Elsevier

Leukemia Research

Volume 38, Issue 5, May 2014, Pages 581-585
Leukemia Research

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

https://doi.org/10.1016/j.leukres.2014.02.005Get rights and content

Abstract

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.

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.

References (18)

There are more references available in the full text version of this article.

Cited by (19)

  • How I treat Philadelphia chromosome–positive acute lymphoblastic leukemia

    2019, Blood
    Citation Excerpt :

    More recent methods such as amplicon-based next generation sequencing of immunoglobulin/T-cell receptor may overcome some of the limitations of RT-PCR and enhance sensitivity.33,34 Other novel and highly sensitive assays such as droplet digital PCR are also in development.35,36 Achieving deep molecular responses using TKI-based regimens has also rekindled interest in autologous SCT with some studies reporting equivalent long-term outcomes for patients undergoing allo-HCT and autologous SCT.15,37,38

  • Droplet Digital PCR Is a Robust Tool for Monitoring Minimal Residual Disease in Adult Philadelphia-Positive Acute Lymphoblastic Leukemia

    2018, Journal of Molecular Diagnostics
    Citation Excerpt :

    Digital PCR is especially powerful in MRD monitoring because it is able to detect rare mutant events in a background of wild-type molecules. In fact, several researchers have endeavored to investigate the reliability of ddPCR in MRD monitoring of hematologic malignancies.37–44 To date, only one study used digital PCR technology for MRD monitoring of Ph+ ALL patients, comparing its performance and sensitivity with the method based on qPCR.37

  • Secondary mutations as mediators of resistance to targeted therapy in leukemia

    2015, Blood
    Citation Excerpt :

    Real-time measurement of differences in the melting properties forms the basis of high-resolution melting curve analysis used in screening of relatively low-frequency mutations such as IDH1, IDH2, and DNMT3A.19 Multiplexing and multianalyte approaches in real-time PCR are gradually emerging to allow simultaneous detection of many single nucleotide variants and translocations at high analytical sensitivity.20,21 In comparison with the nonsequencing technologies listed above, which rely on physical properties of the PCR product or specificity of primer/probe sequences, the sequencing-based technologies such as pyrosequencing (best suited for short stretches of DNA such as codons 12, 13, and 61 in KRAS/NRAS; sensitivity: 5-10%), Sanger sequencing (best suited for genes with nonhotspot mutations such as CEBPA; sensitivity: 10-20%), and recent next-generation sequencing (NGS; sensitivity for clinical use: ∼5%) directly sequence the target DNA to identify changes compared with the reference sequence.

View all citing articles on Scopus
1

These authors contributed equally to the work.

View full text