A new aberrantly spliced BCR‐ABL1 transcript variant (e13a1) identified in routine monitoring using different quantitative reverse transcription polymerase chain reaction techniques in a patient with chronic myeloid leukemia

Abstract Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) of BCR‐ABL1 transcript level is an essential part of routine disease monitoring in patients with chronic myeloid leukemia. One patient sample (e13a2 transcript detected by nested PCR) attracted attention by revealing an aberrantly spliced BCR‐ABL1 transcript variant e13a1. The last 38 base pairs (bp) of BCR exon 13 were replaced by a 37 bp insertion of the ABL1 intron 1–2/exon 1 sequence. The rare aberrant BCR‐ABL1 fusion transcript can cause discrepancies in molecular diagnostics. This scenario highlights the importance of an individual characterization of the BCR‐ABL1 fusion sequence in case of unclear qRT‐PCR results.


INTRODUCTION
The reciprocal translocation between chromosomes 9 and 22 (t(9;22)(q34;q11)) resulting in the Philadelphia chromosome (Ph) and the BCR-ABL1 fusion gene is causal to the development of chronic myeloid leukemia (CML). The occurrence of different BCR-ABL1 mRNA fusion variants (most commonly e13-a2, e14-a2, and e1-a2) results in the expression of an abnormal BCR-ABL1 fusion tyrosine kinase in the majority of the patients. In most cases, the breakpoints occur within the major breakpoint cluster region (M-bcr) within the BCR gene. The breakpoints are less often located in two other breakpoint cluster regions, termed minor (m-bcr) and micro (µ-bcr). Rare  sections, and atypical splicing events were detected [1][2][3][4][5][6][7][8][9][10][11][12]. Here, we report on the occurrence of a novel BCR-ABL1 transcript generating most likely a functional BCR-ABL1 tyrosine kinase in a Ph-positive CML patient where standard diagnostic quantitative reverse transcription polymerase chain reaction (qRT-PCR) procedure showed no amplification of the typical BCR-ABL1 transcripts.

RESULTS AND DISCUSSION
For evaluation of the externally transmitted (e13a2) transcript type, we performed our qualitative in-house multiplex PCR assay as previously described [13].
Since the patient received imatinib therapy over 6 months, the negative multiplex PCR result for any transcript variant could be explainable by a low BCR-ABL1 quotient or a binding failure of primer(s) or probe. Therefore, we performed a nested-PCR assay [16] and we Sanger DNA sequencing of one-step PCR products revealed an atypical BCR-ABL1 transcript variant (e13a1) as shown in Figure 1.
The primer binding site for the BCR sense primer of the TM qRT-PCR method [15] and the qualitative multiplex PCR assay [13] was deleted (38 bp deletion) and replaced by a 37 bp insertion of ABL1 intron 1-2/exon 1 sequence. This sequence exchange resulted in missing PCR

F I G U R E 2 Retrospective measurement of BCR-ABL1/ABL1
quotient with in-house LightCycler (LC) and TaqMan (TM) assays at different time points. Due to the deletion of parts of BCR exon 13, the TM forward primer was not able to bind the target sequence resulting in no signal (orange). The LC assay (using another primer/probe combination) can detect the BCR-ABL1 fusion transcript (blue).
amplicons. Using the primer/probe combination of the LC qRT-PCR method [14], PCR amplicons were detectable ( Figure 2). The binding site of the BCR sense primer was located 73 base pairs further upstream and was, therefore, not affected by the deletion/insertion. investigations. For future monitoring, the qRT-PCR method for this patient has to be performed by LC instead of the TM PCR system.

F I G U R E 3
Fusion sequence of e13a1 on cDNA basis with the respective amino acid code. The deletion/insertion event resulted in an in-frame fusion of the BCR and ABL1 genes. In red bold letters, the G>A mutation in the ABL1 exon 1 sequence insertion is shown resulting in an amino acid chance from glutamine to lysine (E27K).

CONCLUSION
Our scenario highlights the importance of an individual characteri-

FUNDING
For the publication fee, we acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding program "Open Access Publikationskosten" directed by Heidelberg University.

SS received honoraria from Novartis Pharma GmbH, Bristol-Myers
Squibb (BMS), Pfizer, ARIAD, and research funding from Novartis Pharma GmbH and BMS. All other authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
All data that support the findings of this study are included in the manuscript.

ETHICS STATEMENT
The study design adhered to the tenets of the Declaration of