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Chromosomal aberrations and structural variations are a major cause of human genetic diseases. Their detection in clinical routine still relies on standard cytogenetics, karyotyping and CNV-microarrays, in spite of the low resolution of the first one and the inability to detect neither balanced SVs nor to provide the genomic localization or the orientation of duplicated segments, of the latter. We here investigated the clinical utility of high resolution optical mapping by genome imaging for patients carrying known chromosomal aberrations in a context of constitutional conditions.For 85 samples, ultra-high molecular weight gDNA was isolated either from blood or cultured cells. After labeling, DNA was processed and imaged on the Saphyr instrument (Bionano Genomics). A de novo genome assembly was performed followed by SV and CNV calling and annotation. Results were compared to known aberrations from standard-of-care tests (karyotype, FISH and/or CNV-microarray).In total, we analyzed 100 chromosomal aberrations including 7 aneuploidies, 35 translocations, 6 inversions, 2 insertions, 39 copy number variations (20 deletions and 19 duplications), 6 isochromosomes, 1 ring chromosome and 4 complex rearrangements. High resolution optical mapping reached 100% concordance compared to standard assays for all aberrations with non-centromeric breakpoints.Our study demonstrates the ability of high resolution optical mapping to detect almost all types of chromosomal aberrations within the spectrum of karyotype, FISH and CNV-microarray. These results highlight its potential to replace these techniques, and provide a cost-effective and easy-to-use technique that would allow for comprehensive detection of chromosomal aberrations.

Somatic structural variants are important for cancer development and progression. In a diagnostic set-up, especially for hematological malignancies, the comprehensive analysis of all cytogenetic aberrations in a given sample still requires a combination of techniques, such as karyotyping, fluorescence in situ hybridization and CNV-microarrays. We hypothesize that the combination of these classical approaches could be replaced by high-resolution genome imaging.Bone marrow aspirates or blood samples derived from 48 patients with leukemia, who received a clinical diagnoses of different types of hematological malignancies, were processed for genome imaging with the Bionano Genomics Saphyr system. In all cases cytogenetic abnormalities had previously been identified using standard of care workflows. Based on these diagnostic results, the samples were divided into two categories: simple cases (<5 aberrations, n=37) and complex cases (≥5 aberrations or an unspecified marker chromosome, n=11). By imaging the labelled ultra-long gDNA molecules (average N50 >250kb), we generated on average ∼280-fold mapped genome coverage per sample. Chromosomal aberrations were called by Bionano Genomics Rare variant pipeline (RVP) specialized for the detections of somatic variants.Per sample, on average a total of 1,454 high confidence SVs were called, and on average 44 (range: 14-130) of those were rare i.e. not present in the population control database. Importantly, for the simple cases, all clinically reported aberrations with variant allele frequencies higher than 10% were detected by genome imaging. This held true for deletions, insertions, inversions, aneuploidies and translocations. The results for the complex cases were also largely concordant between the standard of care workflow and optical mapping, and in several cases, optical mapping revealed higher complexity than previously known. SV and CNV calls detected by optical mapping were more complete than any other previous single test and likely delivered the most accurate and complete underlying genomic architecture. Even complex chromothripsis structures were resolved. Finally, optical mapping also identified multiple novel events, including balanced translocations that lead to potential novel fusion-genes, opening the potential to discover new prognostic and diagnostic biomarkers.The full concordance with diagnostic standard assays for simple cases and the overall great concordance with (previously likely incompletely understood) complex cases demonstrates the potential to replace classical cytogenetic tests with genome imaging. In addition, this holds the potential to rapidly map new fusion genes and identify novel SVs and CNVs as novel potential leukemia drivers.

Breast cancer treatment depends on human epidermal growth factor receptor-2 (HER2) status, which is often determined using dual probe fluorescence in situ hybridisation (FISH). Hereby, also loss and gain of the centromere of chromosome 17 (CEP17) can be observed ( HER2 is located on chromosome 17). CEP17 gain can lead to difficulty in interpretation of HER2 status, since this might represent true polysomy. With this study we investigated whether isolated polysomy is present and how this effects HER2 status in six breast cancer cell lines and 97 breast cancer cases, using HER2 FISH and immunohistochemistry, DNA ploidy assessment and multiplex ligation dependent probe amplification. We observed no isolated polysomy of chromosome 17 in any cell line. However, FISH analysis did show CEP17 gain in five of six cell lines, which reflected gains of the whole chromosome in metaphase spreads and aneuploidy with gain of multiple chromosomes in all these cases. In patients’ samples, gain of CEP17 indeed correlated with aneuploidy of the tumour (91.1%; p < 0.001). Our results indicate that CEP17 gain is not due to isolated polysomy, but rather due to widespread aneuploidy with gain of multiple chromosomes. As aneuploidy is associated with poor clinical outcome, irrespective of tumour grade, this could improve future therapeutic decision making.