The reason why a few myeloma cells egress from the bone marrow (BM) into peripheral blood (PB) remains unknown. Here, we investigated molecular hallmarks of circulating tumor cells (CTCs) to identify the events leading to myeloma trafficking into the bloodstream. After using next-generation flow to isolate matched CTCs and BM tumor cells from 32 patients, we found high correlation in gene expression at single-cell and bulk levels (r ≥ 0.94, P = 10-16), with only 55 genes differentially expressed between CTCs and BM tumor cells. CTCs overexpressed genes involved in inflammation, hypoxia, or epithelial-mesenchymal transition, whereas genes related with proliferation were downregulated in CTCs. The cancer stem cell marker CD44 was overexpressed in CTCs, and its knockdown significantly reduced migration of MM cells towards SDF1-α and their adhesion to fibronectin. Approximately half (29/55) of genes differentially expressed in CTCs were prognostic in patients with newly-diagnosed myeloma (n = 553; CoMMpass). In a multivariate analysis including the R-ISS, overexpression of CENPF and LGALS1 was significantly associated with inferior survival. Altogether, these results help understanding the presence of CTCs in PB and suggest that hypoxic BM niches together with a pro-inflammatory microenvironment induce an arrest in proliferation, forcing tumor cells to circulate in PB and seek other BM niches to continue growing.

Biocruces Health Research Institute Barakaldo Spain

Campus Bio Medico University of Rome Rome Italy

Cancer Research Center CIBER ONC number CB16 12 00400 Salamanca Spain

Clinica Universidad de Navarra Centro de Investigacion Medica Aplicada CIBER ONC numbers CB16 12 00369 and CB16 12 00489 Pamplona Spain

Complejo Hospitalario de Navarra Pamplona Spain

Department of Clinical and Experimental Medicine Magna Graecia University Catanzaro Italy

Department of Hematooncology University Hospital of Ostrava Ostrava Czech Republic

Faculty of Medicine University of Ostrava Ostrava Czech Republic

Faculty of Science University of Ostrava Ostrava Czech Republic

Hospital Clínico Universitario Lozano Blesa Zaragoza Spain

Hospital Son Espases Palma Spain

Hospital Universitario 12 de Octubre Madrid Spain

Hospital Universitario Virgen de las Nieves Granada Spain

Masaryk University Brno Czech Republic

National Center for Cancer Care and Research Hamad Medical Corporation Doha Qatar

Translational Genomics Research Institute Phoenix AZ USA

University Hospital Brno Brno Czech Republic

Zobrazit více v PubMed

Palumbo A, Anderson K. Multiple myeloma. N Engl J Med. 2011;364:1046–60. PubMed DOI PMC

Rajkumar SV, Dimopoulos MA, Palumbo A, Blade J, Merlini G, Mateos MV, et al. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol. 2015;15:e538–48. DOI

Paiva B, Pérez-Andrés M, Vídriales MB, Almeida J, De Las Heras N, Mateos MV, et al. Competition between clonal plasma cells and normal cells for potentially overlapping bone marrow niches is associated with a progressively altered cellular distribution in MGUS vs myeloma. Leukemia. 2011;25:697–706. PubMed DOI PMC

Sanoja-Flores L, Flores-Montero J, Garcés JJ, Paiva B, Puig N, García-Mateo A, et al. Next generation flow for minimally-invasive blood characterization of MGUS and multiple myeloma at diagnosis based on circulating tumor plasma cells (CTPC). Blood Cancer J. 2018;8:117. PubMed DOI PMC

Kumar S, Rajkumar SV, Kyle RA, Lacy MQ, Dispenzieri A, Fonseca R, et al. Prognostic value of circulating plasma cells in monoclonal gammopathy of undetermined significance. J Clin Oncol. 2005;23:5668–74. PubMed DOI PMC

Bianchi G, Kyle RA, Larson DR, Witzig TE, Kumar S, Dispenzieri A, et al. High levels of peripheral blood circulating plasma cells as a specific risk factor for progression of smoldering multiple myeloma. Leukemia. 2013;27:680–5. PubMed DOI PMC

Gonsalves WI, Rajkumar SV, Dispenzieri A, Dingli D, Timm MM, Morice WG, et al. Quantification of circulating clonal plasma cells via multiparametric flow cytometry identifies patients with smoldering multiple myeloma at high risk of progression. Leukemia. 2017;31:130–5. PubMed DOI PMC

Gonsalves WI, Rajkumar SV, Gupta V, Morice WG, Timm MM, Singh PP, et al. Quantification of clonal circulating plasma cells in newly diagnosed multiple myeloma: implications for redefining high-risk myeloma. Leukemia. 2014;28:2060–5. PubMed DOI PMC

Gonsalves WI, Morice WG, Rajkumar V, Gupta V, Timm MM, Dispenzieri A, et al. Quantification of clonal circulating plasma cells in relapsed multiple myeloma. Br J Haematol. 2014;167:500–5. PubMed DOI PMC

Chakraborty R, Muchtar E, Kumar SK, Jevremovic D, Buadi FK, Dingli D, et al. Serial measurements of circulating plasma cells before and after induction therapy have an independent prognostic impact in patients with multiple myeloma undergoing upfront autologous transplantation. Haematologica. 2017;102:1439–45. PubMed DOI PMC

Cowan AJ, Stevenson PA, Libby EN, Becker PS, Coffey DG, Green DJ, et al. Circulating plasma cells at the time of collection of autologous PBSC for transplant in multiple myeloma patients is a negative prognostic factor even in the age of post-transplant maintenance therapy. Biol Blood Marrow Transplant. 2018;24:1386–91. PubMed DOI PMC

Foulk B, Schaffer M, Gross S, Rao C, Smirnov D, Connelly MC, et al. Enumeration and characterization of circulating multiple myeloma cells in patients with plasma cell disorders. Br J Haematol. 2018;180:71–81. PubMed DOI PMC

Granell M, Calvo X, Garcia-Guiñón A, Escoda L, Abella E, Martínez CM, et al. Prognostic impact of circulating plasma cells in patients with multiple myeloma: implications for plasma cell leukemia definition. Haematologica. 2017;102:1099–104. PubMed DOI PMC

Huhn S, Weinhold N, Nickel J, Pritsch M, Hielscher T, Hummel M, et al. Circulating tumor cells as a biomarker for response to therapy in multiple myeloma patients treated within the GMMG-MM5 trial. Bone Marrow Transplant. 2017;52:1194–8. PubMed DOI PMC

Dingli D, Nowakowski GS, Dispenzieri A, Lacy MQ, Hayman SR, Rajkumar SV, et al. Flow cytometric detection of circulating myeloma cells before transplantation in patients with multiple myeloma: a simple risk stratification system. Blood. 2006;107:3384–8. PubMed DOI PMC

Paiva B, Paino T, Sayagues JM, Garayoa M, San-Segundo L, Martín M, et al. Detailed characterization of multiple myeloma circulating tumor cells shows unique phenotypic, cytogenetic, functional, and circadian distribution profile. Blood. 2013;122:3591–8. PubMed DOI PMC

Masri S, Sassone-Corsi P. The emerging link between cancer, metabolism, and circadian rhythms. Nat Med. 2018;24:1795–803. PubMed DOI PMC

Méndez-Ferrer S, Lucas D, Battista M, Frenette PS. Haematopoietic stem cell release is regulated by circadian oscillations. Nature. 2008;452:442–7. PubMed DOI PMC

Ghobrial IM. Myeloma as a model for the process of metastasis: implications for therapy. Blood. 2012;120:20–30. PubMed DOI PMC

Mishima Y, Paiva B, Shi J, Park J, Manier S, Takagi S, et al. The mutational landscape of circulating tumor cells in multiple myeloma. Cell Rep. 2017;19:218–24. PubMed DOI PMC

Lohr JG, Kim S, Gould J, Knoechel B, Drier Y, Cotton MJ, et al. Genetic interrogation of circulating multiple myeloma cells at single-cell resolution. Sci Transl Med. 2016;8:1–10. DOI

Ledergor G, Weiner A, Zada M, Wang S-Y, Cohen YC, Gatt ME, et al. Single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma. Nat Med. 2018;24:1867–76. PubMed DOI PMC

Flores-Montero J, Flores LS, Paiva B, Puig N, García-Sánchez O, Böttcher S, et al. Next generation flow (NGF) for highly sensitive and standardized detection of minimal residual disease in multiple myeloma. Leukemia. 2017;31:2094–103. PubMed DOI PMC

Puig N, Paiva B, Lasa M, Burgos L, Perez JJ, Merino J, et al. Flow cytometry for fast screening and automated risk assessment in systemic light-chain amyloidosis. Leukemia. 2019;33:1256–67. PubMed DOI PMC

Fu GK, Wilhelmy J, Stern D, Fan HC, Fodor SPA. Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting. Anal Chem. 2014;86:2867–70. PubMed DOI PMC

Cole M, Risso D (2019). scone: Single Cell Overview of Normalized Expression data. R package version 1.8.0. https://doi.org/10.18129/B9.bioc.scone .

Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018;36:411–20. PubMed DOI PMC

Schlitzer A, Sivakamasundari V, Chen J, Sumatoh HR, Schreuder J, Lum J, et al. Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow. Nat Immunol. 2015;16:718–28. PubMed DOI PMC

Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43:e47. PubMed DOI PMC

Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005;102:15545–50. DOI

Raimondi L, Amodio N, Teresa M, Martino D, Altomare E, Leotta M, et al. Targeting of multiple myeloma-related angiogenesis by miR- 199a-5p mimics: in vitro and in vivo anti-tumor activity. Oncotarget. 2014;5:3039–54. PubMed DOI PMC

Alboukadel Kassambara (2019). ggpubr: ‘ggplot2’ Based Publication Ready Plots. R package version 0.2.1. https://CRAN.R-project.org/package=ggpubr .

Jaitin DA, Kenigsberg E, Keren-Shaul H, Elefant N, Paul F, Zaretsky I, et al. Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types. Science. 2014;343:776–9. PubMed DOI PMC

Lavin Y, Kobayashi S, Leader A, Amir ED, Elefant N, Bigenwald C, et al. Innate immune landscape in early lung adenocarcinoma by paired single-cell analyses. Cell. 2017;169:750–7. e17 PubMed DOI PMC

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. PubMed DOI PMC

Therneau T (2015). A Package for Survival Analysis in S. R package version 2.38. https://CRAN.R-project.org/package=survival .

Alboukadel Kassambara and Marcin Kosinski (2019). survminer: Drawing Survival Curves using ‘ggplot2’. R package version 0.4.4. https://CRAN.R-project.org/package=survminer .

McKenzie CW, Craige B, Kroeger TV, Finn R, Wyatt TA, Sisson JH, et al. CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice. Mol Biol Cell. 2015;26:3140–9. PubMed DOI PMC

Martin TA, Harrison G, Mansel RE, Jiang WG. The role of the CD44/ezrin complex in cancer metastasis. Crit Rev Oncol Hematol. 2003;46:165–86. PubMed DOI PMC

Ivetic A, Ridley AJ. Ezrin/radixin/moesin proteins and Rho GTPase signalling in leucocytes. Immunology. 2004;112:165–76. PubMed DOI PMC

Zhu Y, Zhu MX, Zhang XD, Xu XE, Wu ZY, Liao LD, et al. SMYD3 stimulates EZR and LOXL2 transcription to enhance proliferation, migration, and invasion in esophageal squamous cell carcinoma. Hum Pathol. 2016;52:153–63. PubMed DOI PMC

Hsieh Y-H, Chou R-H, Hsieh S-C, Cheng C-W, Lin C-L, Yang S-F, et al. Targeting EMP3 suppresses proliferation and invasion of hepatocellular carcinoma cells through inactivation of PI3K/Akt pathway. Oncotarget. 2015;6:34859–74. PubMed PMC

Dumitru CA, Bankfalvi A, Gu X, Zeidler R, Brandau S, Lang S. AHNAK and inflammatory markers predict poor survival in laryngeal carcinoma. PLoS ONE. 2013;8:e56420.

Davis TA, Loos B, Engelbrecht A-M. AHNAK: the giant jack of all trades. Cell Signal. 2014;26:2683–93. PubMed DOI PMC

Paiva B, Corchete LA, Vidriales M-B, Puig N, Maiso P, Rodriguez I, et al. Phenotypic and genomic analysis of multiple myeloma minimal residual disease tumor cells: a new model to understand chemoresistance. Blood. 2016;127:1896–906. PubMed DOI PMC

Waldschmidt JM, Simon A, Wider D, Müller SJ, Follo M, Ihorst G, et al. CXCL12 and CXCR7 are relevant targets to reverse cell adhesion-mediated drug resistance in multiple myeloma. Br J Haematol. 2017;179:36–49. PubMed DOI PMC

Anreddy N, Hazlehurst LA. Targeting intrinsic and extrinsic vulnerabilities for the treatment of multiple myeloma. J Cell Biochem. 2017;118:15–25. PubMed DOI PMC

Storti P, Marchica V, Giuliani N. Role of galectins in multiple myeloma. Int J Mol Sci. 2017;18:2740. DOI

Glavey SV, Naba A, Manier S, Clauser K, Tahri S, Park J, et al. Proteomic characterization of human multiple myeloma bone marrow extracellular matrix. Leukemia. 2017;31:2426–34. PubMed DOI PMC

Ma Y, Jin Z, Huang J, Zhou S, Ye H, Jiang S, et al. IQGAP1 plays an important role in the cell proliferation of multiple myeloma via the MAP kinase (ERK) pathway. Oncol Rep. 2013;30:3032–8. PubMed DOI PMC

Gocke CB, McMillan R, Wang Q, Begum A, Penchev VR, Ali SA, et al. IQGAP1 scaffold-MAP kinase interactions enhance multiple myeloma clonogenic growth and self-renewal. Mol Cancer Ther. 2016;15:2733–9. PubMed DOI PMC

Aust G, Zhu D, Van Meir EG, Xu L. Adhesion GPCRs in tumorigenesis. Handb Exp Pharmacol. 2016;234:369–96. PubMed DOI PMC

Demchenko YN, Glebov OK, Zingone A, Keats JJ, Bergsagel PL, Kuehl WMClassical. and/or alternative NF-kB pathway activation in multiple myeloma. Cancer. 2010;115:3541–52.

Troppan K, Hofer S, Wenzl K, Lassnig M, Pursche B, Steinbauer E, et al. Frequent down regulation of the tumor suppressor gene A20 in multiple myeloma. PLoS ONE. 2015;10:e0123922. PubMed DOI PMC

Broyl A, Hose D, Lokhorst H, De Knegt Y, Peeters J, Jauch A, et al. Gene expression profiling for molecular classification of multiple myeloma in newly diagnosed patients. Gene Expr. 2010;116:2543–53.

Li D, Wu C, Cai Y, Liu B. Association of NFKB1 and NFKBIA gene polymorphisms with susceptibility of gastric cancer. Tumor Biol. 2017;39:1–6.

Zhang M, Huang J, Tan X, Bai J, Wang H, Ge Y, et al. Common polymorphisms in the NFKBIA gene and cancer susceptibility: a meta-analysis. Med Sci Monit. 2015;21:3186–96. PubMed DOI PMC

Wang Q, Wang X, Liang Q, Wang S, Xiwen L, Pan F, et al. Distinct prognostic value of mRNA expression of guanylate-binding protein genes in skin cutaneous melanoma. Oncol Lett. 2018;15:7914–22. PubMed PMC

Godoy P, Cadenas C, Hellwig B, Marchan R, Stewart J, Reif R, et al. Interferon-inducible guanylate binding protein (GBP2) is associated with better prognosis in breast cancer and indicates an efficient T cell response. Breast Cancer. 2014;21:491–9. PubMed DOI PMC

Thijssen VL, Heusschen R, Caers J, Griffioen AW. Galectin expression in cancer diagnosis and prognosis: a systematic review. Biochim Biophys Acta. 2015;1855:235–47. PubMed PMC

Cheng CL, Hou HA, Lee MC, Liu CY, Jhuang JY, Lai YJ, et al. Higher bone marrow LGALS3 expression is an independent unfavorable prognostic factor for overall survival in patients with acute myeloid leukemia. Blood. 2013;121(Apr):3172–80. PubMed DOI PMC

Albright RA, Chang WC, Robert D, Ornstein DL, Cao W, Liu L, et al. NPP4 is a procoagulant enzyme on the surface of vascular endothelium. Blood. 2012;120:4432–40. PubMed DOI PMC

Xu X, Han K, Zhu J, Mao H, Lin X, Zhang Z, et al. An inhibitor of cholesterol absorption displays anti-myeloma activity by targeting the JAK2-STAT3 signaling pathway. Oncotarget. 2016;7:75539–50. PubMed PMC

Murai T. Cholesterol lowering: role in cancer prevention and treatment. Biol Chem. 2015;396:1–11. PubMed DOI PMC

Silvente-Poirot S, Poirot M. Cancer. Cholesterol and cancer, in the balance. Science. 2014;343:1445–6. PubMed DOI PMC

Cohen Y, Gutwein O, Garach-Jehoshua O, Bar-Haim A, Kornberg A. The proliferation arrest of primary tumor cells out-of-niche is associated with widespread downregulation of mitotic and transcriptional genes. Hematology. 2014;19:286–92. PubMed DOI PMC

Deng Y, Jiang L, Wang Y, Xi Q, Zhong J, Liu J, et al. High expression of CDC6 is associated with accelerated cell proliferation and poor prognosis of epithelial ovarian cancer. Pathol Res Pract. 2016;212:239–46. PubMed DOI PMC

Avigdor A, Goichberg P, Shivtiel S, Dar A, Peled A, Samira S, et al. CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+ stem/progenitor cells to bone marrow. Blood. 2004;103:2981–9. PubMed DOI PMC

Dimitroff CJ, Lee JY, Rafii S, Fuhlbrigge RC, Sackstein R. CD44 is a major E-selectin ligand on human hematopoietic progenitor cells. J Cell Biol. 2001;153:1277–86. PubMed DOI PMC

Kodama H, Murata S, Ishida M, Yamamoto H, Yamaguchi T, Kaida S, et al. Prognostic impact of CD44-positive cancer stem-like cells at the invasive front of gastric cancer. Br J Cancer. 2017;116:186–94. PubMed DOI PMC

Chanmee T, Ontong P, Kimata K, Itano N. Key roles of hyaluronan and its CD44 receptor in the stemness and survival of cancer stem cells. Front Oncol. 2015;5:180. PubMed DOI PMC

Palumbo A, Avet-Loiseau H, Oliva S, Lokhorst HM, Goldschmidt H, Rosinol L, et al. Revised International Staging System for Multiple Myeloma: A Report From International Myeloma Working Group. J Clin Oncol. 2015;33:2863–9. PubMed DOI PMC

Storti P, Marchica V, Airoldi I, Donofrio G, Fiorini E, Ferri V, et al. Galectin-1 suppression delineates a new strategy to inhibit myeloma-induced angiogenesis and tumoral growth in vivo. Leukemia. 2016;30:2351–63. DOI

Takagi S, Tsukamoto S, Park J, Johnson KE, Kawano Y, Moschetta M, et al. Platelets enhance multiple myeloma progression via il-1b upregulation. Clin Cancer Res. 2018;24:2430–9. PubMed DOI PMC

Leblanc R, Peyruchaud O. Metastasis: new functional implications of platelets and megakaryocytes. Blood. 2016;128:24–31. PubMed DOI PMC

Dongre A, Weinberg RA. New insights into the mechanisms of epithelial–mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol. 2019;20:69–84. PubMed DOI PMC

Batlle E, Clevers H. Cancer stem cells revisited. Nat Med. 2017;23:1124–34. DOI

Zhang H, Brown RL, Wei Y, Zhao P, Liu S, Liu X, et al. CD44 splice isoform switching determines breast cancer stem cell state. Genes Dev. 2019;33:1–14. DOI

Misra S, Heldin P, Hascall VC, Karamanos NK, Skandalis SS, Markwald RR, et al. Hyaluronan-CD44 interactions as potential targets for cancer therapy. FEBS J. 2011;278:1429–43. PubMed DOI PMC

Mueller N, Wicklein D, Eisenwort G, Jawhar M, Berger D, Stefanzl G, et al. CD44 is a RAS/STAT5-regulated invasion receptor that triggers disease expansion in advanced mastocytosis. Blood. 2018;132:1936–50. PubMed DOI PMC

Bjorklund CC, Baladandayuthapani V, Lin HY, Jones RJ, Kuiatse I, Wang H, et al. Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications. Leukemia. 2014;28:373–83. PubMed DOI PMC

Yan Y, Zuo X, Wei D. Concise review: emerging role of CD44 in cancer stem cells: a promising biomarker and therapeutic target. Stem Cells Transl Med. 2015;4:1033–43. PubMed DOI PMC

Natoni A, Smith TAG, Keane N, McEllistrim C, Connolly C, Jha A, et al. E-selectin ligands recognised by HECA452 induce drug resistance in myeloma, which is overcome by the E-selectin antagonist, GMI-1271. Leukemia. 2017;31:2642–51. PubMed DOI PMC

Lytle NK, Barber AG, Reya T. Stem cell fate in cancer growth, progression and therapy resistance. Nat Rev Cancer. 2018;18:669–80. PubMed DOI PMC

Roccaro AM, Mishima Y, Sacco A, Moschetta M, Tai Y-T, Shi J, et al. CXCR4 regulates extra-medullary myeloma through epithelial-mesenchymal-transition-like transcriptional activation. Cell Rep. 2015;12:622–35. PubMed DOI PMC

Peacock CD, Wang Q, Gesell GS, Corcoran-Schwartz IM, Jones E, Kim J, et al. Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc Natl Acad Sci USA 2007;104:4048–53. PubMed DOI PMC

Matsui W, Huff CA, Wang Q, Malehorn MT, Barber J, Tanhehco Y, et al. Characterization of clonogenic multiple myeloma cells. Blood. 2004;103:2332–6. PubMed DOI PMC

Thiago LS, Perez-Andres M, Balanzategui A, Sarasquete ME, Paiva B, Jara-Acevedo M, et al. Circulating clonotypic B cells in multiple myeloma and monoclonal gammopathy of undetermined significance. Haematologica. 2014;99:155–62. PubMed DOI PMC

Li H, Zhong A, Li S, Meng X, Wang X, Xu F, et al. The integrated pathway of TGFβ/Snail with TNFα/NFκB may facilitate the tumor-stroma interaction in the EMT process and colorectal cancer prognosis. Sci Rep. 2017;7:4915. PubMed DOI PMC

Manzi M, Bacigalupo ML, Carabias P, Elola MT, Wolfenstein-Todel C, Rabinovich GA, et al. Galectin-1 controls the proliferation and migration of liver sinusoidal endothelial cells and their interaction with hepatocarcinoma cells. J Cell Physiol. 2016;231:1522–33. PubMed DOI PMC

Beyond the marrow: insights from comprehensive next-generation sequencing of extramedullary multiple myeloma tumors

More Than 2% of Circulating Tumor Plasma Cells Defines Plasma Cell Leukemia-Like Multiple Myeloma

Involvement of Small Non-Coding RNA and Cell Antigens in Pathogenesis of Extramedullary Multiple Myeloma

LncRNAs LY86-AS1 and VIM-AS1 Distinguish Plasma Cell Leukemia Patients from Multiple Myeloma Patients