Proposed Algorithm for the Best Detection of Different bcr-abl Gene Fusion Transcripts in Molecular Diagnostics Laboratories: Experience of a Major Referral Center

Abstract

Aims:

Detection of bcr-abl transcripts is important both in diagnosis as well as in prognostication and treatment modalities of different types of leukemia, both chronic and acute. However, the techniques employed are variable and different among laboratories. Our aim was to share with other labs a strategy/algorithm that we find highly useful for implementation to best detect all bcr-abl fusion transcripts for proper patient management.

Methods:

We have used two techniques for the detection of bcr-abl transcripts, an in-house developed polymerase chain reaction and a real-time quantitative commercial polymerase chain reaction (PCR) kit and tested 849 patients referred for initial screening for bcr-abl.

Results:

Out of 849 cases, 146 (17.2%) were positive for bcr-abl. Around 92.11% of the total bcr-abl positive cases (N = 76) detected by the real-time quantitative technique were also positive by the gel-based PCR assay; however, six cases (around 7.89%) were missed by the real-time assay and detected by the other technique in chronic myelogenous leukemia-proven cases.

Conclusion:

We highly encourage other laboratories to perform testing using a simple and inexpensive gel-based PCR screening assay followed by a real-time quantitative assay for a baseline bcr-abl expression level. This combination will enable laboratories to detect all the reported fusion transcripts in accordance with the clinical presentation of the patient as well as other laboratory tests for the best use of this genetic test in patient management and care.

Introduction

Chronic myelogenous leukemia (CML) is characterized in more than 99% of cases by the presence of Philadelphia chromosome (Ph), a shortened chromosome 22, which is due to a translocation t(9;22)(q34;11). This translocation is also found in 25%-40% of adult acute lymphoblastic leukemia cases (Zhang et al., 2007). As a result of this translocation, the proto-oncogene c-ABL on chromosome 9 fuses with the BCR (breakpoint cluster region) gene on chromosome 22 to produce the BCR-ABL gene. This gene codes for the production of different proteins that differ in size depending on the breakpoint in the BCR gene.

Three breakpoint cluster regions were identified in the BCR gene: M-BCR (major), m-BCR (minor), and μ-BCR (micro) (Chasseriau et al., 2004). M-BCR falls between exons 13 and 15; it is 2.9 kb long and is found in most of the CML cases. It is associated with two types of mRNA molecules: e13a2 and e14a2 (also known as b2a2 and b3a2). These mRNA molecules code for the formation of a p210 protein.

m-BCR is located between exons 1 and 2; it is 55 kb long and is mostly found in Ph-positive acute lymphoblastic leukemia (ALL) cases. It is associated with the e1a2 mRNA molecule, which codes for a p190 fusion protein.

μ-BCR is a 1 kb sequence in intron 19; it is found in Ph-positive neutrophilic-CML cases, classical CML cases, and acute myeloid leukemia (AML) cases. μ-BCR is associated with e19a2 mRNA (a low-incidence junction), which codes for a p230 protein (Boeckx et al., 2005).

Several methods are used for the detection of the BCR-ABL translocation including Southern blot, fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR, and others. These methods differ in the degree of sensitivity, the limit of detection, and the capability of detecting the translocation. For example, Southern blot and FISH techniques are capable of detecting all the BCR-ABL translocations, whereas RT-PCR is limited to certain breakpoints due to the dependence of the technique on the location of the primers and the probes. However, RT-PCR has a lower limit of detection than FISH or Southern blot (Jinawath et al., 2009).

The complexity in detecting the translocation is mainly attributed to the arbitrary fusion between the genes on chromosomes 9 and 22 and to the presence of three breakpoints in the BCR gene (Chasseriau et al., 2004). These complications have been overcome by introducing new techniques for the characterization of the transcript. Some of the modified methods are

Multiplex PCR using primers coupled to different fluorochromes, which can be followed by the use of an optical system of a sequencer. When negative results were achieved using this technique, the transcripts were identified using agarose gel electrophoresis and sequencing (Chasseriau et al., 2004).

Ligation-Mediated PCR used in a study done in The Netherlands to detect the rare e19a2 junction (Boeckx et al., 2005).

Multiplex RT-PCR for the detection of numerous BCR-ABL rearrangements, followed by real-time quantitative (RQ)-PCR for the detection of each type using specific primers and then by a nested RT-PCR (when RQ-PCR yielded a negative result) (Goh et al., 2006).

In different instances, and based on our experience at the American University of Beirut Medical Center, which is a major tertiary care center and referral diagnostic laboratory for molecular testing, especially for bcr-abl in CML and ALL, we have encountered cases that are negative for bcr-abl by using the quantitative real-time PCR technique but which turned out to be positive by regular PCR (on agarose electrophoresis). These cases are typically CML by morphology and clinical presentation; thus, we address in this report the strategy that we implement for the best detection of all bcr-abl fusion gene transcripts by utilizing a combination of two techniques.

Materials and Methods

Samples and RNA extraction

This study was conducted at the American University of Beirut Medical Center, which is a tertiary-care Lebanese center. The extraction of the RNA material was done using the TRIZOL-Chloroform technique for around 5 × 10⁶ cells and genomic material stored at −80°C. This RNA material originated from samples belonging to patients referred for BCR-ABL fusion transcripts detection in our center to help in the diagnosis of chronic myeloid leukemia.

bcr-abl mRNA and the LightCycler—t(9;22) Quantification Kit

The LightCycler t(9;22) Quantification Kit (Roche) is designed for the detection of BCR-ABL fusion transcripts, resulting from M-BCR and m-BCR breakpoints. The analysis of expression levels of BCR-ABL mRNA is done in association with that of the housekeeping gene glucose-6-phosphate dehydrogenase (G6PDH). Using a two-step RT-PCR, mRNA or total RNA undergoes RT, and the generated cDNA is amplified. The amplicon is detected by fluorescence using a specific pair of hybridization probes. One probe is labeled at the 5′-end with LightCycler-Red 640 and modified at the 3′-end by phosphorylation to avoid extension. The other probe is labeled at the 3′-end with fluorescein. Fluorescence is emitted after hybridization to the template DNA, and it is measured by the LightCycler Instrument. BCR-ABL and G6PDH are amplified. The G6PDH reaction product serves as a control for the procedure and a reference for the quantification of BCR-ABL. This technique detects fusion transcripts resulting from the breakpoints b3a2, b2a2, b2a3, b3a3, and e1a2. The values for BCR-ABL and G6PDH for each sample are calculated by the LightCycler software by comparing the crossing points to the standard curve. A normalized target value (the ratio BCR-ABL/G6PDH) is then derived by dividing the amount of BCR-ABL by the amount of G6PDH.

Gel-based in-house multiplex technique (RT-PCR)

The starting material in this technique is mRNA. In RT-PCR, the first step consists of RT of mRNA into cDNA and then application of a regular PCR technique using specific oligonucleotides. RNAsin and reverse transcriptase are used to get cDNA. After getting the DNA material, a thermocycler program is introduced to amplify the suspected BCR-ABL fragment. The primer sequences that were used for bcr-abl amplification consisted of 5′-AGATACTCAGCGGCATTG-3′, 5′-CGGTTGTCGTGTCCGAGG-3′, and 5′-AGCTTCTCCCTGACATCCGTG-3′. The thermocycler program consists of an initial denaturation step of 94°C for 11 min, followed by 14 cycles of 94°C for 30 s, 64°C for 1 min, and 72°C for 1 min, then 29 cycles of 94°C for 30 s, 50°C for 1 min, and 72°C for 1 min, and a final extension step of 72°C for 5 min. Finally, the products are run on a 2% agarose/TBE (Tris/borate/EDTA) gel and visualized under ultraviolet light. This technique helps in detecting p190 and p210 breakpoint products. Housekeeping genes were amplified in every run for best sample quality and result interpretation. The sequences of the internal housekeeping gene used (abl gene) were 5′-AGATACTCAGCGGCATTG-3′ and 5′-TTCAGCGGCCAGTAGCATCTGACTT-3′.

Results

Table 1 shows the distribution of the 849 patient screening results based on the two utilized techniques. Out of 849 patients, 327 were initially screened using the real-time PCR technique and 522 were initially screened using the gel-based technique. Of the total screened cases by both techniques, 146 cases (17.2%) were positive for bcr-abl fusion transcript. Out of the latter positive cases, all of the 70 cases screened by gel assay were correctly identified. However, out of the 76 cases screened by real-time PCR, 6 cases (7.89%) were missed by this technique, though confirmed to be positive by gel-based assay.

Table 1.

Distribution of the (A) 849 Patient Screening Results and (B) the 146 Positive Patient Results and the Rate of Detection Based on the Two Utilized Techniques

	Positive reactions		Negative reactions
A	N	%	N	%	Total
bcr-abl	146	17.2	703	82.8	849

		bcr-abl
		Missed	Detected
B	N	N (%)	N (%)
Screening by real-time	76	6 (7.89)	70 (92.11)
Screening by gel assay	70	0 (0)	70 (100)

Discussion

Gel-based assays and internationally standardized quantitative real-time RT-PCR are commonly utilized, respectively, for the initial diagnosis of bcr-abl positive leukemias, both in the acute and chronic phases as well as in molecular monitoring of residual disease after initiation of treatment or allogeneic stem cell transplantation (Neumann et al., 2003; Müller et al., 2007).

Different laboratories employ different strategies for the detection of bcr-abl fusion transcripts including the use of variable techniques such as southern blot, FISH, PCR, RT-PCR, quantitative RT-PCR, and so on.

In our tertiary care referral center, we mainly utilize two techniques, a gel-based PCR assay (developed in-house) and a quantitative real-time RT-PCR assay (LightCycler BCR-ABL Detection Kit, ROCHE Diagnostics) to initially assess the presence or absence of bcr-abl and then perform a baseline detection of bcr-abl expression levels for future use in minimal residual monitoring and follow up on the response of the patient to treatment. A total of 849 patients were screened for a possible presence of bcr-abl; however, only 149 cases were positive and confirmed the diagnosis of CML or bcr-abl positive ALL. The remaining 703 negative results did not miss a CML or ALL but were rather part of a panel of tests that were run to exclude other translocations in addition to bcr-abl. For example, our ALL genetic panel testing includes screening for t(1;19), t(4;11), and t(12;21) as well as t(9;22).

Based on our experience, the gel-based bcr-abl assay did not miss a single CML or bcr-abl positive ALL case (as confirmed by karyotyping results and morphology on bone marrow aspirate). However, and to our surprise, in six cases, the result of bcr-abl testing turned out to be negative when the quantitative real-time RT-PCR assay was used as initial screening for the referred cases that were clinically, highly suspicious for CML and that were then checked by the gel-based assay and turned out to be positive for bcr-abl. The most likely explanation for the lack of detection of bcr-abl transcripts in some cases by the real-time PCR method is the presence of mutations at the sites where the probes used are going to complement their target DNA sequence; and, hence, amplification may take place but the probe-dependent quantification phase will be missing. These six cases reflected missing up to 7.89% of the positive cases by the real-time RT-PCR assay, which may really be a major problem in laboratories that solely rely on this technique for bcr-abl detection.

Although it is well known that different techniques can detect different bcr-abl fusion product breakpoints, we highly recommend relying on more than one molecular technique for the best detection of bcr-abl. Our strategy-to-share of starting with a gel-based assay, for initial diagnosis and confirmation of CML, followed by a baseline expression level of bcr-abl using real-time RT-PCR, provided us with a highly sensitive detection rate and accurate result reporting, so far, not missing any bcr-abl positive case, regardless of the breakpoint site.

Disclosure Statement

No competing financial interests exist.

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