Next generation flow for minimally-invasive blood characterization of MGUS and multiple myeloma at diagnosis based on circulating tumor plasma cells (CTPC)

Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the disease. Overall, 508 samples from 264 newly diagnosed PCN patients, were studied. CTPC were detected in PB of all active multiple myeloma (MM; 100%), and smoldering MM (SMM) patients (100%), and in more than half (59%) monoclonal gammopathy of undetermined significance (MGUS) cases (p < 0.0001); in contrast, CTPC were present in a small fraction of solitary plasmacytoma patients (18%). Higher numbers of CTPC in PB were associated with higher levels of BM infiltration and more adverse prognostic features, together with shorter time to progression from MGUS to MM (p < 0.0001) and a shorter survival in MM patients with active disease requiring treatment (p <= 0.03). In summary, the presence of CTPC in PB as assessed by NGF at diagnosis, emerges as a hallmark of disseminated PCN, higher numbers of PB CTPC being strongly associated with a malignant disease behavior and a poorer outcome of both MGUS and MM

Characterization of MxA-expressing VACV recombinants.

<p>Virus recombinants W-MxA and WI-MxA contain the MxA gene under the control of a synthetic early/late promoter in the genetic background of virus strain WR or WI [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181459#pone.0181459.ref016" target="_blank">16</a>] respectively. A) Standard 2-day VACV plaque assay under liquid medium. B) Mean area of virus plaques made by different parental and recombinant viruses (n = 30). C) Immunoblot on cell extracts of cells infected by VACV (WR) or the VACV recombinants W-MxA or WI-MxA, revealed with antibodies to MxA or to the VACV late protein F13. D) Coinfection of VSV with VACV recombinants expressing MxA. To achieve some MxA expression before VSV replication, VSV infection was delayed with respect to VACV infection for the times indicated in the x axis. VSV titers from cultures coinfected with control VACV (W-BFP, dark grey bars) or MxA-expressing virus (W-MxA, light grey bars) are shown. Hatched bar corresponds to the VSV titer obtained in the absence of coinfecting VACV.</p

MxA expression in MxA-293T does not inhibit VACV replication.

<p>Cells were incubated for 18h with medium containing 1 μg/ml tetracycline and subsequently infected with vaccinia virus strain WR (WR) or VSV-GFP recombinant (VSV-g) at an MOI of 3 pfu/cell. A) Virus production at 24 h. Total VACV titers (cell+medium) or VSV titers (medium) were determined by plaque infectivity assay. C-293T are the parental FITR-293T cells. As a positive control, a similar cell line expressing a flag-tagged version of PKR (FlagPKR-293T) was included. Note: this tagged version of PKR was less active in VSV inhibition than the non-tagged version (M.L., unpublished). B) Extracts obtained from the cells indicated above were subjected to immunoblot with the antibodies indicated on the right of each panel. Note the drastic decrease in GFP expression in cells infected with VSV-g as a consequence of MxA expression, and the normal expression of a VACV late protein (F13).</p

Isolation and characterization of the MxA-293T cell line.

<p>A) Scheme showing the insertion site for the MxA gene. Insertional recombination of the plasmid results in a functional Hygromycin resistance gene, and places MxA gene under the control of a CMV promoter / 2xTet operator cassette (PCMV/2xTetO₂). Insert shows a western blot on cell extracts showing MxA accumulation after induction with medium containing 1 μg/ml tetracycline for 18h (+Tet). B) Subcellular distribution of MxA. DNA (Hoechst) staining and MxA staining are shown for uninduced (-Tet) and induced (+Tet) cell cultures.</p

Intracellular localization of MxA in cells infected with recombinant virus VV-MxA.

<p>Fluorescence microscopy images obtained by widefield (A) or confocal (B) microscopy in C-293T cells infected with VV-MxA for 18 hours. The images shown correspond to MxA, F13 and DNA staining. In the Merge panel, MxA, F13 and DNA monochrome images were pseudocolored as green, red and blue and combined together. The position of viral factories (F) is indicated by arrows. The brighter DNA staining corresponds [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181459#pone.0181459.ref005" target="_blank">5</a>] to cell nuclei (N).</p

Lack of an anti-MxA trans-acting factor in Orthopoxviruses.

<p>A) Coinfection of VSV and VACV strain WR in parental control cells (C-293T), cells inducibly expressing MxA (MxA-293T) or cells inducibly expressing Flag-tagged PKR (FlagPKR-293T). Titers of VSV in the culture medium at 24 h are shown. B) Uninduced (-) or Tet induced (+) MxA-293T cells were infected with the Orthopoxviruses indicated, and incubated for 24 h. Orthopoxvirus production in cell lysates is shown. C, D) growth of VSV and Poxviruses in coinfected MxA-293T cells. For each infected culture, infection was allowed for 24 hours, and titers of VSV were obtained from the culture medium (C) and Poxvirus titers from cell lysates. Note the slight increase in replication of the Poxviruses when the competing VSV is inhibited by MxA. E) Flow cytometry analysis of VSV-g GFP fluorescence in cells coinfected with Orthopoxviruses. The Orthopoxviruses used were VACV strains WR, MVA and IHD-J, and CPXV strains BR and EP4.</p

Subcellular distribution of MxA in infected cells.

<p>Cells were infected for 18 hours with VACV recombinant expressing RFP- tagged A3 protein to label viral factories and VACV Intracellular Mature Virions (A, C) or VACV strain WR (B,D). Cells were stained with To-Pro3 to visualize DNA, anti-MxA antibody or anti-F13 antibody, as indicated. Tet induction (“+” rows) was allowed for 18 hours before infection with medium containing 1 μg/ml tetracycline. A and B are widefield microscopy images, C and D are confocal images.</p