Next-Generation Optical Mapping Reveals Numerous Previously Unrecognizable Structural Variants in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in western countries. Although a number of genetic aberrations (complex karyotype, del(17p), del(11q), unmutated IGVH status, TP53 mutations) having predictive and prognostic significance in CLL was reported, there is still a lack of information about large structural variants, repetitive regions, copy number variations, and inversions in the CLL genome due to limitations of current methods (NGS, cytogenetics).

Aim: Using novel next-generation optical mapping technology, we analyzed leukemic cells from peripheral blood of CLL patients at different stages of the disease.

Methods and Patients: High-molecular weight DNA was isolated from peripheral blood (>90% CD5+/CD19+/CD20+/CD23+ B-cells) from two CLL patients (P1: 70-y-old male, diagnosis of CLL in 2018, Binet Stage B, unmutated IGVH, untreated; P2: 59-y-old male, diagnosis of CLL in 2005, Binet Stage A, unmutated IGVH, on ibrutinib (2nd line) since 4/2018). For optical mapping, DNA was labeled by DLS chemistry and visualized on Saphyr system (BioNano Genomics). The optical genome maps (480 Gbp, coverage 124X) were aligned to a human reference genome (GRCh38) and the detected structural variants (SVs) were compared against the database of healthy controls; only rare de novo variants were analyzed. In both patients, cytogenetic analysis (karyotype, FISH, and arrayCGH) and mutational analysis by NGS (TP53, ATM, BIRC3, NOTCH1, SF3B1, POT1 genes) were available.

Results: For both CLL patients, the optical mapping method identified 97% of chromosomal abnormalities previously reported by standard cytogenetic methods such as deletions on 6q, 13q, 11q, 17p and trisomy 12. In both patients, same large deletions of 17.5 kbp in 9q21 and 7.1 kbp in 14q21 chromosome region were detected, undetectable by standard methods. Both patients also carried intra-chromosomal translocation t(13;13) located in 13q14 region. Additionally, P1 genome carried 2 kbp deletion in oncogene EGFR and 500 bp insertion affecting tumor suppressor TCF4, both genes previously associated with CLL. Moreover, numerous additional copy number variations and large somatic SVs, including on average tens of deletions, insertions, inversions, duplications, and translocations were detected in both patients.

Conclusion: Our study revealed numerous novel genomic rearrangements in CLL genome, unrecognizable by standard methods. These data highlights a high potential of optical maps for refinement of genomic variability in CLL, mainly for detection of large structural variations and copy number variants. The study on larger patient cohort and longer follow-up of patients may identify genetic aberrations associated with the clinical course and therapy response.

Support: MZ ČR VES16-32339A, MZ ČR - RVO (FNOl, 00098892), IGA UP_2019_014, IGA UP_2019_01