Annals of Clinical & Laboratory Science 38:338-343 (2008)
© 2008 Association of Clinical Scientists
Rapid Detection of Prognostically Important Childhood Acute Lymphoblastic Leukemia Chimeric Transcripts Using Multiplex SYBR Green Real-Time Reverse Transcription PCR
Kamariah Ibrahim,
Siti Sarah Daud,
Yeow-Liang Seah,
Allen E. Yeoh,
Hany Ariffin for the Malaysia-Singapore Leukemia Study Group
Department of Paediatrics, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
Address correspondence to Dr Hany Ariffin, Department of Paediatrics, University of Malaya, 59100 Kuala Lumpur, Malaysia; tel 603 7949 2065; fax 603 7955 6114; e-mail hany{at}um.edu.my.
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Abstract
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Childhood acute lymphoblastic leukaemia (ALL) is a heterogenous disease in which oncogene fusion transcripts are known to influence the biological behaviour of the different ALL subtypes. Screening for prognostically important transcripts is an important diagnostic step in treatment stratification and prognostication of affected patients. We describe a SYBR-Green real-time multiplex PCR assay to screen for transcripts TEL-AML1, E2A-PBX1, MLL-AF4, and the two breakpoints of BCR-ABL (p190 and p210). Validation of the assay was based on conventional karyotyping results. This new assay provides a rapid, sensitive, and accurate detection method for prognostically important transcripts in childhood ALL.
Keywords: acute lymphoblastic leukemia, oncogene fusion transcripts, real-time PCR
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Introduction
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Childhood precursor B-cell acute lymphoblastic leukemia (cALL) is a highly heterogenous disease in which prognostically important oncogene chimeric transcripts can be identified in up to 63% of cases [1]. The most frequent and important of these transcripts are t(12;21)/TEL-AML1, t(1;19)/ E2A-PBX1, t(9;22)/BCR-ABL, and t(4;11)/MLL-AF4. These transcripts influence behaviour of the leukemic blasts and thus are valuable for risk-stratification of treatment as they are predictors of prognosis [2]. In addition, different treatment approaches may be useful for particular subgroups. For example, ALL patients with TEL-AML1 may benefit from intensive asparaginase therapy [3] while patients with BCR-ABL have shown improved outcome following aggressive chemotherapy and bone marrow transplantation [4]. The latter patients may also benefit from the addition of imatinib, which specifically targets the BCR-ABL-positive leukemic cells [5]. Infants positive with infantile associated leukemic transcript MLL-AF4 may improve their outcome with high dose cytarabine and allogeneic bone marrow transplantation [6,7].
We previously described a multiplex nested reverse-transcription PCR assay for detecting these prognostically significant transcripts [8]. Although this technique is simple and relatively inexpensive, it is time- and sample-consuming. In addition, carry-over contamination may occur during the second nested PCR step and post-PCR manipulation, leading to erroneous results.
Siraj et al [9] reported a multiplex real-time PCR method that is able to detect simultaneously all the prognostically important fusion transcripts in a single reaction using the fluorogenic dye, SYBR Green. This system is superior to the previously reported Taqman technology where dual-labeled fluorogenic-specific oligonucleotides are used in individual reactions [9,10]. Also, hydrolysis probes are much more expensive than SYBR Green.
In the present study, our main aim was to develop and evaluate an improved assay for detecting the prognostically important chimeric transcripts in cALL patients using SYBR Green real-time RT-PCR.
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Materials and Methods
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Patient samples.
This study included a total of 90 (83 de novo and 7 relapse) patients who were diagnosed with precursor B-cell ALL from January 2006 to April 2008 at the University of Malaya Medical Centre. All de novo patients were treated using a joint Malaysia-Singapore (Maspore ALL2003) chemotherapy protocol, which is an AEIOP-BFM based regimen. Parental signed informed consent and institutional ethical committee approval were obtained. The patients included 52 males and 38 females. The median age at diagnosis for de novo patients was 6 yr, 4 mo. Bone marrow aspirates were taken at diagnosis and collected in EDTA tubes. In several patients who had hyperleucocytosis with predominant blasts, peripheral blood was used.
RNA extraction and cDNA synthesis.
Mononuclear cells from BM aspirates and peripheral blood were isolated using Ficoll-hypaque density gradient ultracentrifugation. Total RNA was extracted from mononuclear cells using TRIZOL reagent (Invitrogen Life Tech Inc, Carlsbad, CA, USA) by means of acid guanidium thiocyanate-phenol-chloroform extraction. RNA was kept at –80°C prior to use. One µg of total RNA was reverse transcribed into cDNA using Omniscript reverse transcriptase (Qiagen, Germany) and 10 µM oligo-dT (New England Biolabs, USA). Reverse transcriptase minus control was included in all runs to exclude DNA contamination.
Conventional RT-PCR.
During the initial PCR optimization, conventional singleplex RT-PCR was used. The singleplex PCR was performed using leukemic cell lines containing specific chromosomal translocations; REH with t(12;21)/ TEL-AML1, K562 with t(9;22)/major breakpoint BCR-ABL (p210)Sup15 with t(9;22)/minor breakpoint BCR-ABL (p190), 697 with t(1;19)/E2A-PBX1, and RS4 with t(4;11)/MLL-AF4. These cell line cDNAs served as the positive controls while HL60 cell line cDNA served as the negative control. Primers used were as published by Siraj et al [9]. We designed primers for BCR-ABL p190 and p210. Amplification was initiated at 95°C for 15 min, followed by 94°C (15 sec), 60°C (30 sec), and 72°C (30 sec) for a total of 40 cycles. After ensuring that all primers were able to amplify the specific chimeric transcript accurately, a multiplex system was developed. Amplicons were run on 3% gel electrophoresis and visualized using ethidium bromide to check for specific products.
Multiplex real-time RT-PCR.
We used the RotorGene 3000 apparatus (Corbett Instruments, Australia), a rotor-based real-time PCR cycler. Detection of amplified PCR products was done using SYBR Green master mix (Qiagen, Germany). Each PCR reaction was carried out in 25 µl containing 10 µM of each primer, SYBR Green master mix, and water. Real-time PCR parameters consisted of 15 min of Taq polymerase activation, followed by 15 sec denaturation at 94°C, 30 sec annealing at 60°C, and 30 sec elongation at 72°C. DNA amplification proceded to 40 cycles. We monitored the fluorescence emission spectra and determined the specificity of the amplicon by melting peak analysis. Each patient sample was run in duplicate and experiments were repeated on separate occasions to ensure reliability and reproducibility of the results.
We optimized our method of screening for the common ALL-related chromosomal translocations using cell line cDNA. Subsequently we multiplexed the primers following the method proposed by Siraj et al [9], where detection of 3 different fusion transcripts (TEL-AML1, E2A-PBX1, and MLL-AF4) was feasible in a single PCR reaction. Specificity of a product was observed by the specific temperature melting peak. The primers used are shown in Table 1
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Up to 40% of BCR-ABL positive ALL patients have a p210 altered BCR-ABL fusion transcript [2,10]. Therefore, we also included primers for the major breakpoint (p210) BCR-ABL in our assay. Missing out this particular group during screening at initial diagnosis has great implications for patient management and prognosis. We combined the primer pairs specific for minor BCR-ABL (p190) and major BCR-ABL (p210) transcripts together in one reaction.
For each run, positive control, negative control, blank, and RT minus negative control were included. Hypoxanthylperoxyl transferase (HPRT) gene was used as the internal control (housekeeping) gene. HPRT is an excellent choice for a control gene during multiplex PCR as its amplicon has a different melting peak from the fusion transcripts [9]. It is invariably expressed in most cancer cells [11], making it one of the best housekeeping genes.
Identification of chromosomal translocation by karyotyping.
Concomitant karyotyping results from diagnostic marrow samples were available in 38 patients. Karyotyping analysis was performed at the cytogenetics laboratory at National University Hospital, Singapore. Bone marrow samples were kept in RPMI solution during transportation to provide suitable media for leukemia blast survival. Karyotype results served to validate the sensitivity and accuracy of our multiplex real-time RT-PCR assay as well as to rule out false positives.
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Results
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Identification of specific chimeric transcripts by real-time PCR.
Using cell line cDNA, we were able to detect simultaneously 3 different transcripts (TEL-AML1, E2A-PBX1, and MLL-AF4) in a single reaction with each PCR product producing a separate melting peak (Fig. 1). The 2 BCR-ABL transcripts were combined in a separate reaction tube and a specific melting peak was produced by each BCR-ABL isoform (Fig. 2).
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Fig. 1. Validation of multiplex real time PCR of 3 transcripts using specific cell line cDNA: REH cell line cDNA for TEL-AML1, 697 cell line cDNA for E2A-PBX1, and RS4 cell line cDNA for MLL-AF4. HL60 cell line was used as negative control. (Panel A): RT-PCR products demonstrated on agarose gel electrophoresis. Lanes 1 to 4: E2A-PBX1 (275bp), TEL-AML1 (181bp), MLL-AF4 (358bp), and HPRT (164bp). (Panel B): Melting peaks (Tm) of individual fusion transcripts; TEL-AML1 = 85.5°C, MLL-AF4 = 82.5°C, E2A-PBX1 = 92.5°C, and HPRT housekeeping gene = 78.0°C.
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Fig. 2. Fluorescence melting curve for SYBR Green-based multiplex real time PCR for simultaneous screening of two different BCR-ABL fusion transcript isoforms. K562 cell line cDNA was used for detecting BCR-ABL p210 and SupB15 cell line cDNA for BCR-ABL p190. (Panel A): Lane 1 = 298bp amplicon of BCR-ABLp190, Lane 2 = 250 bp amplicon of BCR-ABLp210, and Lane M = 100bp DNA ladder (Fermentas, USA). (Panel B): Melting peaks (Tm) of individual fusion transcripts; BCR-ABL p190 (90.8°C), and BCR-ABL p210 (84.2°C).
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Screening chimeric transcripts using multiplex SYBR Green RQ RT-PCR.
We tested and validated the assay with diagnostic bone marrow aspirates or peripheral blood samples from 90 pediatric patients with pre-B cell ALL. Each patient was screened for 5 different transcripts separated into 2 reaction tubes with internal control gene HPRT included. However, we noted that the ‘blank’ for the minor and major BCR-ABL breakpoints panel produced primer-dimer artifacts after 37 cycles. To eliminate this problem, we reduced the PCR cycles to 37 only.
We found that 50 patients were negative for fusion transcripts while 25 (27.8%) had TEL-AML1, five (5.5%) had BCR-ABL p190, two (2.2%) had BCR-ABL p210, seven (7.7%) had E2A-PBX1, while one infant was positive for MLL-AF4.
Validation of RQ-PCR assay by karyotyping.
As part of validation, we compared the molecular screening results in 38 patients who had karyotyping data (Table 2). The concordance was 100% between conventional karyotyping and our multiplex real-time RT-PCR assay. In addition, there were no false positives that might have arisen from contamination. The TEL-AML1 translocation is not readily detected via conventional karyotyping. Hence, multiplex real time RT-PCR is more sensitive in detecting the cryptic translocation TEL-AML1 and in cases where metaphase cells were inadequate for analyses. Although 7 patients were found to have E2A-PBX1 transcript, none of them had successful karyotyping performed and thus, validation of the RQ-PCR assay was done using cDNA from cell line 697, which is known to contain the E2A-PBX1 fusion transcript [12].
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Discussion
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Detection of specific chimeric transcripts in childhood ALL at the time of diagnosis is crucial since it plays an important role for accurate risk stratification and treatment management. Arguably, the BCR-ABL-positive subjects and, to some extent, those with MLL-AF4 are the patients that truly benefit from this assay since potentially curative bone marrow transplantation can be employed to prolong clinical remission.
For medical centers with a high ALL patient load, an accurate and sensitive method with rapid turnaround time is needed for determination of these important chimeric transcripts. We previously used multiplex nested RT-PCR for screening fusion transcripts [8]. However, we found that it became too time consuming and tedious as our workload increased.
In 2002, Siraj et al [9] reported a method using monochromatic SYBR Green RQ-PCR to detect 4 clinically important fusion transcripts. However, they did not include the major BCR-ABL primer in their assay. To address this problem, we developed an improved detection of the 4 important cALL oncogene fusion transcripts using a SYBR Green platform on the Rotorgene 3000 real-time PCR apparatus, with modifications of the previously published method.
The multiplex SYBR Green RQ-PCR assay is a promising tool for rapid and accurate identification of different chimeric transcripts. Analysis of specific amplicons via melting temperature (Tm) protects the analyst from the risks of handling carcinogenic substances such as ethidium bromide and avoids the tedious preparation of agarose gel. Chances of carry-over contamination are eliminated because no post-PCR manipulation is involved. This platform is able to provide an accurate fusion transcript marker for subsequent minimal residual study without requiring expensive probes [10].
We found a consistent isoform of TEL-AML1 in our positive patients where the melting temperature of the amplicon Tm was 85.3 ±0.5°C. This is different from the previous observation in non-Asian populations where several different TEL-AML1 isoforms were found [9].
We have shown that multiplex SYBR Green real-time RT-PCR can detect all important chimeric transcripts including the cryptic t(12;21), which is nearly impossible to detect using conventional karyotyping. However, this does not imply that molecular screening can wholly replace karyotype analysis in the diagnostic work-up of childhood ALL. The two methods complement each other. Karyotyping allows detection of other abnormalities involving chromosome number and morphology, while the molecular method is especially useful when there are inadequate cells for karyotype analysis.
In conclusion, we believe that the SYBR Green real-time multiplex RT-PCR assay is useful in the screening of childhood ALL because it provides rapid, sensitive, and accurate detection of clinically important oncogene chimeric transcripts.
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Acknowledgements
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Karyotyping analysis was performed by Sok-Peng Chua and Jean Chen of the National University Hospital, Singapore. This work was partially funded by the Childhood Cancer Research Fund, University of Malaya, and the Ministry of Higher Education, Malaysia.
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Abstract
Introduction
