Background. Approximately 98% of CML patients express e13a2 and/or e14a2 BCR::ABL1 mRNA isoforms resulting from genomic breakpoints that fall within a ~2.9 Kb region in BCR and a ~140 Kb region in ABL1. Rarer isoforms result from breakpoints in other parts of BCR and/or ABL1.Additional BCR::ABL1-associated abnormalities have also been described that may be prognostically relevant. We set out to assess the ability of long read nanopore sequencing to detect genomic BCR::ABL1 fusions and any BCR::ABL1-associated rearrangements or other structural variants in patients with CML.

Methods. DNA from patients with CML (n=30) were fragmented to 5-10 Kb and prepared for sequencing using the Oxford Nanopore Technologies (ONT) ligation sequencing kit v14. Adaptive sampling was used to target a panel of 240 genes recurrently involved in hematological malignancies in all 30 cases (37.1 Mb total), with 11kb of padding sequence at either end of each gene to maximise coverage. The full lengths of chr9 and chr22 (223.9 Mb total) was also included for 25/30 cases. Sequencing was performed on the MinION Mk1b, Dorado (ONT) and minimap2 were used for basecalling and alignment, SV were called with sniffles2. Selected rearrangements were confirmed by targeted PCR and sequencing. BCR::ABL1 transcript type was known for all cases and full karyotype for 18 cases.

Results. Median on-target sequencing depth was 38X, representing a 7-fold enrichment over non-targeted sequence. Median read N50 was 7.8 Kb. Evidence for BCR::ABL1 was called by sniffles2 in 27/30 cases. In the remaining 3 cases, the fusion call was initially filtered in 2 samples due to a low fraction of fusion reads (0.16 and 0.17), and only 2 fusion supporting reads in 1 sample. Evidence for the fusion was found upon manual review. BCR::ABL1 transcript type was concordant with the genomic breakpoints in all cases, including 3 with atypical fusions (e14a2, n=14; e13a2, n=10; e14a2/e13a2, n=3; e1a2, n=2; e14a3, n=1).

Cytogenetic results were available for 18/30 cases and a Ph chromosome was seen in 12/18 of these, including 11 where it was seen as the sole abnormality. Two of these 11 cases (cases 15 and 30) had unexpected additional rearrangements detected by nanopore sequencing: case 15 had a t(1;9)(q25.3;q34.12) with a 6.1 Kb inversion at chr9:130,709,000 within 1Kb of the t(9;22) reciprocal breakpoint. Case 30 had an additional t(3;22)(p21.31;q11.23). One patient (case 26) had a Ph chromosome plus complex additional chromosome abnormalities that were partially resolved by nanopore sequencing. A normal karyotype was seen in 1/18 cases, with ABL1 inserted into BCR as determined by FISH. A 2.17 Mb deletion between chr9:130,842,023-133,012,328 was detected by nanopore sequencing, with breakpoints for BCR::ABL1 and the reciprocal fusion found at either end.

Complex rearrangements involving 9q34 and/or 22q11 were detected in 5/18 samples by cytogenetics. Genomic breakpoints were detected by nanopore sequencing in all 5 cases, 2 of which were found to have additional complexity. Case 1 had a 46,XY,t(9;22;9)(q34;q11;q13) by karyotyping, but nanopore suggested 2 distinct t(9;22) events, with the additional t(9;22) revealed to have breakpoints at 9q21 and 9q34, separated by a 58.5 Mb duplication. Case 3 had a 46,XY,t(3;9)(p14;q34) by cytogenetics but nanopore sequencing indicated a t(3;9)(p21.31;q34.12), t(3;9)(p21.2;q34.13) and an additional t(3;22)(22q11.23;p21.2). Of the 12 cases that did not have cytogenetic results, 2 had evidence for additional rearrangements. Case 11 had a t(7;9)(q21.2;q34.12) and t(7;22)(q21.2;q11.23) whereas case 17 had a 154 bp inversion close to the BCR::ABL1 junction and a reciprocal breakpoint approximately 1 Mb downstream of BCR in intron 19 of CABIN1.

Conclusions. This study demonstrates nanopore sequencing is able to successfully detect typical and atypical BCR::ABL1 genomic breakpoints and can revealthe underlying complexity in samples with BCR::ABL1-associated rearrangements, as well as other structural variants. Further work is required to determine the clinical significance of this additional genomic complexity in CML.

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2025
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