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

Blood, vol. 140, pp. 3450–3451

Abstract

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.

Authors 7

1. Martina Margrit Crysandt Aachen 1Department of Hematology Faculty of Medicine 2Department of Hematology Medical Faculty 3Department of Internal Medicine IV

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   1Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, University Hospital RWTH Aachen, Aachen, Germany

   2Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany

   3Department of Internal Medicine IV, University Hospital RWTH Aachen, Aachen, Germany

   4Center for Integrated Oncology Aachen Bonn Cologne Düsseldorf (CIO ABCD), Aachen Bonn Cologne Düsseldorf, Germany

2. Pia Krüger Aachen 5Institute for Human Genetics and Genomic Medicine

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   5Institute for Human Genetics and Genomic Medicine, University Hospital RWTH Aachen, Aachen, Germany

3. Steffen Koschmieder Aachen 6Department of Hematology Faculty of Medicine 8Department of Medicine (Hematology Department of Medicine (Hematology

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   6Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine, University Hospital of Aachen, Aachen, Germany

   7Center for Integrated Oncology Aachen Bonn Cologne Düsseldorf (CIO ABCD), Aachen, Germany

   8Department of Medicine (Hematology, Oncology, Hemostaseology, and SCT), RWTH Aachen University, Aachen, Germany

   Department of Medicine (Hematology, Oncology, Hemostaseology, and SCT), RWTH Aachen University, Aachen, Germany

4. Ingo Kurth Aachen 9Institute of Human Genetics

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   9Institute of Human Genetics, University Hospital, Technical University (RWTH) Aachen, Aachen, Germany

5. Tim H. Brümmendorf Aachen 10Department of Hematology and Oncology Center for Integrated Oncology

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   10Department of Hematology and Oncology, Center for Integrated Oncology (CIO), Uniklinik RWTH Aachen, Aachen, Germany

   7Center for Integrated Oncology Aachen Bonn Cologne Düsseldorf (CIO ABCD), Aachen, Germany

6. Florian Kraft Aachen 11Institute for Human Genetics and Genomic Medicine

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   11Institute for Human Genetics and Genomic Medicine, University Hospital RWTH Aachen, Aachen, Germany

7. Edgar Jost Aachen 1Department of Hematology Faculty of Medicine

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   12Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), Aachen, Germany

   1Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, University Hospital RWTH Aachen, Aachen, Germany