Circular extrachromosomal DNA (ecDNA) is a type of genomic focal amplification frequently found in tumors and is rapidly becoming a foundational frontier in cancer biology. The discovery that genomic focal amplifications can arise by different mechanisms revealed that ecDNA in particular leads to worse patient survival and enhanced tumor evolution. Consequently, there is an urgent need for methods and tools to study ecDNA and related genomic amplifications.

Gene editing-based cell and gene therapy strategies often involve the delivery of a foreign DNA to the cell product or directly to the patient. For the efficacy and safety of such therapy strategies, we need to determine this DNA’s 1) on-target integration in the host 2) potential off-target integrations across the entire host genome 3) copy number of the integrated insert at each detected locus. Current NGS-based methods, relying on the sequencing of PCR enriched target DNA, fail to capture all types of integrations and cannot detect concatemerized integrations. Bionano Genomics Optical DNA mapping technology overcomes these barriers, by mapping long intact DNA molecules and by evaluating the entire genomic DNA directly without any PCR enrichment. We report its application in detecting a reporter cassette integration in embryonic stem cell lines and determination of the copy number of the integrated inserts.

A well-known problem of induced Pluripotent StemCell (iPSC) culture is the spontaneous acquisition of genetic abnormalities during culture. In order to check the health of our iPSC banks, we require an accurate and reliable QC solution to verify the health of our cells.

We have investigated multiple different techniques and services to assess the cytogenetic health of our cells, ranging from G banding through array-based PCR solutions to next generation technologies. We provide here a summary of our findings and why we have settled on the Bionano Saphyr as the best solution for cytogenetic QC.

In a subset of pediatric cancers, the causative germline cancer predisposition mutations remain unidentified because they are hidden in the dark matter of the genome. As consequence, genetic counseling and predictive testing in those families are seriously hampered.

Optical genome mapping: hidden structural variants in families with inherited retinal diseases Kornelia Neveling1,3, Zeinab Fadaie1,2, Suzanne de Bruijn1,2, Tuomo Mantere1, Ronny Derks1, Lonneke Haer-Wigman1, Amber den Ouden1, Michael Kwint1, Luke O'Gorman1, Dyon Valkenburg2,4, Carel B. Hoyng2,4, Christian Gilissen1,5, Lisenka E.L.M. Vissers1, Marcel Nelen1, Frans P.M. Cremers1,2, Susanne Roosing1,2, Alexander Hoischen1,3

1 Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

2 Donders Institute for Brain, Cognition and Behavior, Nijmegen, The Netherlands

3 Radboud Institute of Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands

4 Department of Ophthalmology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, The Netherlands

5 Radboud Institute of Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands

6 Department of Internal Medicine and Radboud Center for Infectious Diseases (RCI), Radboud University Medical Center, Nijmegen, The Netherlands

The lack of molecular diagnoses in rare genetic diseases can be explained by limitations of current standard genomic technologies. Upcoming long-read techniques have complementary strengths to overcome these limitations, with a particular strength in identifying structural variants (SVs). By using optical genome mapping (Bionano Genomics) and long-read HiFi genome sequencing (Pacific Biosciences), we aimed to identify the pathogenic variant in families with molecularly unsolved inherited retinal diseases. Here, we will show a family with the X-linked progressive eye disease Choroideremia, a patient with Usher syndrome (syndromic blindness and deafness) and a patient with Senior Løken syndrome (syndromic kidney disease and eye disease). All three cases had been unsolved for many years. Optical genome mapping finally helped us to identify the different pathogenic structural vari

Undifferentiated small round cell sarcomas (USRCS) are highly aggressive neoplasms that predominantly affect children and young adults. Recent molecular findings allowed reclassification of these tumors that were formerly included in a wastebasket for different entities. Tumor types within this category comprises Ewing sarcoma (ES) and other neoplasms formerly designated “Ewing-like” sarcomas (ELS). USRCS are characterized by chromosomal translocations that generate oncogenic gene fusions, which constitute the driver alteration of the disease. However, secondary alterations at diagnosis are rare, as USRCS present a very low mutation rate. Therefore, little is known about the biological factors determining the risk of relapse or progression, thus precluding accurate patient stratification and risk-adapted therapeutic approaches. Despite USRCS were thought to be silent at genomic level, several studies have revealed that they can present genomic structural variations (SVs) such as copy number alteration in specific chromosomal regions, with an impact in disease progression. Besides, translocations themselves could represent a source of SV. Herein, we profiled SVs with optical genome mapping in a series of USRCS comprising 8 cases of ES (EWSR1-FLI1), and 2 cases of ELS (BCOR-CCNB3 and EWSR1-NFATC2). We detected the pathognomonic translocation in all the cases. The reciprocal translocation FL1-EWSR1 was detected in all ES samples excepting one. Additional chromoplectic translocation events were found in several ES samples that were obtained from patients with poor clinical outcome. Interestingly, two pairs of matched ES samples -primary and lung metastatic tumors-showed a similar SV profile, suggesting a common clonal origin for both lesions. Finally, the case with EWSR1-NFATC2 translocation showed amplification of the two loci reflecting the generation of a ring chromosome, along with clustered rearrangements that could correspond to a chromotriptic event.

Malignant pleural mesothelioma (MPM) is a slow growing fatal cancer whose origins begin in the pleural lining of the thoracic cavity. MPM can be classified into three main types of histology, epithelioid, biphasic, and sarcomatoid. Although common mutations have been documented for this pathology such as CDKN2A, TP53, and MDM2, the landscape of alterations to chromosomes and what translocations are present in this cancer has not been fully documented. Here we apply Bionano optical mapping and the rare variant analysis pipeline to 45 separate mesothelioma tumors. We generated a total of 47 trillion bases of molecules maps with an average length of 279.6 kbp. Analysis of the optical mapping with Bionano access software reveled a largely altered chromosomal landscape including whole chromosome translocations, copy number changes and whole chromosome amplifications/deletions. Total events with a deletion threshold greater than 500kp varied greatly across the samples ranging from 1,400 to 2,700 observed events. Hallmark genes observed with large scale complex changes which would have been missed using short read technology include HNF1A,MDM2,ERBB4. This research reveals a highly complex chromosomal landscape for mesothelioma and produces the first global view of complex mutations for this cancer.

Brain tumors are a leading cause of cancer-related mortality in children. Research shows that structural variants (SVs) play an important role in tumorigenesis and growth. However, most genetic studies have relied on short-read based exome/genome sequencing (SRS) that have limited ability to detect SVs. Optical genome mapping (OGM) utilizes ultra-long DNA molecules to construct and evaluate much longer genomic fragments, making it effective in identifying large SVs. We utilized OGM for the detection of novel clinically relevant SVs in pediatric brain tumors. Ultra high molecular weight DNA was extracted from a cohort of pediatric brain tumors and matched normal samples (n=50). OGM was performed using nanochannel chip arrays on a Saphyr instrument and SVs (large insertions/deletions, inversions, translocations) were identified. We then used nanotatoR to subclassify SVs into functional pathways and determined which genes impact specific cell developmental functions. Tumor-associated, low population frequency SVs were identified in all samples. The most common SV events were deletions and translocations. Nearly all samples had a SV that overlapped with a gene known to have clinical significance. CDKN2A/B deletions were most common in high grade tumors. OGM also detected clinically relevant, previously unknown fusions (i.e. NTRK2-KANK1, C11orf95-NCOA1 fusion) and deletions (NF1, ATRX) that may have affected therapeutic management. We also discovered novel SVs overlapping genes and miRNAs that could impact tumorigenesis. OGM effectively identifies novel clinically relevant SVs in pediatric brain tumor samples, including SVs that were not discovered other methods of testing. This is a promising new method to map the full SV spectrum of pediatric brain tumors. Future studies will expand testing to a larger cohort of samples to better understand the full spectrum of SVs and begin clinical analysis to determine the effect of SVs on response to treatment/prognosis.

Breast cancer is the most common malignancy among women and the contribution of hereditary susceptibility to its development has been well recognized. However, the known genetic risk factors still explain less than half of the familial risk of the disease. Here, the planned research aims to elucidate genetic risk factors of breast cancer by utilizing optical mapping technology for genome-wide and high-resolution detection of structural variants. The study consists of ~100 high-risk breast cancer cases that have undergone diagnostic gene panel and/or research-based exome sequencing without any pathogenic variants identified in the well-established breast cancer susceptibility genes. The main aim of the project is to assess the frequency of hidden structural variants in the known breast cancer genes and to investigate whether a more complete picture of structural genomic variation in unexplained high-risk breast cancer cases could reveal novel susceptibility genes.

Optical genome mapping (OGM) was key for the diagnosis of a patient with mediastinal mass and pleural effusion hospitalized in an insensitive care unit at the Hospital Infantil Universitario Niño Jesús. Given the severity state of the patient, the mass could not be biopsied and the pleural effusion was extracted as part of her treatment. The analysis of cell population from pleural effusion by flow cytometry reveled a specific population CD56+CD45- compatible with a solid tumor. In order to determine the type of pediatric solid tumor, CD56+ cells were analyzed by OGM. Among the structural variants described, the interchromosomal translocation t(2;13) generating the PAX3·-FOX1 novel fusion protein was key since this translocation is a diagnostic factor for alveolar rhabdomyosarcoma. Other structural variations linked to this type of sarcoma was described such as the gain of MYCN. Once the clinical state of the patient improved, the diagnosis of an alveolar rhabdomyosarcoma was corroborated by the analysis of mediastinal mass by routine diagnosis protocols such as histopathology studies, molecular pathology analysis and FISH. Therefore, we show that OGM could be key for the diagnosis of some solid tumours in critical situations.

The Cancer Cytogenetics Laboratory at the University Health Network (Toronto) started a pilot program nearly one year ago to test the feasibility of replacing karyotyping and FISH assays with Optical Genome Mapping as a front line test. Our goal was to test several different types of hematolgic malignancy and compare the results seen with Optical Genome Mapping (OGM) with conventional cytogenetics (CC). To that end, we tested more than 25 prospective samples referred for our Eosinophilic FISH panel (PDGFRA, PDGFRB and FGFR1 FISH), an additional five samples from the Princess Margaret Tissue Bank and the KG-1 Cell Line (known FGFR1 translocation) were used as positive controls. Our results showed that OGM was concordant to FISH and in some cases more clearly identified complex rearrangements not clearly delineated by FISH. We further tested greater than 40 samples with acute leukemia that showed high concordance between OGM and CC. Additional testing of cases with failed CC showed that OGM was able to generate a result in many cases where CC had failed. Further, OGM was able to identify previously unidentified translocations and structural variations of clinical significance. The era of precision medicine, especially in hematologic malignancies, demands that structural variation analysis be performed at high resolution, cost effectively and with a fast enough turn around time that clinical management decisions can be made. OGM has demonstrated its compatibility as a front line test in public health care setting.

A recent national multi-institutional study assessed the performance, in a real-world setting, of optical genome mapping (OGM) in a cohort of 100 AML cases. The results revealed that OGM effectively recovers clinically relevant balanced structural variants (SVs) and copy number changes found by standard of care methods such as karyotyping, fluorescence in situ hybridization (FISH) and chromosomal microarray analysis (CMA). Importantly, OGM identified clinically relevant SVs in 11% of cases that had been missed by the routine methods and refined the underlying genomic structure reported by traditional cytogenomic testing in 13% of cases. The ability to detect cryptic translocations in an unbiased manner was a highlighted by the study with 3 cases reported with normal karyotypes shown to have cryptic translocations involving gene fusions. Some of these findings would have altered recommended clinical management or rendered the case eligible for clinical trials. The ability of OGM to yield results in a single assay analogous to those attainable from three separate assays (karyotyping, FISH, CMA) demonstrated that OGM can potentially eliminate the requirement for multiple labor-intensive cytogenetic tests while concomitantly maximizing diagnostic detection through a standardized workflow.

Technical and clinical validation of optical genome mapping @RadboudUMC Kornelia Neveling,1,2 Tuomo Mantere,1,3,4 Susan Vermeulen,1 Michiel Oorsprong,1 Ronald van Beek,1 Ellen Kater-Baats,1 Marc Pauper,1 Guillaume van der Zande,1 Dominique Smeets,1 Su Ming Sun,5 Brigitte Verbeek,5 Simone Wezenberg,1 Stijn Ghesquiere,5 Lisenka Vissers,1 Daniel Olde Weghuis,1 Marian JPL Stevens-Kroef,1 Alexander Hoischen1,3,6 1Department of Human Genetics, Radboud university medical center, Nijmegen, 6500 HB, The Netherlands, 2Radboud Institute of Health Sciences, Radboud University Medical Center, Nijmegen, The Netherlands, 3Radboud Institute of Medical Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands, 4Laboratory of Cancer Genetics and Tumor Biology, Cancer and Translational Medicine Research Unit and Biocenter Oulu, University of Oulu, Oulu, Finland, 5Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, The Netherlands, 6Department of Internal Medicine and Radboud Center for Infectious Diseases (RCI), Radboud University Medical Center, 6532 GA Nijmegen, The Netherlands. Somatic structural variants (SVs) are important drivers of cancer development and progression, and are relevant for the prognosis and a determinant in treatment decision making. In a diagnostic set-up, especially for hematological malignancies, the comprehensive SV analysis still requires a combination of cytogenetic techniques (karyotyping, FISH, CNV-microarrays). We hypothesize that the combination of these classical approaches could be replaced by optical genome mapping (OGM). To investigate this hypothesis, samples from 52 patients with a clinical diagnosis of a hematological malignancy and an abnormal cytogenetic result were analyzed as part of a retrospective technical validation study. Detailed technical comparison with standard-of-care tests showed high analytical validity of OGM, resulting in a sensitivity of 100%, with a positive predictive value.

Optical Genome Mapping for diagnosis of paediatric leukaemia Cytogenetics has led to the discovery of multiple chromosomal anomalies associated with hemato-oncologic diseases in children and adolescents. The Hospital Infantil Universitario Niño Jesús is a Spanish reference center for pediatric leukemia. To date, the clinical cytogenetic analyses are a combination of karyotyping (KT), fluorescence in situ hybridization (FISH) and PCR to detect genetic biomarkers of the disease. We show that optical genome mapping (OGM) arises as a new technology of one step with a higher resolution than karyotype, FISH and CNV-microarrays able to detect structural variants and copy number variation clinically relevant for pediatric leukemia. We analyzed 26 samples from patients with acute lymphoblastic leukemia and 7 samples from patients with acute myeloid leukemia by OGM and the results were compared with the results obtained with the traditional diagnosis techniques. We obtained 100% of concordance between conventional and OGM technologies for both types of leukaemia, and not only that, but also, we demonstrated that OGM refined the structural variant found and defined new abnormalities. All together, these data demonstrate the improvement of leukaemia diagnosis by OGM as an alternative for clinical cytogenetic.

Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are prevalent hematopoietic malignancies that are subdivided into distinct disease entities based on clinical, immunophenotypic and genomic features. Cytogenetic analysis (karyotyping and Fluorescent In-Situ Hybridization) together with Multiple Ligation-dependent Probe Amplification (MLPA) and PCR-based methods form the backbone of routine ALL and AML diagnostics. This approach is labor-intensive, time-consuming and costly. New molecular technologies now exist that can detect structural variants (SVs) and copy number alterations in one test.

Complex karyotype (CK) predicts poor prognosis in chronic lymphocytic leukemia (CLL). CK detection is routinely assessed by chromosome banding analysis (CBA) or chromosomal microarrays (CMA), although results obtained by both techniques are not equivalent. In this setting, our group has assessed 42 CLL patients by optical genome mapping (OGM) with the aim to analyze the utility of this a novel technology in the cytogenetic assessment of CLL patients. Besides the global concordance of OGM with current techniques, the talk will be focused in some examples of the new information from previously known abnormalities underscored by OGM.

Acute lymphoblastic leukemia (ALL) is the most prevalent type of cancer occurring in children. ALL is characterized by structural and numeric genomic aberrations that strongly correlate with prognosis and clinical outcome. Usually, a combination of cyto- and molecular genetic methods (karyotyping, array-CGH, FISH, RT-PCR, RNA-Seq) is needed to identify all aberrations relevant for risk stratification. We investigated the feasibility of optical genome mapping (OGM), a DNA-based method, to detect these aberrations in an all-in-one approach. As proof of principle, twelve pediatric ALL samples were analyzed by OGM, and results were validated by comparing OGM data to results obtained from routine diagnostics. All genomic aberrations including translocations (e.g., dic(9;12)), aneuploidies (e.g., high hyperdiploidy) and copy number variations (e.g., IKZF1, PAX5) known from other techniques were also detected by OGM. Moreover, OGM was superior to well-established techniques for resolution of the more complex structure of a translocation t(12;21) and had a higher sensitivity for detection of copy number alterations. Importantly, a new and unknown gene fusion of JAK2 and NPAT due to a translocation t(9;11) was detected. We demonstrate the feasibility of OGM to detect well-established as well as new putative prognostic markers in an all-in-one approach in ALL. We hope that these limited results will be confirmed with testing of more samples in the future.

In the following study, plasma cell dyscrasias were examined using OGM. FISH and aCGH were used as a comparison. The study was intended to clarify how reliable OGM is compared to conventional systems with regard to clinical diagnostics. A total of 24 samples were analyzed, 6 of them had to be excluded due to failed quality controls and polyploidy. With the remaining samples, a total of 62 relevant aberrations were found with FISH and aCGH. With Optical Genome Mapping, samples were first analyzed with Solve and Bionano Access 1.6, 60 of these aberrations were detected. Later with version 1.7 all aberrations could be detected.

Enhanced Structural Variation Detection with Optical Genome Mapping in Constitutional Genetic Disorders.

Identification and detection of structural variations (SVs), copy number variations (CNVs), and tandem repeat expansions/contractions has been the standard of care (SOC) for the detection and diagnosis of constitutional genetic disorders (prenatal and postnatal setting). Current SOC methods have limitations that include low resolution (karyotyping), inability to interrogate the whole genome (FISH), and inability to detect balanced SVs and orientation of duplicated segments of genome (chromosomal microarrays). Other molecular methods utilized for repeat expansion/contraction disorders are time-consuming and laborious (southern blotting) or have a low dynamic range (PCR, NGS). Optical genome mapping (OGM) has emerged as a technology that enables the detection of several classes of SVs, CNVs and repeat array analyses in a single workflow. At Augusta University, we performed a clinical evaluation and validation of OGM for constitutional genetic disorders in 196 samples that included 85 prenatal specimens (cultured amniocytes) and 106 postnatal specimens (peripheral blood/cryopreserved or cultured cell lines). Our results were 100% concordant with SOC and demonstrated a smooth laboratory workflow for different sample types. The inter- and intra-run studies were performed on a subset of samples and the reproducibility data was 100% concordant.

Giemsa banding (GTG-Banding) is a conventional cytogenetic analysis that allows for the identification of balanced and unbalanced structural and numerical chromosome abnormalities with a diagnostic yield of about 3%. Fluorescence in situ hybridization (FISH) is targeted to a particular chromosomal region or regions specifically indicated by the patient’s phenotype. Chromosome microarray array (CMA) aids in the identification of copy number variants (CNVs) with a diagnostic yield of about 10%-15%. Currently, CMA is a tier-1 test for aneuploidy and unbalanced structural chromosome abnormalities (SVs; CNVs), some types of ploidy and regions of homozygosity (ROH). However, it will not detect balanced SVs like translocations and inversions. Optical Genome Mapping (OGM) is a complementary genomic tool to sequencing, produces a genome “map” with all balanced and unbalanced SVs and CNVs circumventing the routine cytogenetics manual workflow (cell culture and karyotyping).

The Northern Ireland Regional Molecular Diagnostics Service (RMDS) has relied traditionally on cytogenetic techniques such as karyotyping, fluorescent in situ hybridisation (FISH) and SNP Array to identify and report chromosomal aberrations in constitutional and acquired cytogenetic patients. A pilot study is underway in RMDS to evaluate the clinical utility of optical genome mapping (OGM) using The Saphyr system from Bionano Genomics in prenatal, suspected aneuploidy and complex constitutional cases. Patient samples previously processed using current cytogenetic methods and with known chromosomal aberrations were analysed using OGM and the results from both routes compared for concordance and any additional findings. These cases include chromosomal abnormalities such as trisomies, translocations, copy number deletions and duplications previously identified by QFPCR, karyotyping, FISH or SNP array. Cases will be presented that have been assessed using traditional methodologies and the Bionano Saphyr system and further work is ongoing to assess the additional findings generated from this higher resolution platform.

Apparently balanced reciprocal translocations are one of the most common genetic causes of male infertility. The commonly accepted underlying pathophysiological mechanism is that they impair meiosis but few studies have been conducted and not for each reported translocation. Here, we sought to investigate the hypothesis of an altered expression of spermatogenesis genes located at the breakpoints, at least in some cases, as already described in other constitutional and somatic diseases.

We included 13 unrelated infertile men carrying apparently balanced reciprocal translocations that were identified by karyotyping. We performed optical genome mapping (OGM) according to the Bionano protocol and using the Saphyr® instrument and Bionano Solve® and Access® software for data analysis. Gene content at the breakpoints and flanking regions was investigated. Twelve out of 13 translocations could be characterized by OGM. The undetected translocation had breakpoints in the heterochromatic regions of both chromosome Y and 16 long arms. Preliminary analysis of gene content at the breakpoints revealed the disruption of at least one gene involved in spermatogenesis or sperm function and a possible positional effect for another patient.

In conclusion, this study underlines the added-value of using OGM to characterize apparently balanced structural variations in infertility. These analyses could allow to identify new genes involved in reproductive disorders and refine genotype-phenotype correlations.