpoint cluster region protein-tyrosine-protein kinase ABL1 (BCR-ABL1) and ABL1 and compared the results with qPCR. cDNA samples (n = 230) from patients with chronic myeloid leukemia were analyzed using both procedures. A second, commercially developed ddPCR assay for BCR-ABL1 was also evaluated. ABL1 and BCR-ABL1 transcript levels were similar with all assays, but the proportion of deep molecular responses was lower with ddPCR than with qPCR. The EAC ddPCR assay had a false-positive rate of 4% using a cutoff of three BCR-ABL1 copies per duplicate, compared with 2% without cutoff for the commercial ddPCR. The detection rate for molecular response 4.5 was 100, and a shift toward more minimal residual disease was seen in patient samples. In conclusion, using the EAC protocol for BCR-ABL1 quantification with ddPCR is feasible and shows low intra-assay and interassay variation but requires a cutoff that reduces sensitivity. The commercial ddPCR assay is highly sensitive and specific for BCR-ABL1. The use of either ddPCR assay resulted in a shift to lower molecular response classes compared with qPCR aligned to international scale.

Franke GN ，Maier J ，Wildenberger K ，Cross M ，Giles FJ ，Müller MC ，Hochhaus A ，Niederwieser D ，Lange T ... -  《-》