Single cell genotyping of exome sequencing-identified mutations to characterize the clonal composition and evolution of inv(16) AML in a CBL mutated clonal hematopoiesis☆
Introduction
The knowledge about the clonal composition or architecture of acute myeloid leukemia (AML) allows conclusions regarding its pathogenesis. This is of particular interest in patients who have had a clonal hematopoiesis without an evident phenotype prior to development of the leukemia, i.e. clonal hematopoiesis of unknown significance or of indeterminate potential [1], [2], [3], [4].
In recent years, next generation sequencing (NGS) has been increasingly used to gain more information about the clonal composition of hematologic malignancies as well as solid tumors. For this, the cell populations that share common mutations, i.e. clones, are identified from the variant allele frequencies (VAF) of the detected mutations. However, as this information is bioinformatically derived and might just yield an approximation of the definite clonal architecture at best, single cell genotyping is still required to verify the co-existence of mutations in a given cell and to derive reliable information about the clonal composition and evolution of a disease. Only few studies have yet investigated the clonal evolution of AML through the analyses of gene mutations in single cells [5], [6], [7], and, to our knowledge, no such study has yet been conducted in the context of a clonal hematopoiesis of indeterminate potential.
We recently described the first case of evolution of an inv(16) positive AML on a clonal hematopoiesis background due to a germline CBL mutation (defining the CBL syndrome) [8], [9], [10], and we identified possibly cooperating mutations by exome sequencing [11]. In the present study, we further characterized the clonal composition and evolution of the AML through the integrative analysis of the genetic data retrieved from the analysis of bulk specimens and those from single cell genotyping.
Section snippets
Assessment of genetic aberrations in the bulk sample
Whole exome sequencing, CytoScan HD array (Affymetrix)-based assessment of copy number variations (CNV) and metaphase karyotyping of the bulk specimens were previously described; germ line or somatic origin of the gene mutations was verified in skin fibroblasts [11]. Written informed consent was obtained prior to sampling. The AML cell line Kasumi-1 was analyzed for KIT exon 17 mutations as previously reported [12], and by the Human SNP Array 6.0 (Affymetrix) according to the manufacturer’s
Genetic aberrations in AML and estimation of the proportion of affected cells
We first performed an integrative analysis of the genetic data retrieved from the chromosome banding, microarrays and exome sequencing experiments in order to estimate the fraction of AML cells in the bone marrow which harbored a specific aberration (Table 1). Based on the detection of the inv(16) in all 22 metaphases analyzed, we concluded that inv(16) was present in all cells. The microarray patterns showing loss of heterozygosity of chromosome 11q (LOH 11q) and the additional gain of 11q
Discussion
We recently described the development of an inv(16) positive AML on background of a CBL mutated clonal hematopoiesis, and we characterized the genetic aberrations associated with the disease course [11]. In the present study, we provide further insights into the clonal composition and evolution of the AML and show that mutations in CAND1, PTPRT, and DOCK6 co-exist within the same clone, and that the PTPRT mutation was likely acquired after the mutations in CAND1 and DOCK6.
Briefly summarized, in
Authorship
Conception and design: C.N., N.R., N. B.-D., K.Y., J.D., R.C., S.O., M.L., H.B. Collection and assembly of data: C.N., N.R., S.B., N. B.-D., C.G., K.Y., D.P., M.F., M.L., H.B. Data analysis and interpretation: C.N., N.R., K.Y., D.P., M.F., R.C., J.D., S.O., M.L., H.B. Drafting of the manuscript: C.N., M.L., H.B. Critical review and final approval of the manuscript: C.N., N.R., S.B., N. B.-D., C.G., K.Y., D.P. M.F., J.D., R.C., S.O., M.L., H.B.
Conflict of interest
The authors declare no potential conflict of interest.
Acknowledgements
We thank Klaus Geiger of the Core Facility of the Department of Internal Medicine I, University Freiburg—Medical Center, for his support with the cell sorting.
The research was supported in part by the Research Committee of the University Freiburg, Germany [BEC1058/15 (H.B.)], the German Cancer Aid [DKH 111210 (H.B.)], the EHA and ASH Translational Research Training in Hematology (H.B.), the SUCCESS program of the Department of Medicine I, Medical Center—University of Freiburg, Freiburg, Germany
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Presented in part at the 57th Annual Meeting of the American Society of Hematology (ASH), Orlando, USA, December 4–8, 2015.