Next-Generation Karyotyping Outperforms Conventional Cytogenetics for Prognostication of AML

Compared with conventional cytogenetic analysis (CCA), next generation sequencing (NGS)–based karyotyping offered greater insight into the prognosis of patients with intensively treated acute myeloid leukemia (AML), particularly through its ability to better detect copy number alterations (CNAs). These findings suggest that NGS-based approaches, given their increased precision and sensitivity, could replace CCA for karyotyping “but warrants special caution for lowly expressed fusions,” said lead investigator Sylvain Mareschal, PhD, from the Karolinska Institute in Sweden.

The study, published in Blood Advances, included 281 patients who were diagnosed with AML in Sweden between 1997 and 2014. Clinicians collected bone marrow or peripheral blood at time of diagnosis. Patients who did not have acute promyelocytic leukemias started treatment with standard chemotherapy, and 57 patients underwent allogeneic hematopoietic cell transplantation.

CCA was performed as the routine diagnostic, and the investigators compared these findings to those from a combination of two NGS-based techniques. They used fluorescence in situ hybridization (FISH) to validate any conflicting results and disparities between CCA and shallow whole-genome sequencing (sWGS).

The investigators sequenced total RNA after ribosomal RNA depletion, producing a median of 69.3 million paired-end 126-base pair reads for each patient. Total genomic DNA was also sequenced, providing a median of 11.5 million single-end 51-bp reads per patient. A total of 225 patients underwent ligation-dependent reverse transcription polymerase chain reaction.

According to the investigators, the use of sWGS identified 901 genomic segments with DNA CNAs in 43% of the cohort (n=12). Comparisons between sWGS and CCA in detecting CNAs were made at the aberration level. Both techniques described 672 aberrations. Approximately 52% of the 118 aberrations detected by sWGS were shorter than those normally detected by CCA.

Overall, sWGS performed better than CCA at detecting European LeukemiaNet (ELN)–defining CNAs. CCA overestimated monosomies and was not an optimal method for assessing karyotype complexity, the investigators found. Concordance between CCA and sWGS for all ELN CNA-related criteria averaged 96%, but each technique reported aberrations that weren’t confirmed by the other.

The investigators found that 1 million reads per patient was sufficient for the accurate assessment of ELN-defining CNAs. Total genomic loss, characterized by a total loss of 200 megabases or greater by sWGS, was a better marker of genetic complexity and worse prognosis than a CCA-based definition of complex karyotype.

The concordance between CCA and whole transcriptome sequencing (WTS) for fusion detection was 99%. Additionally, WTS offers a broader coverage of fusions in AML that might be missed by cytogenetic analysis. For instance, the WTS technique featured a better sensitivity for the identification of inv(16) and KMT2A rearrangements.

One issue that arises with the use of NGS-based and other new techniques with improved resolution and coverage is increased identification of genetic aberrations with unknown significance. Specifically, the techniques lack the ability to provide clues on how to approach these aberrations, and whether they are clinically relevant. “However, this is a situation that we already face when CCA detects aberrations with unknown significance or when DNA mutation panels detect mutations with unknown prognostic value,” the authors wrote.

The ability of NGS techniques to provide resolution and sensitivity superior to CCA emphasizes the need for future research to investigate and develop new NGS-based prognostic markers, the researchers added, and this may also improve individualized AML treatment.

Study authors report no relevant conflicts of interest.


Mareschal S, Palau A, Lindberg J, et al. Challenging conventional karyotyping by next-generation karyotyping in 281 intensively treated patients with AML. Blood Adv. 2021;5:1003-1016. doi:10.1182/bloodadvances.2020002517