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.

• MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• exprese genu genetika   MeSH
• genetická transkripce genetika   MeSH
• hypoxie genetika   patologie   MeSH
• kostní dřeň patologie   MeSH
• lidé MeSH
• mnohočetný myelom genetika   patologie   MeSH
• nádorové buněčné linie MeSH
• nádorové cirkulující buňky patologie   MeSH
• nádorové kmenové buňky patologie   MeSH
• nádorové mikroprostředí genetika   MeSH
• pohyb buněk genetika   MeSH
• prognóza MeSH
• proliferace buněk genetika   MeSH
• zánět genetika   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. >20years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG and IgA subclasses for a broad range of laboratory assays, except for flow cytometry. Here we analyzed the performance of commercially available Ab clones to detect IgG and IgA subclasses in memory B-cells and plasma cells (PCs) by flow cytometry. In a first step, 28 Ab clones were evaluated in peripheral blood from healthy donors. Only 17/28 clones showed reactivity against IgG and IgA subclasses expressed on the B-cell and PC surface membrane, including Ab clones for IgG1 (SAG1, HP6188, HP6001 and HP6186), IgG2 (SAG2, HP6014 and HP6002), IgG3 (SAG3, HP6095 and HP6050), IgG4 (SAG4), IgA1 (SAA1, H69-11.4 and B3506B4) and IgA2 (SAA2, 2E2, and A9604D2). In a second step, for each Ig subclass a single clone was selected according to its specificity and fluorescence intensity (resolution power), for further more detailed validation (SAG1, SAG2, SAG3, SAG4, SAA1 and SAA2). This validation process was carried out in 4 different laboratories by testing the selected Ab clones in human peripheral blood, bone marrow and tonsil samples, using different staining protocols (e.g. surface membrane and/or cytoplasmic staining). All selected Ab clones displayed strong positivity, high specificity and optimal resolution between negative and positive cells. Alternative Ab clones were also validated. Thus, our results show the feasibility of using the validated Ig subclass Ab clones in combination with other B cell-associated markers for detailed dissection of the memory B-cell and PC compartments that express distinct Ig subclasses in different human tissues.

• MeSH
• B-lymfocyty imunologie   MeSH
• dospělí MeSH
• imunofenotypizace metody   MeSH
• imunoglobulin A analýza   MeSH
• imunoglobulin G analýza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• monoklonální protilátky * MeSH
• plazmatické buňky imunologie   MeSH
• předškolní dítě MeSH
• průtoková cytometrie metody   normy   MeSH
• senioři MeSH
• specificita protilátek * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH

Fluorochrome selection is a key step in designing multi-color antibody panels. The list of available fluorochromes is continuously growing, fitting current needs in clinical flow cytometry to simultaneously use more markers to better define multiple leukocyte subpopulations in a single tube. Several criteria guide fluorochrome selection: i) the fluorescence profiles (excitation and emission), ii) relative brightness, iii) fluorescence overlap, iv) fluorochrome stability, and v) reproducible conjugation to antibodies. Here we used 75 samples (45 bone marrow and 30 blood) to illustrate EuroFlow strategies for evaluation of compatible fluorochromes, and how the results obtained guide fluorochrome selection as a critical step in the antibody-panel building process. Our results allowed identification of optimal fluorescence profiles (e.g. higher fluorescence intensity and/or resolution with limited fluorescence overlap into neighbor channels) for brilliant violet (BV)421 and BV510 in the violet laser and allophycocyanin (APC) hilite 7 (H7) or APC C750 in the red laser vs. other candidate fluorochromes generally applied for the same detectors and here evaluated. Moreover, evaluation of the same characteristics for another group of fluorochromes (e.g. BV605, BV650, PE CF594, AF700 or APC AF700) guided selection of the most appropriate fluorochrome conjugates to be combined in a multi-color antibody panel. Albeit this is a demanding approach, it could be successfully applied for selection of fluorochrome combinations for the EuroFlow antibody panels for diagnosis, classification and monitoring of hematological malignancies and primary immunodeficiencies. Consequently, sets of 8-, 10- and 12-color fluorochrome combinations are proposed as frame of reference for initial antibody panel design.

• MeSH
• fluorescenční barviva * MeSH
• imunofenotypizace metody   MeSH
• lidé MeSH
• průtoková cytometrie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH