Whole exome sequencing of CK- and NK-AML patients

Acute myeloid leukemia (AML) is a heterogeneous disease that based on the cytogenetic profile of the tumour cells can be divided into normal karyotype (NK) and complex karyotype (CK) AML. The latter is associated with a particularly poor prognosis. In spite of the undisputed significance of CK-AML, it remains relatively underinvestigated due to the scarcity of CK-AML patient samples. We have characterized a cohort of 33, mostly newly diagnosed, CK-AML patients from two German clinics (German Cancer Research Center in Heidelberg and Technical University in Dresden), in parallel with 2 NK-AML patients from the same consortium and 2 healthy references (Agilent OneSeq male and female DNA).TP53 mutations were present in 17 patients, mostly affected the DNA binding domain and were frequently classified as dominant negative. The TP53 mutations were typically associated with a deletion of the other allele or a second hit mutation. The presence of TP53 mutations was connected with older age, higher count of karyotype abnormalities, increased expression of CD34, and shorter overall survival, even within the CK-AML cohort with an already poor prognosis. Among the patients with TP53 mutation, a high variant allele frequency (VAF) was linked with short survival, whereas a much weaker or no association was observed between overall survival and the blast fraction. Analysis of CNV and VAF data for the TP53 locus made it possible to deduce a likely order of events for patients with a mutation and deletion. Our data are consistent with the scenario that patients typically acquire a dominant negative mutation in TP53 first, which is rapidly followed by a deletion of the second allele at this locus, and then by other karyotype aberrations, presumably due to TP53 pathway impairment after the initial hit.The samples originated from mononuclear cell fractions of whole blood or bone marrow and were obtained as frozen cellular pellets, cryopreserved cells, or isolated genomic DNA. Five CK-AML samples were subjected to FACS to separate the CD34+ and CD34- cellular fractions, which were sequenced separately. Genomic DNA was extracted using DNeasy Blood & Tissue Kit or Monarch Genomic DNA Purification Kit. Whole Exome NGS libraries were prepared using the SureSelectXT HSQ kit. Target enrichment was done using OneSeq 300kb BB+Human All Exon V7 kit with baits targeting exons, high minor allele frequency SNP regions, and ClinGen-defined disease-associated regions, and enriched libraries were PCR-amplified. NGS was performed with Illumina NextSeq or NovaSeq 6000 platforms using S1 Reagent Kit, with 2 x 100 cycles and paired-end mode. The libraries were sequenced with 50 million or 100 million reads per sample, aiming for an average depth of 100x in the covered regions.