BCR-ABL1 Quantification Research Articles

Comparison of an international scale method and a log reduction method for monitoring of early molecular response in chronic myeloid leukemia patients.

TO THE EDITOR: The International Randomized Interferon versus STI-571 study was the first randomized trial to demonstrate the prognostic significance of monitoring BCR-ABL1 transcripts by real-time quantitative polymerase chain reaction (RT-PCR) [1], and defined a major molecular response (MMR) after treatment as a 3-log10 reduction from the baseline value in the level of measured BCR-ABL1 transcripts [1]. On the other hand, an international scale (IS) was proposed as an absolute value of 0.1% BCR-ABL1, which corresponds to a 3-log10 reduction from the baseline value, for MMR, in an effort made by international experts to standardize BCR-ABL1 quantification [2,3].

MRD Monitoring Using Minor-BCR-ABL1 Genomic Breakpoint in Childhood ALL Identifies a Subgroup with Distinct Biology and a Very Poor Prognosis

▪Background:The exact role of minimal residual disease (MRD) testing in childhood BCR-ABL1+ ALL is still unclear and MRD levels are used rather for stem cell transplantation (SCT) indication than for frontline treatment stratification. There are two standard targets used for MRD monitoring in these ALLs: Ig/TR rearrangements and the fusion transcript quantification. However, despite similar sensitivity of the methods, we previously showed a poor correlation between them in some patients. Overall, 20% of samples negative by Ig/TR MRD were BCR-ABL1 positive (even at high levels). By demonstrating presence of BCR-ABL1 within the non-lymphoid (Ig/TR-negative) population in the non-correlating samples we showed that multilineage involvement is at least partly responsible for the discrepancy (Zaliova, Leukemia 2009). Here we established MRD monitoring based on the quantification of the patient-specific genomic (intronic) BCR-ABL1 breakpoint sequence, compared MRD levels obtained from different approaches and analyzed their prognostic significance.Methods:Our cohort totals 35 children diagnosed with minor-BCR-ABL1+ ALL. The BCR-ABL1 genomic breakpoint was found using multiplex long distance DNA PCR. The patient-specific intronic fusion sequences were used for MRD quantification by qPCR. We quantified MRD levels in 386 bone marrow (BM) samples. The results of genomic BCR-ABL1 quantification were compared with Ig/TR and BCR-ABL1 transcript levels. Samples with >1 log difference in MRD levels were considered non-correlating. For correlation analysis of methods, double-negative (DN) samples were excluded. Overall survival (OS) rates were calculated according to Kaplan-Meier and compared by log-rank test.Results:Analysis of MRD in BM samples confirmed poor correlation between Ig/TR and BCR-ABL1 mRNA quantification (Spearman correlation coefficient R=0.70; n=166 non-DN samples). Moreover, we also saw poor correlation when comparing the two DNA methods (Ig/TR vs. BCR-ABL1 DNA, R=0.69; n=254 non-DN samples) with 21% of samples positive by BCR-ABL1 (at levels up to 10e-1) while negative by Ig/TR. While in some patients the correlation of the DNA-based methods was very good, others had several subsequent follow-up samples with poor correlation (> 1 log difference) suggesting distinct biology of the disease. MRD levels determined by BCR-ABL1 DNA quantification had the best predictive value for overall survival (the best separation of survival curves was at week 12 of treatment with 10e-3 cut-off: BCR-ABL1 DNA, p=0.0003; Ig/TR, p=0.017; BCR-ABL1 mRNA, not significant).The proportion of samples with BCR-ABL1 DNA levels higher than Ig/TR by more than 1 log increased with time on treatment (day 15: 10% (2/20); day 33: 26% (7/27); week 12: 39% (7/18); week 22: 50% (7/14)). Importantly, presence of samples with this poor correlation was prognostically relevant as patients with BCR-ABL1 levels higher than Ig/TR by more than 1 log at the end of induction treatment (day 33) had significantly worse outcome (2 years OS 84+/-9% (n=21) vs. 28+/-23% (n=6) for correlating and non-correlating patients, respectively; p=0.004).Conclusion:The BCR-ABL1 quantification at genomic level probably provides the most accurate measurement of leukaemic burden. Due to multi-lineage involvement in some Ph+ ALL as well as occasional presence of subclones with different Ig/TR markers, quantification of the BCR-ABL1 gene breakpoint is more reliable than Ig/TR monitoring. Moreover, in contrast to the BCR-ABL1 transcript, measurement of the gene is not influenced by possible variability in expression between cell subtypes or during concurrent treatment. Importantly, the MRD levels determined by BCR-ABL1 DNA-based testing have higher predictive value than Ig/TR DNA or BCR-ABL1 transcript quantification. The poorer outcome observed in the subset of patients in whom BCR-ABL1 DNA-based MRD levels were higher than Ig/TR-based MRD detection possibly reflects multilineage involvement, suggesting, that some cases with minor-BCR-ABL1+ ALL are in fact CML cases identified in a lymphoid blast crisis. In view of their poor outcome, these cases might be candidates for early SCT or alternative treatments. The most reliable method for MRD detection in BCR-ABL1+ ALL needs to be further investigated within a large therapeutic protocol.Support: GAUK 554214; MH CZ-DRO UH Motol 00064203; NHMRC Australia APP1057746. DisclosuresMachova Polakova:Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

DNA Based Detection and Quantification of BCR-ABL1 Gene Using Patient-Specific qPCR and Droplet Digital PCR Tests in Chronic Myeloid Leukemia

Introduction - Targeted therapy of chronic myeloid leukemia (CML) using tyrosine kinase inhibitors (TKI) very effectively suppresses the growth of leukemic clone to a clinically safe level of the disease. Therefore sensitive monitoring of molecular response (MR) based on standardized measurements of BCR-ABL1 transcript levels plays an important role for prediction either of optimal response or progression. Thus, its sensitive and accurate detection is very important for the individualized therapy, especially for a TKI stopping treatment management in patients achieving long-term deep MR. While the transcript levels may not exactly correspond to the number of leukemic cells, BCR-ABL1 quantification at the DNA level using patient-specific assays may bring more accurate information about residual disease. Moreover, BCR-ABL1 DNA quantification may contribute to a stratification of patients for whom the cessation of TKI therapy may be safe.Objectives - This work is focused on the comparison of BCR-ABL1 quantity at DNA and mRNA level in CML patients after TKI initiation until the MR achievement, and after TKI discontinuation within the EURO-SKI clinical trial (Europe Stops TKI in CML), respectively, and on determination of differences on BCR-ABL1 positivity/negativity of samples analyzed. Additionally, the BCR-ABL1 DNA data obtained from the measurements by qPCR and droplet digital PCR (ddPCR) were compared.Methods - Our study included analyses of 232 samples of 13 patients with CML in chronic phase (CP), who achieved deep MR during TKI treatment. Four of 13 patients (total of 85 samples analyzed) discontinued the treatment within the EURO-SKI study. The levels of BCR-ABL1 mRNA were determined by standardized RT-qPCR method and DNA levels were detected by qPCR with developed patient-specific qPCR assays after BCR-ABL1 genomic fusions characterization. The ddPCR method was performed on 72 samples using QX-200 Droplet Digital PCR System (Bio-Rad). The determined ratios of BCR-ABL1/GUSB (mRNA) and BCR-ABL1/albumin (DNA level) of follow-up samples of every patient were related to the diagnostic sample (considered as 100% level) for the most appropriate comparison of mRNA and DNA levels.Results - Standardized RT-qPCR and patient-specific qPCRs were able to detect the mRNA and DNA BCR-ABL1 levels, respectively, reduced by more than 5 logs from the levels at the time of diagnosis. We observed a significant correlation between the mRNA and DNA levels by comparing 148 paired BCR-ABL1 positive results (correlation coefficient r2=0.9055; P˂0.0001). When comparing the frequency of the BCR-ABL1 negative/positive results at the DNA vs. mRNA levels, BCR-ABL1 positivity at DNA level was found in 21 samples which were BCR-ABL1-negative at mRNA level. BCR-ABL1 mRNA was found either negative or positive at MR5.0 in 3/4 EURO-SKI patients within the period between MR achievement until the TKI discontinuation (19 samples analyzed). The level of BCR-ABL1 DNA was continuously positive in this period before the TKI cessation. The molecular relapse (i.e. MMR loss) was observed in all those 3 patients several months after the therapy cessation. The levels of BCR-ABL1 DNA detected by ddPCR method correlated with DNA qPCR results (r2=0.9684; P˂0.0001) and the obtained values were not significantly different.Conclusion - The levels of the BCR-ABL1 mRNA and DNA in peripheral blood (PB) correlated significantly, thus both consistently reflecting the course of CP-CML at the molecular level. Although much more patients with TKI cessation need to be tested for the presence of BCR-ABL1 DNA, our data suggest that quantification at the DNA level appears to be (at least in some cases) more sensitive method for detection of residual leukemic cells in PB in comparison to the mRNA quantification. The BCR-ABL1 DNA measurement may have a potential prognostic significance in deciding for eventual withdrawal of TKI therapy, which is currently being investigated within the clinical trials. Digital PCR enables absolute quantification without a need of patient-specific calibration curves and with the potentially higher sensitivity and accuracy for BCR-ABL1 DNA quantification.This work was supported by the project 15-31540A of the Czech Health Research Council, GAUK 554214 and EURO-SKI Research Consortium. DisclosuresKlamova:Novartis: Consultancy, Honoraria, Research Funding; Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding. Saussele:ARIAD: Honoraria; Pfizer: Honoraria, Other: Travel grant; BMS: Honoraria, Other: Travel grant, Research Funding; Novartis Pharma: Honoraria, Other: Travel grant, Research Funding. Mahon:Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Machova Polakova:Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Clinical Evaluation of Xpert® BCR-ABL Ultra, an Automated and Standardized Cartridge-Based Assay for the Quantification of BCR-ABL1

▪Assays used to monitor BCR-ABL levels in CML patients on TKI therapy should be sensitive enough to reliably detect BCR-ABL transcript levels at 4.5 logs below baseline for optimal clinical utility. Here we describe the clinical comparison of Xpert® BCR-ABL Ultra (V2) and Xpert® BCR-ABL Monitor (V1). V1 uses 200uL of peripheral blood. V2 uses 4mLs to enhance sensitivity. Both assays are currently standardized to the IS.V2 was compared to V1 using prospectively acquired CML patient specimens (N=165) in EDTA, NaCitrate, and PAXgene for a potential 495 paired comparisons (Phase I). Linear regression, Bland-Altman, and concordance at MMR and CMR4.0 analyses were performed. Using blinded precision materials (Ph+ cultured cells containing BCR-ABL e14a2 diluted into Ph- normal donor blood to produce specimens at targeted BCR-ABL1 transcript levels spanning the dynamic range from 10% to 0.001% IS), V1 and V2 were repeat tested by two operators at each of 4 sites 8 times on different days (N = 2 x 8 x 4 = 64 replicates per day; 5 sites participated on Day 1, N=80), for a total N = 272 (Phase II).96.2% of V1, 93.7% of V2, and 99.6% of external control specimens were successful on the first attempt. Across all types of anticoagulant, linear regression shows a slope of 1.083 +/- 0.028 (95% CI) with intercept of 0.31 +/- 0.032 (95% CI) and an R2 = 94.5% for V2 log10 BCR-ABL/ABL% IS (Y) versus V1 log10 BCR-ABL/ABL% IS (X). Bland-Altman analyses reveal a mean bias across the dataset of 0.27. Positive Percent Agreement (PPA) for V2 vs. V1 was 96.4% (298/309) and Negative Percent Agreement (NPA) for V2 vs V1 was 66.9% (87/130). 39/43 specimens detected by V2 but not V1 were below 0.01% (IS). Concordance studies for MMR showed an Overall Percent Agreement (OPA = [# comparisons with both assays <0.01%] plus [# with both assays >0.01%]/total comparisons) > 97.0% for all comparisons and 98.0%, 93.9%, and 89.4%, respectively, for EDTA, NaCitrate, and PAXgene for CMR4.0. In Phase II, 100% (N=203) of samples at 1%-0.01% were positive by V2; 50.8% (32/63) at 0.001% were positive by V2.In conclusion, the Xpert BCR-ABL Ultra (V2) assay demonstrates concordance with version 1 of the Xpert BCR-ABL assay across a wide dynamic range in both clinical and contrived specimens. Higher discordance is noted in the range 0.01-0.001% (IS) likely due to the enhanced sensitivity of BCR-ABL Ultra (V2). DisclosuresMauro:Novartis Pharmaceutical Corporation: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Pinilla-Ibarz:Pfizer: Consultancy, Other: Consulting & Advisory Role, Research Funding, Speakers Bureau; ARIAD Pharmaceuticals, Inc.: Consultancy, Other: Consulting & Advisory Role, Research Funding; Novartis: Consultancy, Other: Consulting & Advisory Role, Research Funding; BMS: Consultancy, Honoraria, Other: Consulting & Advisory Role, Speakers Bureau; Teva: Consultancy, Speakers Bureau. Uy:Novartis: Research Funding.

International Scale (IS)-Standardized BCR-ABL1 Digital Polymerase Chain Reaction (dPCR) Assays Using ABL1, BCR, and GUS Control Genes for Measuring Deep Molecular Response (MR) in Chronic Myeloid Leukemia (CML)

▪Background: The evolution of BCR-ABL1 tyrosine kinase inhibitors (TKIs), from imatinib to newer agents such as nilotinib, has led to progressively lower levels of disease burden in patients with CML receiving TKI therapy. Currently, real-time quantitative PCR (RQ-PCR) is the most commonly used test for assessing MR in patients with CML and is able to detect BCR-ABL1 transcript levels down to MR4.0 to MR4.5 on the IS. It has been reported that dPCR can detect BCR-ABL1 with greater sensitivity and precision than RQ-PCR. In addition, unlike RQ-PCR, dPCR is able to perform absolute copy number quantification without the use of calibrators; thus, it should be free from calibrator biases. To date, validation of dPCR BCR-ABL1 assays with multiple control genes and standardization to the World Health Organization (WHO) IS reference material have not been reported. Standardization to the IS is critically important to enable appropriate assessment of patient response to therapy and to establish a standard of care. In this study, we describe the development, validation, and IS standardization of a sensitive BCR-ABL1 dPCR assay using 3 different control genes. We also tested the comparability of 3 different dPCR platforms.Methods: Four experiments were included in this study: (1) Three BCR-ABL1 dPCR assays were developed using ABL1, BCR, and GUS as control genes on the Bio-Rad dPCR platform. The ABL1 assay was standardized to the IS using the WHO Reference Panel. The BCR and GUS IS alignments are currently in process. All 3 assays were validated for linearity, precision, accuracy, limit of detection, and limit of blank, following in vitro diagnostics industry best practices and Clinical and Laboratory Standards Institute (CLSI) guidelines. (2) Multiple BCR-ABL1 and ABL1 primer/probe sets were tested on both RQ-PCR and dPCR for the selection of the best primer/probe design. (3) BCR-ABL1 and ABL1 quantifications were compared between 3 dPCR platforms: Bio-Rad QX200, RainDance RainDrop System, and Applied Biosystems QuantStudio3D. (4) Sensitivity of the BCR-ABL1 dPCR assay will be compared in CML PAXgene vs K2EDTA blood tube samples to assess the potential impact of blood-collection tube type on deep MR measurement in patients with CML.Results: (1) The BCR-ABL1/ABL1 dPCR assay WHO IS alignment resulted in a conversion factor of 1. Using the WHO IS-calibrated BCR-ABL1/ABL dPCR assay, MR in blood samples from patients with CML were quantified with sensitivity beyond MR4.5 to levels between MR5.0 and MR5.5 (Figure 1). Combining multiple dPCR wells, which enables analysis of thousands to millions of reactions together, improved the sensitivity and precision of the assay (Figure 2). (2) Evaluation of 10 BCR-ABL1 and 9 ABL1 primer/probe sets using the Bio-Rad dPCR platform resulted in < 2-fold differences in copy number detection compared with the 16- to 32-fold difference using RQ-PCR. This is likely due to dPCR measurement being an endpoint PCR reaction. (3) Less than 2-fold differences in both detected copy number and BCR-ABL1/ABL1 ratios were observed using 1 selected primer/probe set when a contrived sample panel representing key BCR-ABL1 levels from 10% to 0.0032% IS was tested across 3 different dPCR platforms, suggesting good cross-platform robustness of dPCR (Table). (4) The PAXgene vs K2EDTA blood tube comparison study is ongoing; data will be presented upon completion of the study.Conclusion: Three dPCR BCR-ABL1 assays were developed. The BCR-ABL1 dPCR assay demonstrated detection capability at levels below MR4.5, down to MR5.0 to MR5.5 in contrived samples from patients with CML. This increased sensitivity relative to RQ-PCR may aid future comparisons of deep MR rates across different CML therapies. dPCR assay performance is also more robust against primer/probe design changes than RQ-PCR, thus requiring less assay optimization. Our results demonstrate that dPCR offers robust BCR-ABL1 quantification on the WHO IS across commonly used control genes without requiring calibrators or extensive assay optimization. [Display omitted] [Display omitted] [Display omitted] DisclosuresHuang:Novartis: Employment. Salcius:Novartis: Employment, Equity Ownership. Wong:Novartis: Employment, Equity Ownership. Wong:Novartis: Employment. Wang:Novartis: Employment, Equity Ownership.

Performance of two commercially available BCR-ABL1 quantification assays that use an international reporting scale.

Quantifying the BCR-ABL1 rearrangement is important for monitoring chronic myelogenous leukemia (CML). To standardize BCR-ABL1 quantification, the World Health Organization (WHO) established the first international genetic reference panel. Here, we compared the BCR-ABL1 levels determined using international scale (IS)-based commercially available assays. BCR-ABL1 transcripts were quantified using two IS-based assays. 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 dilutions of the b3a2 positive RNA were used for evaluating linearity, precision, and limit of detection. Correlation of the assay was evaluated by using DNA obtained from CML patients carrying the BCR-ABL1 b3a2 and b2a2 types. Both Ipsogen and Asuragen assays showed fine linearity with reasonable %CV. LOD of each assay was calculated as 0.003% for Ipsogen, and 0.005% for Asuragen. By comparing the results that were lower than 10% by either one of the assay, Ipsogen and Asuragen results showed an overall good linear correlation with a tendency for the Ipsogen assay to show slightly higher levels than the Asuragen assay for b3a2 transcript. For b2a2, the tendency was opposite, with Asuragen showing higher values than the Ipsogen. Two commercially available IS-based BCR-ABL1 assays showed an overall good quantitative correlation. It should be taken into consideration that each assay tended to produce higher values than the other, depending on the BCR-ABL1 subtypes, suggesting that a separate conversion factor for each subtype can be more helpful when BCR-ABL1 transcript levels are converted into IS.

Quantification of the Minimal Residual Disease in Pediatric Philadelphia-Positive Acute Lymphoblastic Leukemia

Philadelphia (Ph) chromosome in acute lymphoblastic leukemia (ALL) is found nearly in 20-30% adults and 5% children. Ph chromosome results from the reciprocal translocation t(9;22)(q34;q11) joining the tyrosine kinase c-ABL1 proto-oncogene on chromosome 9 to BCR gene on chromosome 22. The majority of Ph breakpoint in ALL occurs in the minor breakpoint cluster region at first intron of the BCR gene, and results in the fusion of BCR exon 1 to ABL1 exon 2 (e1a2) generating the oncoprotein p190BCR-ABL1. Ph+ ALL patients present an aggressive disease with a poor prognosis. Current treatment includes potent chemotherapy and tyrosine kinase inhibitors, such as imatinib. The aim of this study was to quantify BCR-ABL1 transcripts in ALL Ph+ patients at different stages of treatment in order to evaluate the kinetics of the tumor clone in response to treatment.We reported a preliminary analysis of the clinical trial GATLA LLA-Ph+2011 (Grupo Argentino de Tratamiento de la Leucemia Aguda LLA-Ph+) that includes patients from 1 to 21 years old newly diagnosed Ph+ ALL. Written informed consent was obtained from all subjects. Diagnosis of Ph+ALL was confirmed by the presence of Ph chromosome and/or positive molecular analysis for detection of BCR-ABL1 fusion transcripts. All patients received induction treatment consisting of vincristine, daunorubicin, prednisolone, asparaginase, and metrotexate. Imatinib was administered 300 mg/m2/day from day 15 of induction until the initiation of conditioning for alloHSCT or to the end of maintenance therapy. The clinical protocol consist in an induction IA (33 days) and IB (28 days), consolidation RA1, RA2 and RA3, re-induction (Protocol II A and B, 36 days each) and maintenance to complete 2 years of treatment. Eligible patients are transplanted after the third block.Minimal residual disease (MRD) monitoring was performed by one-step quantitative RT-PCR assays in bone marrow and/or peripheral blood, using RotorGene® Qiagen; at the end of induction AI, previous RA1, RA2, RA3 and protocol II Fase IIA, IIB and during maintenance or post-alloHSCT. BCR-ABL1 quantification was expressed relative to the amount of ABL1 mRNA. We studied a control group of pediatrics positive P190 isoform bone marrow samples at diagnosis in order to establish our basal reference to estimate the logarithmic reduction of BCR-ABL1 transcripts for our cohort. The obtained mean value of BCR-ABL1(e1a2)/ABL1 in the control group was 55.5% (range 29% - 92%). The molecular response was defined as optimal (OMR, OMR-undetectable) if the BCR-ABL1/ABL1 ratio was lower than 0.055%.A total of 25 subjects (male: female = 16: 9) were enrolled into the study from November 2011 to July 2014. The median age was 7 (range 2 - 19) years old. Type of BCR breakpoint was minor (e1a2) in 96% of patients, and only one present a rearrangement corresponding to major BCR (b3a2). During the follow up (median: 15 months), five patients (20%) died: 3 by sepsis, 1 by post alloHCT complications, and 1 due to disease progression. Two patients had to rotate to dasatinib because of disease relapse: one in central nervous system and the other post alloHCT. Our preliminary results showed that the overall survival was 79%±10% and the survival free of event (death, molecular and disease relapse) was 67%±10% at 12 months.We serially measured BCR-ABL1 at different trial stage (AI, RA1, RA2, RA3, Protocol II) and OMR was 13.3%, 41.2%, 33.3%, 45.5% and 76.9%, respectively. Among 17 evaluable patients on maintenance and post alloHCT, 88% (15/17) obtained OMR values, and among them, 87% (13/15) presented OMR-undetectable values.We evaluated the kinetics of the tumor clone under treatment regarding prognostic factors. Patients older than 10 years showed a higher rate of OMR at RA1 (83% vs 27%, p=0.05). However, all evaluated younger patients reached an OMR at protocol II stage (100% vs 33%, p=0.045), showing a sustained and deeper molecular response. Other evaluated parameters did not show a relationship with OMR at different stages.Our preliminary data showed that BCR-ABL1 quantification may be a good marker to evaluate response to treatment in Ph+ positive ALL patients. BCR–ABL1 transcripts dropped to undetectable levels in 76% (13/17) of evaluable cases at the later stage of treatment. Also, 77% of patients achieved an OMR at Protocol II not only might provide a better chance for receiving alloHSCT but also might contribute to durable remissions. DisclosuresNo relevant conflicts of interest to declare.

Calibration of BCR–ABL1 mRNA quantification methods using genetic reference materials is a valid strategy to report results on the international scale

ObjectivesUntil recently, diagnostic laboratories that wanted to report on the international scale had limited options: they had to align their BCR–ABL1 quantification methods through a sample exchange with a reference laboratory to derive a conversion factor. However, commercial methods calibrated on the World Health Organization genetic reference panel are now available. We report results from a study designed to assess the comparability of the two alignment strategies. Design and methodsSixty follow-up samples from chronic myeloid leukemia patients were included. Two commercial methods calibrated on the genetic reference panel were compared to two conversion factor methods routinely used at Saint-Louis Hospital, Paris, and at Lille University Hospital. Results were matched against concordance criteria (i.e., obtaining at least two of the three following landmarks: 50, 75 and 90% of the patient samples within a 2-fold, 3-fold and 5-fold range, respectively). ResultsOut of the 60 samples, more than 32 were available for comparison. Compared to the conversion factor method, the two commercial methods were within a 2-fold, 3-fold and 5-fold range for 53 and 59%, 89 and 88%, 100 and 97%, respectively of the samples analyzed at Saint-Louis. At Lille, results were 45 and 85%, 76 and 97%, 100 and 100%, respectively. Agreements between methods were observed in the four comparisons performed. ConclusionOur data show that the two commercial methods selected are concordant with the conversion factor methods. This study brings the proof of principle that alignment on the international scale using the genetic reference panel is compatible with the patient sample exchange procedure. We believe that these results are particularly important for diagnostic laboratories wishing to adopt commercial methods.

Use of a high sensitive nanofluidic array for the detection of rare copies of BCR-ABL1 transcript in patients with Philadelphia-positive acute lymphoblastic leukemia in complete response

Monitoring of minimal residual disease (MRD) by quantification of BCR-ABL1 transcript levels has become a main part of the management of patients with BCR-ABL1-positive acute lymphoblastic leukemia (ALL) in treatment with tyrosine kinase inhibitors (TKIs). The failure to achieve molecular negativity shortly after starting TKI has been demonstrated to be predictive of relapse, suggesting that an accurate measurement of low BCR-ABL1 levels may have a role in preventing hematological relapse. Despite the big efforts made by many European laboratories within the European Study Group, at the time of writing a standardized procedure to quantify and express results is still missing for BCR-ABL1-positive ALL. In this study, in order to detect with high sensitivity low levels of BCR-ABL1 transcripts, we used a new technology and a new molecular approach based on microfluidic digital polymerase chain reaction (dPCR) using Taqman chemistry and we compared obtained results with those generated by the conventional method based on reverse transcriptase PCR reaction (RQ-PCR) for BCR-ABL1 and total ABL1, with TaqMan chemistry and with Applied Biosystems instrument. We demonstrated the dPCR is high-sensitive (able to detect a single copy of BCR-ABL1) and reliable (results are comparable to those obtained by BCR-ABL1 quantification with conventional technology), allowing an accurate monitoring of BCR-ABL1-positive ALL patients in complete remission.

Comparative study of BCR-ABL1 quantification: Xpert assay, a feasible solution to standardization concerns

The level of BCR-ABL1 reached after treatment with tyrosine kinase inhibitors is an effective marker of the therapeutic response and a good survival predictor in chronic myeloid leukemia (CML) patients. However, no agreement has yet been achieved about either the standardization of the technique to determine BCR-ABL1 or the interpretation of the results. The aim of this study was to compare the method currently recommended by the European Leukemia Net, which includes the application of a conversion factor to express the results in international scale, with an automated method (Xpert BCR-ABL™, Cepheid). BCR-ABL1 transcript quantification was performed in 117 samples from CML patients in two different laboratories by both methods, and the results were compared by statistical procedures. A high linear correlation was obtained in the results between the two methods. The concordance at logarithmic intervals reached 62 %. When the major molecular response (MMR) was analyzed, 85 % agreement was achieved. The automated method provides reproducible results and does not show significant differences compared with the traditional method. As a clinical tool, Xpert correctly classified the patients in MMR and can be considered a useful alternative for the molecular follow-up of CML patients.

BCR-ABL1 transcript levels increase in peripheral blood but not in granulocytes after physical exercise in patients with chronic myeloid leukemia

In chronic myeloid leukemia (CML) treatment response is determined by measurements of BCR-AB1L transcripts in peripheral blood by quantitative real-time PCR (qRT-PCR) and a 2–5 fold increase is considered a warning sign. The BCR-ABL1 gene is mainly expressed in myeloid cells whereas quantification of BCR-ABL1 is performed on the nucleated cell fraction of peripheral blood. Hence, leukocyte composition of the nucleated cell fraction may affect the result of BCR-ABL1 quantification. The aim of this study was to investigate if changes in leukocyte composition of peripheral blood had any effect on BCR-ABL1 transcript levels in CML patients. Six CML patients in complete cytogenetic remission (CCgR) performed a maximal physical exercise test. Blood samples were collected before exercise, at maximal exhaustion and after exercise. A biphasic increase in leukocyte count was observed and the relative proportion of granulocytes in peripheral blood changed significantly after exercise compared with baseline (p < 0.001). The BCR-ABL1 transcript level increased significantly following exercise, in nucleated cell fraction of peripheral blood (p < 0.05) but not in isolated granulocytes. In the nucleated cell fraction, the mean BCR-ABL1 transcript level was 3.3-fold (range 0.7–6.8) higher 180 min after exercise compared with baseline (p < 0.01). In conclusion, physical exercise induced significant increases in BCR-ABL1 transcript levels concomitant with changes in leukocyte content of peripheral blood. We therefore suggest that variations in leukocyte composition of peripheral blood, causing pre-analytic variations that affect BCR-ABL1 quantification, have to be accounted for. Consequently, small variations in BCR-ABL1 transcript levels should be interpreted cautiously in CML patients in CCgR.