Clonal Evolution at First Sight: A Combined Visualization of Diverse Diagnostic Methods Improves Understanding of Leukemic Progression

Abstract

Patients with myeloid neoplasia are classified by the WHO classification systems. Besides clinical and hematological criteria, cytogenetic and molecular genetic alterations highly impact treatment stratification. In routine diagnostics, a combination of methods is used to decipher different types of genetic variants. Eight patients were comprehensively analyzed using karyotyping, fluorescence in situ hybridization, array-CGH and a custom NGS panel. Clonal evolution was reconstructed manually, integrating all mutational information on single nucleotide variants (SNVs), insertions and deletions (indels), structural variants and copy number variants (CNVs). To allow a correct integration, we differentiate between three scenarios: 1) CNV occurring prior to the SNV/indel, but in the same cells. 2) SNV/indel occurring prior to the CNV, but in the same cells. 3) SNV/indel and CNV existing in parallel, independent of each other. Applying this bioinformatics approach, we reconstructed clonal evolution for all patients. This generalizable approach offers the possibility to integrate various data to analyze identification of driver and passenger mutations as well as possible targets for personalized medicine approaches. Furthermore, this model can be used to identify markers to assess the minimal residual disease.

Reconstructing Clonal Evolution—A Systematic Evaluation of Current Bioinformatics Approaches

“…Several issues underlie this observation: First, a majority of the tools just allow for defining the underlying copy number for every SNV and not the fraction of cells characterized by a certain copy number. Second, the scenario of overlap is not considered despite having a major influence on the genotype and requiring an adjusted calculation of the CCF (formula provided in [ 14 ]). Third, none of the considered tools are capable of jointly clustering CNVs and SNVs in the case of lacking overlap.…”

“…This includes the number of overlapping SNVs, the affected clone as well as the scenario of overlap. We distinguish four scenarios: CNV first, SNV first affected, SNV first un-affected, and parallel [ 14 ] (the influence of the different scenarios on the genotype is summarized in Appendix A.1 . clevRsim, Table A1 ).…”

“…clevRsim, Table A1 ). According to the formula provided in [ 14 ], the VAF is adjusted with respect to the scenario and CNV type. For example, in the case of a deletion present in the scenario ‘SNV first un-affected’, we expect to observe the cells characterized by SNV (genotype AB) and SNV+CNV (genotype B).…”

“…However, the resolution is considerably lower compared to scDNA-seq (100–200 interphase nucleoli for FISH, 10–25 metaphases for karyotyping). However, the valid reconstruction of clonal evolution is only possible if the data on both small and large variants are integrated and jointly evaluated [ 14 , 15 ].…”

clevRvis: visualization techniques for clonal evolution

“…Commonly, various sources of data are considered to allow for valid detection of these variants, for example, karyotyping, fluorescence in situ hybridization, microarrays like single-nucleotide polymorphism (SNP) arrays or array-CGH (aCGH), next-generation sequencing, and Sanger sequencing. By integrating all of these data, clonal evolution may be reconstructed [ 7 ].…”