Contributions
Abstract: EP402
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Despite recent advances in the understanding of the molecular biology of acute myeloid leukemia (AML), the outcome of patients in the high risk subgroup, especially of AML with myelodysplasia-related changes (AML-MRC), remains poor. Newly developed therapeutic options such as CPX-351 offer the possibility to more refined treatment approaches. However, this requires fast detection of cytogenetic abnormalities that define AML-MRC. Conventional karyotyping is still the golden standard in routine AML diagnostics, but this analysis is labor and time consuming. To select the best available therapeutic option, it is crucial to determine copy number and structural variations in the shortest time possible.
Aims
To establish and validate karyotyping based on low coverage whole genome sequencing (WGS) of AML patients using long read Oxford Nanopore technology (ONTseq) that offers the possibility for diagnostic testing within a short timeframe of 48-72 hours.
Methods
Cytogenetically characterized diagnostic samples from peripheral blood or bone marrow of AML patients with MRC, such as complex karyotype or monosomal karyotype, were sequenced on a GridION sequencer within a ~20 hours sequencing run. Sequencing was performed using SQK-LSK109 kits according to the manufacturer’s instructions. Analysis of sequencing reads and generation of profiles was performed using an ‘in house’ bioinformatics pipeline based on publicly available tools, such as nglmr aligner, samtools, IGVTools, R package ACE, and a custom script for transforming graphical genome profiles to karyotype formula. For the current analysis copy number variations by ONTseq were only considered if ≥30% of a chromosome arm was aberrant.
Results
Sequencing of the first 47 cases achieved a 3.1-fold genome coverage (range: 1.9-5.0) and established copy number profiles of all samples at a high resolution of ≤0.1 Mbp. To check reproducibility, n=10 AML cases were sequenced and profiled at two medical centers. All 83 aberrations of these cases with gain or loss >10 Mbs were identified by both laboratories, and the fraction gained/lost was highly concordant (R²=0.998). Considering all 47 cases, the median number of unbalanced aberrations found by cytogenetics were 4 (range 1-11) and by ONT-profiling were 3 (0-14) per patient. Additional 59 and 49 aberrations were identified by ONTseq that affected 10-30% and 5-10% of the chromosome arm, respectively, that were not considered here. Comparing the copy number status of all chromosome arms for all patients we found a 91% concordance, while 5% of chromosome arms were aberrant by ONT and 4% by cytogenetics only. Those discrepancies were expected and could be explained on the one hand by a higher resolution of ONT profiling, and on the other hand by the fact that ‘marker chromosomes’ often contain the genomic material that is classified as lost in conventional cytogenetics. Of note, ONT profiles captured also very complex rearrangements, such as chromothripsis and focal amplifications, at high resolution.
Conclusion
Our retrospective analysis of a well characterized cohort of cytogenetically aberrant AML patients demonstrates the feasibility of ONTseq-based low-coverage WGS with excellent reproducibility and resolution. Thus, ONTseq provides an opportunity to rapidly and reliably delineate AML-MRC within 48-72 hours and thereby advance genotype-based treatment strategies.
AD and CL, LB and MH contributed equally to this work.
Keyword(s): Acute myeloid leukemia, Complex aberrant karyotype
Abstract: EP402
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Despite recent advances in the understanding of the molecular biology of acute myeloid leukemia (AML), the outcome of patients in the high risk subgroup, especially of AML with myelodysplasia-related changes (AML-MRC), remains poor. Newly developed therapeutic options such as CPX-351 offer the possibility to more refined treatment approaches. However, this requires fast detection of cytogenetic abnormalities that define AML-MRC. Conventional karyotyping is still the golden standard in routine AML diagnostics, but this analysis is labor and time consuming. To select the best available therapeutic option, it is crucial to determine copy number and structural variations in the shortest time possible.
Aims
To establish and validate karyotyping based on low coverage whole genome sequencing (WGS) of AML patients using long read Oxford Nanopore technology (ONTseq) that offers the possibility for diagnostic testing within a short timeframe of 48-72 hours.
Methods
Cytogenetically characterized diagnostic samples from peripheral blood or bone marrow of AML patients with MRC, such as complex karyotype or monosomal karyotype, were sequenced on a GridION sequencer within a ~20 hours sequencing run. Sequencing was performed using SQK-LSK109 kits according to the manufacturer’s instructions. Analysis of sequencing reads and generation of profiles was performed using an ‘in house’ bioinformatics pipeline based on publicly available tools, such as nglmr aligner, samtools, IGVTools, R package ACE, and a custom script for transforming graphical genome profiles to karyotype formula. For the current analysis copy number variations by ONTseq were only considered if ≥30% of a chromosome arm was aberrant.
Results
Sequencing of the first 47 cases achieved a 3.1-fold genome coverage (range: 1.9-5.0) and established copy number profiles of all samples at a high resolution of ≤0.1 Mbp. To check reproducibility, n=10 AML cases were sequenced and profiled at two medical centers. All 83 aberrations of these cases with gain or loss >10 Mbs were identified by both laboratories, and the fraction gained/lost was highly concordant (R²=0.998). Considering all 47 cases, the median number of unbalanced aberrations found by cytogenetics were 4 (range 1-11) and by ONT-profiling were 3 (0-14) per patient. Additional 59 and 49 aberrations were identified by ONTseq that affected 10-30% and 5-10% of the chromosome arm, respectively, that were not considered here. Comparing the copy number status of all chromosome arms for all patients we found a 91% concordance, while 5% of chromosome arms were aberrant by ONT and 4% by cytogenetics only. Those discrepancies were expected and could be explained on the one hand by a higher resolution of ONT profiling, and on the other hand by the fact that ‘marker chromosomes’ often contain the genomic material that is classified as lost in conventional cytogenetics. Of note, ONT profiles captured also very complex rearrangements, such as chromothripsis and focal amplifications, at high resolution.
Conclusion
Our retrospective analysis of a well characterized cohort of cytogenetically aberrant AML patients demonstrates the feasibility of ONTseq-based low-coverage WGS with excellent reproducibility and resolution. Thus, ONTseq provides an opportunity to rapidly and reliably delineate AML-MRC within 48-72 hours and thereby advance genotype-based treatment strategies.
AD and CL, LB and MH contributed equally to this work.
Keyword(s): Acute myeloid leukemia, Complex aberrant karyotype