Lenka Tomášiková

Failure of molecular diagnostics in chronic myeloid leukemia: an aberrant form of e13a2 BCR–ABL transcript causing false-negative results by standard polymerase chain reaction

In 95% of patients suffering from chronic myeloid leukemia (CML), and in 25% of patients suffering from acute lymphoblastic leukemia (ALL), the leukemic cells are characterized by the presence of the Philadelphia chromosome (Phþ), which results from the reciprocal translocation t(9;22)(q34;q11). The resultant BCR–ABL fusion gene encodes oncogenic proteins that vary in size, depending on the breakpoint within the BCR gene. A majority of patients with Phþ CML (*90%) have the most common type of breakpoint, denoted as M-bcr (major), resulting in e13a2 and/or e14a2 fusion transcripts. A minor portion of cases with Phþ CML (*10%) are associated with two other types of breakpoint, termed m-bcr (minor) and m-bcr (micro), involving e1a2 and e19a2 junctions, respectively [1,2]. For the abovementioned fusions (M-bcr, m-bcr, m-bcr), powerful molecular diagnostic tools based on reverse-transcriptase polymerase chain reaction (RT-PCR) have been purposefully used in routine practice. Considering the fact that the majority of patients with Phþ CML and ALL harbor the typical BCR–ABL fusion transcript, many modern molecular diagnostic techniques for the qualitative detection of BCR–ABL fusions are based on a multiplex RT-PCR method reported by Cross et al. in the early 1990s [3]. These authors described a rapid and simple PCR method capable of identifying the common BCR–ABL breakpoints, a technique that was also expected to distinguish all the theoretical in-frame mRNAs, including e19a2, e13a3, and e1a3 transcripts. Unfortunately, to date, atypical types of BCR–ABL fusion genes have also been reported. Involvement of the BCR gene breakpoints outside the cluster regions [4–7], insertion of small sequences [8,9], or genomic breakpoints within individual exons [10,11] represent a risk of misinterpretation during routine investigation at diagnosis. To reduce the possibility of misinterpretation, an improved RT-PCR assay for the simultaneous detection of common (M-bcr, m-bcr, m-bcr) and also rare BCR–ABL fusion transcripts has been recently reported [12]. It is well known that the common strategy for follow-up of patients with Phþ CML on a molecular level is based on characterization of the BCR–ABL fusion transcript at the time of diagnosis (by RT-PCR) and its subsequent quantitative assessment (by real-time quantitative RT-PCR). Therefore, it is evident that exact fusion determination plays a crucial role in the long-term monitoring of minimal residual disease (MRD) in these patients. Here we report on a ‘illustrative’ case where the molecular diagnostics covering mainly common BCR–ABL fusions failed. Moreover, within this case report, we highlight the importance of precise qualitative BCR–ABL fusion detection before quantitative monitoring.