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
Introduction
The BCR-ABL1 transcript resulting from the t(9;22) chromosome translocation known as the Philadelphia (Ph) chromosome is the most frequent genetic abnormality associated with adult ALL. Treatment strategies based on TKIs of first and second generation have substantially improved overall treatment results, with rapid and complete response (CR) rates in 95–100% of patients [1], [2], [3], [4], [5], [6]. Nevertheless, the majority of them experience hematological relapse in a short time, also after hematopoietic stem cell transplantation (SCT) [7]. The presence of BCR-ABL1 transcripts after alloSCT in the pre-imatinib era was indicative of minimal residual disease (MRD) and predicted a relapse in patients with BCR-ABL1-positive ALL [8]. Thereafter in the TKI era, the failure to achieve molecular negativity shortly after starting imatinib was predictive of relapse [9], [10]. Moreover, BCR-ABL1 levels lower than 10−3 at day 85 have been demonstrated to correlate with higher disease-free survival compared with patients who never reached these levels during induction of dasatinib treatment [6]. Therefore, the detection of residual BCR-ABL1 transcript levels by quantitative polymerase chain reaction (qPCR) provides relevant clues to detect an early relapse during TKI treatment therapy allowing a prompt switch of therapy before hematological relapse. The monitoring of residual BCR-ABL1 transcript levels has been recently well standardized for p210 quantification in chronic myeloid leukemia (CML) [11]. The establishment of a laboratory-specific conversion factor using a process initiated by the Adelaide laboratory allows to report own molecular results on an international scale, which standardizes quantitative BCR-ABL1 measurements across tests and laboratories, allowing multiple laboratory studies, patient management, and a harmonized definition of treatment response [12]. In contrast to p210, there is less standardization for p190 quantification. The European Study Group's is currently performing twice annual quality control rounds to define a pan-European standard, but at the time of writing there is still variation in methodology and reporting results among different participating laboratories. In recent years, new technologies have emerged to provide a very sensitive detection of very low levels of disease by microfluidic digital PCR (dPCR). In dPCR single molecules are isolated by dilution and individually amplified by PCR. The partitioning of the sample prior to PCR amplification in chambers containing 0 or 1 copy of target DNA allows that each product is analyzed separately. During analysis a Poisson correction is applied to the results to account for chambers that contain more than one molecule, and an absolute target sequence quantity is estimated [13]. Among different technologies, the Biomark system from Fluidigm (Fluidigm Corporation, South San Francisco, CA) has been recently demonstrated to have good analytical sensitivity and to be highly reproducible [13]. In this study, we assessed the dPCR methodology to detect and quantify residual and rare BCR-ABL1 copies in BCR-ABL1-positive ALL patients, and we compared obtained molecular results with those generated by conventional qPCR using ABI PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).
Section snippets
Patients and methods
60 BCR-ABL1-positive ALL samples in hematologic and cytogenetic remission (42 positive for the p190 BCR-ABL1 isoform and 18 for the p210) were analyzed. Total cellular RNA was extracted from cells using the RNeasy total RNA isolation kit (QIAGEN, Valencia, CA) according to the instructions of the manufacturer and 1 μg was used for cDNA synthesis in the reverse transcriptase reaction (RT), as previously described [14]. For real time PCR analysis we used 5 μL of cDNA (corresponding to 100 ng of
Results and discussion
Sixty samples from ALL patients in complete hematological and cytogenetic response were firstly analyzed by RQ-PCR with conventional method based on TaqMan chemistry and ABI PRISM 7900HT Fast Real-Time PCR System technology (Applied Biosystems). PCR results were expressed as BCR-ABL1/ABL1 ratio% and they ranged between 0 and 0.39 (median 0.01). More in details, analyzed samples showed a ratio ≤0.001 in 36.67% (22/60), ≤0.01 in 18.33% (11/60), ≤0.1 in 36.67% (22/60) and >0.1 in 8.33% (5/60) (
Conflict of interest
The authors have no conflicts of interest.
Contributors
Conception and design: Ilaria Iacobucci, Giovanni Martinelli. Provision of study materials or patients: Annalisa Lonetti, Claudia Venturi, Anna Ferrari, Emanuela Ottaviani, Stefania Paolini, Paola Bresciani, Leonardo Potenza, Sarah Parisi, Federica Cattina, Simona Soverini. Data analysis and interpretation, manuscript writing: Cristina Papayannidis, Ilaria Iacobucci, Maria Chiara Abbenante, Domenico Russo, Mario Luppi. Final approval of manuscript: Ilaria Iacobucci, Giovanni Martinelli.
Acknowledgments
This work was supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Programma di Ricerca Regione–Università 2007–2009, PRIN 2010-2011, NGS-PTL project, grant agreement number 306242, funded by the EC Seventh Framework Programme theme FP7-HEALTH-2012-INNOVATION-1.
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These authors contributed equally to the work.