Optical Genome Mapping as a Tool to Unveil New Molecular Findings in Hematological Patients with Complex Chromosomal Rearrangements

Abstract

Standard cytogenetic techniques (chromosomal banding analysis-CBA, and fluorescence in situ hybridization-FISH) show limits in characterizing complex chromosomal rearrangements and structural variants arising from two or more chromosomal breaks. In this study, we applied optical genome mapping (OGM) to fully characterize two cases of complex chromosomal rearrangements at high resolution. In case 1, an acute myeloid leukemia (AML) patient showing chromothripsis, OGM analysis was fully concordant with classic cytogenetic techniques and helped to better refine chromosomal breakpoints. The OGM results of case 2, a patient with non-Hodgkin lymphoma, were only partially in agreement with previous cytogenetic analyses and helped to better define clonal heterogeneity, overcoming the bias related to clonal selection due to cell culture of cytogenetic techniques. In both cases, OGM analysis led to the identification of molecular markers, helping to define the pathogenesis, classification, and prognosis of the analyzed patients. Despite extensive efforts to study hematologic diseases, standard cytogenetic methods display unsurmountable limits, while OGM is a tool that has the power to overcome these limitations and provide a cytogenetic analysis at higher resolution. As OGM also shows limits in defining regions of a repetitive nature, combining OGM with CBA to obtain a complete cytogenetic characterization would be desirable.

Keywords: chromothripsis; complex karyotype; cytogenetics; hematologic neoplasms; optical genome mapping.

Figure 1

(A) Karyotype of case 1 determined via GTG-banding and FISH analysis with WCP probes specific to chromosomes 5, 7 and 16. (B) Karyograms by GTG-banding analysis of the two clones detected in case 2, bearing a der(19) and a der(15) chromosome, respectively, and reconstruction of the two cellular clones detected using data derived from FISH analyses.

Figure 2

(A) Circos plot related to OGM analysis for case 1. The lines indicate the regions involved in the rearrangement. (B) Summary of rearrangements detected with OGM and reconstruction of the derivative chromosomes observed in case 1. The green boxes indicate the deleted regions. The blue arrows indicate genomic fusions. The analysis failed to highlight some fusions because they were present in repeated regions. (C) Circos plot of case 2 analyzed with stringent filters. (D) Circos plot derived from the release of the filter on the masked regions. Most of the fusions found through the analysis affected centromeric and telomeric regions. The arrow indicates the fusion between chromosome 8 and chromosome 15 and the t(8;15) translocation detected in case 2. (E) Mapping of one of the fusion molecules generated following the t(8;15) translocation in case 2. (F) Immunohistochemical preparation for Myc protein analysis of case 2: note the nuclear staining of the neoplastic cells.