Abstract
The genetic hallmark of chronic myeloid leukemia (CML) is a reciprocal translocation t(9;22) that results in a BCR-ABL1 fusion gene. The most common BCR-ABL1 breakpoint, the major breakpoint (p210), has exons 13 or 14 of BCR fused with exon 2 of ABL1 (named b2a2 or b3a2 respectively) and accounts for the majority of CML cases. Other well characterized BCR-ABL1 breakpoints, such as the minor (p190) or micro (p230) breakpoints, which result from exon 1 or exon 19 of BCR fusing with exon 2 of ABL1, are rare in CML. Alternate or novel breakpoints, which do not occur at the intron/exon boundary or include insertions or deletions, are usually described as case reports [Liu (2003) American Journal of Hematology 74:268-272; Huet (2015) Genes, Chromosomes and Cancer 54:595-605; Crampe (2017) Hematol Oncol Stem Cell Ther 10:85-88] and make up a minority of CML cases. Here, we describe a rare example of a novel breakpoint.
A bone marrow biopsy performed 11/30/2015 in a patient presenting with leukocytosis was consistent with a diagnosis of chronic phase CML. Cytogenetic analysis revealed the Philadelphia chromosome in 20 of 20 metaphases and the FISH analysis detected the BCR-ABL1 translocation in 95.5% of cells. Molecular testing performed with a commercial assay was negative for BCR-ABL fusion. BCR-ABL1 breakpoint analysis performed at the BloodCenter of Wisconsin showed a novel breakpoint, thus explaining the negative result using the standard assay. Sequencing of the patient's fusion breakpoint uncovered a variation of the classical major (e13a2) transcript. The breakpoint was determined to be within BCR exon 13 with deletion of the last 42 nucleotides and insertion of 63 nucleotides from intron 1 of the ABL1 gene, fused to ABL1 exon 2, which remains in-frame. The BloodCenter of Wisconsin's unique quantitative BCR-ABL1 test did detect this novel breakpoint and was used for monitoring patient response to tyrosine kinase inhibitor (TKI), as shown in Figure.
The patient had a gradual response to dasatinib, and on a sample collected a year after the start of treatment, a 2-log fold reduction was achieved. In February 2017, dasatinib was discontinued because of recurrent pleural effusion. After four months off all TKI, the patient began bosutinib and had an additional 1-log fold reduction on bosutinib.
Quantitative BCR-ABL1 testing is performed using reverse transcription of total RNA, followed by real-time polymerase chain reaction to measure BCR-ABL1 fusion transcripts, using gene-specific primers and hydrolysis probes. Our unique design incorporates two BCR primers into a multiplex reaction and allows for detection of b3a2, b2a2, e1a2, and some novel breakpoints. Endogenous ABL1 is used as a reference to report a percent relative ratio of BCR-ABL1 to ABL1 as well as to assess RNA quality. The results are converted to the International Scale (IS) using an external calibrator.
BCR-ABL1 breakpoint is performed using reverse transcription of total RNA, followed by amplification using gene-specific primers and is determined by analyzing the size of the BCR-ABL1 amplicon using capillary electrophoresis. The novel breakpoint product was sequenced using BigDye terminator chemistry.
Note: First and second authors contributed equally to this work.
No relevant conflicts of interest to declare.
