Whole-genome optical mapping of bone-marrow myeloma cells reveals association of extramedullary multiple myeloma with chromosome 1 abnormalities

www.nature.com/scientificreports OPEN Whole‑genome optical mapping of bone‑marrow myeloma cells reveals association of extramedullary multiple myeloma with chromosome 1 abnormalities Eva Kriegova1*, Regina Fillerova1, Jiri Minarik2, Jakub Savara1,3, Jirina Manakova1, Anna Petrackova1, Martin Dihel1, Jana Balcarkova2, Petra Krhovska2, Tomas Pika2, Petr Gajdos3, Marek Behalek3, Michal Vasinek3 & Tomas Papajik2 Extramedullary disease (EMM) represents a rare, aggressive and mostly resistant phenotype of multiple myeloma (MM). EMM is frequently associated with high-risk cytogenetics, but their complex genomic architecture is largely unexplored. We used whole-genome optical mapping (Saphyr, Bionano Genomics) to analyse the genomic architecture of CD138+ cells isolated from bone-marrow aspirates from an unselected cohort of newly diagnosed patients with EMM (n = 4) and intramedullary MM (n = 7). Large intrachromosomal rearrangements (> 5 Mbp) within chromosome 1 were detected in all EMM samples. These rearrangements, predominantly deletions with/without inversions, encompassed hundreds of genes and led to changes in the gene copy number on large regions of chromosome 1. Compared with intramedullary MM, EMM was characterised by more deletions (size range of 500 bp–50 kbp) and fewer interchromosomal translocations, and two EMM samples had copy number loss in the 17p13 region. Widespread genomic heterogeneity and novel aberrations in the high-risk IGH/IGK/IGL, 8q24 and 13q14 regions were detected in individual patients but were not specific to EMM/MM. Our pilot study revealed an association of chromosome 1 abnormalities in bone marrow myeloma cells with extramedullary progression. Optical mapping showed the potential for refining the complex genomic architecture in MM and its phenotypes. Abbreviations EMM Extramedullary multiple myeloma MM Multiple myeloma BM Bone marrow HMW DNA High molecular weight DNA FISH Fluorescence in situ hybridization NGS Next-generation sequencing BMMC Bone marrow mononuclear cell SV Structural variant VAF Variant allele frequency CNV Copy number variation 1 Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Hnevotinska 3, 779 00 Olomouc, Czech Republic. 2Department of Hemato‑Oncology, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Olomouc, Czech Republic. 3Department of Computer Science, Faculty of Electrical Engineering and Computer Science, VŠB-Technical University of Ostrava, Ostrava, Czech Republic. *email: Scientific Reports | (2021) 11:14671 | https://doi.org/10.1038/s41598-021-93835-z 1 Vol.:(0123456789) www.nature.com/scientificreports/ FICTION Fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms CT Chromosome territory Multiple myeloma (MM) is a clonal plasma cell proliferative disorder usually limited to a bone marrow (BM) microenvironment. Rarely, patients present with extramedullary disease (EMM), in which myeloma cells spread to other organ s ystems1–3. This aggressive and mostly treatment-resistant sub-entity of MM can either accompany a newly diagnosed disease, occurring at a frequency of 3–18%4,5, or develop with disease progression or relapse, with a frequency of 6–20%4,6. Currently, little is known about the mechanisms leading to the development of EMM, stroma-independent growth and the survival of myeloma cells at extramedullary sites or the reasons for poor treatment responses. There is growing evidence that genetic factors may contribute to EMM pathogenesis and evolution1,4,5. Genetic studies have shown that high-risk abnormalities, such as 1q21 gain and del(1p32) (detected in > 55% of EMM patients), t(4;14) (~ 52%), MYC overexpression (~ 38%), del(17p13) (~ 35%) and del(13q14) (~ 31%), are commonly associated with E MM1,4,5. The disruption of the TP53 gene by del(17p) and/or mutations seems to be a crucial driver of EMM (EMM vs MM: 34.5% vs 11.9%)7,8. Mutations in the RAS9, KRAS, PIK3CA, ATM and NFKB21 genes have also been associated with the presence of EMM, including CRBN mutations leading to treatment r esistance10. Other important aberrations in EMM include the activating mutations in the NF-κB pathway genes and the homozygous deletion of the genes encoding inhibitors of this pathway11. The resulting constitutive activation of NF-κB enhances the expression of adhesion molecules, such as integrin VLA-4, CD-44, P-selectin and numerous chemokines/receptors6,12, leading to the migration and stroma-independent growth of myeloma cells11. Additional genetic aberrations may occur in patients with extramedullary mass due to clonal evolution7,13. However, the complex genetic architecture in MM and EMM is still poorly understood, likely due to its complexity and heterogeneity. Therefore, we applied novel whole-genome optical mapping to investigate the complex genomic architecture of BM myeloma cells in newly diagnosed MM and EMM patients. This method has an advantage in detecting small and large structural rearrangements as well as complex rearrangements across the whole genome that are undetectable by traditional methods, such as sequencing and c ytogenetics14. The characterisation of genetic architecture in EMM could significantly contribute to the understanding of EMM pathogenesis with the potential to discover new prognostic and diagnostic biomarkers and improve the outcome of this MM entity. Moreover, a comparison of MM and EMM may help to elucidate genetic events, allowing the dissemination of myeloma cells from BM to blood and distant tissues. Materials and methods Subject enrolment. BM aspirates were obtained from an unselected cohort of 11 newly diagnosed MM patients with EMM presentation (n = 4; median age: 77 years, min–max: 51–79; M/F: 3/1) and without EMM (MM, n = 7; 75 years, 62–82; 5/2). Patients were diagnosed according to the International Myeloma Working Group criteria15. The only criteria for patient enrolment were sampling at diagnosis and a sufficient number of sorted cells to perform all genetic analyses (≥ 2 million myeloma cells). In our patients, all EMM sites were bone related, with two in the thoracic spine and two in the pelvis (one in the iliac bone and one in the acetabulum). Patient’s clinical and demographic data are summarised in Table 1 and Table S1. For all patients, karyotype, FISH (fluorescence in situ hybridization, Table S2), arrayCGH (Table S3) and next-generation sequencing (NGS) for mutations in the TP53, KRAS, NRAS and BRAF genes (Table S2) were available. All patients provided written informed consent about the usage of BM for this study, which was performed in accordance with the Helsinki Declaration and approved by the ethics committee of the University Hospital and Palacký University Olomouc. Collection of BM aspirates. BM aspirates (2.5–10 ml) were collected in a 5 ml RPMI-1640 medium (Sigma-Aldrich, (...truncated)