Nanopore Sequencing-Based High-Resolution Karyotyping Compared to Conventional Karyotyping in Patients with Acute Myeloid Leukemia

Introduction: Classification and risk stratification of patients suffering from acute myeloid leukemia (AML) is usually based on cytogenetic and molecular genetics results according to WHO and European LeukemiaNet (ELN) criteria. Next generation sequencing (NGS) and PCR represents rapid methods for the detection of mutations and translocations within a few days. In contrast, turnaround time for standard karyotype analysis is one to two weeks, due to the indispensable metaphase preparation from hematopoietic cells. Additionally, conventional karyotyping has substantial failure rates and is highly operator-dependent. Technical advances are needed to improve confidence and turnaround time of structural variation (SV) detection.

Methods: We utilized long-read nanopore sequencing to overcome the time-consuming karyotyping to increase resolution and accelerate SV analysis for AML classification. In detail, low-coverage whole genome sequencing (LC-WGS; 7-10x) was carried out on 25 fresh or cryopreserved AML bone marrow or blood samples containing more than 20 % of blasts. LC-WGS data was used to identify classification-relevant SV in AML, such as translocations, inversions and tandem duplications. Furthermore, genome-wide copy number variation (CNV) analysis was carried out to identify larger deletions or duplications.

Results: All 25 AML samples could be analyzed successfully by nanopore sequencing whereas in five out of 25 cases, conventional cytogenetic analysis failed. In 10 of the 20 successful cytogenetic analysis, an insufficient number of < 20 metaphases could only be analyzed. In 20 cases with conventional and molecular cytogenetic findings, the chromosomal aberrations were confirmed. In 10 samples, additional large scale structural variations or CNV were detected in comparison with karyotyping and FISH analysis. Five of these additionally detected aberrations had an impact on risk stratification. Furthermore, nanopore sequencing was able to determine a cytogenetic risk profile in all 5 patients where conventional cytogenetic analysis failed. Between the 10 cytogenetic profiles with an insufficient number of metaphases, LC-WGS detected additional abnormalities in 6 patients. All classification-relevant translocations, inversions, and CNVs were identified at single base resolution. Furthermore, FLT3-ITD and KMT2A-PTD, usually addressed by molecular genetics methods, were reliably detected with the nanopore sequencing LC-WGS approach. Moreover, the procedure from DNA isolation to final results required not more than 48h and was operator independent. The method was also able to generate an accurate karyotype from 7 cryopreserved blood or bone marrow samples.

Conclusion: We show that nanopore sequencing can accelerate and improve comprehensive analysis of genome-wide chromosomal and specific molecular aberrations in AML patients. Rapid classification according to WHO classification and ELN risk stratification is able to immediately impact on targeted and risk adapted treatment of AML patients. Due to the low coverage, detection of small subclones will not be possible by LC-WGS. However, in comparison to conventional cytogenetic analysis with a high failure rate, nanopore sequencing was successful in all fresh and cryopreserved samples. Today, NGS is standard for the detection of point mutations for classification and risk stratification of AML. However, technical advances in nanopore sequencing are promising to more robustly detect point mutations and methylation aberrations in a near future.

Crysandt:Astra Zeneca: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Koschmieder:RWTH Aachen University: Patents & Royalties: BET Inhibitor; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Karthos: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Alexion: Other: Travel support; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; PharmaEssentia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; CTI Biopharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Geron: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding. Brümmendorf:Merck: Consultancy, Other: travel support; Pfizer: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Other: travel support; Novartis: Consultancy, Honoraria, Other: travel grant, Research Funding. Jost:Jazz: Honoraria; BMS Celgene: Honoraria.