Background Acute leukemia is the most common malignancy of childhood. Most of pediatric acute leukemias are derived from B-cell precursor, and about 15% are from T-cell origin and lead to T-cell acute lymphoblastic leukemia (T-ALL). Despite recent progress in treatment, the prognosis of T-ALL patients is worse, and ~30% of cases relapse (Pui CH, Seminars in hematology 2009). Despite the need for targeting molecules to treat resistant and high risk pediatric T-ALLs, in vitro drug testing may not be predictive of efficacy in vivo due to the complexity of the signalling network and to the cell heterogeneity within the individual's sample. High-resolution analysis of signalling profile at single-cell level may represent an innovative tool in the comprehension of signalling network and its role in the response to drugs (Bodenmiller B et al, Science signaling 2010). Currently mass cytometry (CyTOF) technique allows the measurement of more than 30 parameter per single cell (Bendall SC et al, Science 2011), rendering this approach an ideal method to investigate the complex biology of T-ALL (Girardi T et al, Blood. 2017) and to assay drug candidates for cellular targeting. Methods Using single-cell mass cytometry we developed panel of 39 metal-labeled monoclonal antibodies (moAbs) identifying T-ALL blasts and non-malignant T-cells. Using phospho-specific moAbs and moAbs targeting proliferation and apoptosis we detected signal transduction upon in vitro treatment of 17 diagnostic and 5 relapse T-ALL samples with IL-7 (Jak/STAT5 pathway activator) , BEZ-235 (inhibitor of PI3K and mTOR), and Pervanadate (inhibitor of tyrosine phosphatases). We have used sample barcoding by anti-CD45 antibodies to unify sample preparation and acquisition for all treatment conditions and we resolved the cells of interest by manual gating. We evaluated both, the individual change sin p-kinases and the overall changes using dimensionality reduction approach. Results T-ALL cells showed constitutive activation of various signaling pathways as well as proliferation markers compared to residual bone marrow T-cells and T-cells isolated from healthy donors. Up-regulated activity of PI3K-mTOR pathway (p4E-BP1, pAkt, pS6), proliferation rate (pRb, Ki-67), MAPK pathway (p-p38, pErk1/2), translation (pCREB) in T-ALL cells was detected, whereas lower levels of anti-apoptotic protein Bcl2 in T-ALL cells were detected. Interleukin 7 (IL-7) activates three main signalling pathways such as STAT5, PI3K/Akt/mTOR and MEK/Erk, leading to the promotion of leukemia cell viability, cell cycle progression and growth. Thus we interrogated T-ALLs in their ability to respond to IL -7 in vitro . We used an hierarchical clustering analysis (with Euclidean distance metrics and an average linkage) and we were able to divide T-ALL samples in IL-7 responder (6 out of 17) and IL-7 non responder (11 out of 17). Of note, no significant differences in IL7Ra (CD127) expression was observed between the two groups. Interestingly IL-7 responders had higher levels of pRb and Ki-67 proliferation markers as compared to both IL-7 non-responders and non-malignant T-cells. Moreover IL-7 non-response correlate with poor response in vivo to prednisone and higher level of minimal residual disease (MRD) at day15 of remission induction treatment. Finally T-ALL cells were treated ex vivo with PI3K/AkT/mTOR dual inhibitor BEZ-235. Of the 17 T-ALL patients tested, 9 responded to BEZ-235 but not to IL-7 , by contrast 4 did not respond to BEZ-235 being IL-7 responders, two patients responded both to IL-7 activation and BEZ-235 inhibition. One single patient did not respond neither to IL-7 nor to BEZ-235 Conclusions In summary we characterized pediatric T-ALL samples demonstrating the feasibility of CyTOF-based single-cell profiling of signal transduction pathways in this setting. We detected constitutively active pathways in T-ALL blasts as compared to residual non-malignant T-cells. Importantly we identified by functional read outs distinct clusters of IL-7 and BEZ-235 T-ALL responders patients, supporting the notion of a mutual exclusivity between JAK-STAT (or Ras) pathway genomic alterations and PI3K-AKT pathway alterations (Liu et al. Nat Genet. 2017). Our observation can contribute to the better understanding of the complex signalling network governing T-ALL behaviour and response to therapy. Disclosures No relevant conflicts of interest to declare.

This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.

Monitoring immune responses to solid cancers may be a better prognostic tool than conventional staging criteria, and it can also serve as an important criterion for the selection of individualized therapy. Multiparametric phenotyping by mass cytometry extended possibilities for immunoprofiling. However, careful optimization of each step of such method is necessary for obtaining reliable results. Also, with respect to procedure length and costs, sample preparation, staining, and storage should be optimized. Here, we designed a panel of 31 antibodies which allows for identification of several subpopulations of lymphoid and myeloid cells in a solid tumor and peripheral blood simultaneously. For sample preparation, disaggregation of tumor tissue with two different collagenases combined with DNase I was compared, and removal of dead or tumor cells by magnetic separation was evaluated. Two possible procedures of barcoding for single-tube staining of several samples were examined. While the palladium-based barcoding affected the stability of several antigens, the staining with two differently labeled CD45 antibodies was suitable for cells isolated from a patient's blood and tumor. The storage of samples in the intercalation solution for up to two weeks did not influence results of the analysis, which allowed the measurement of samples collected within this interval on the same day. This procedure optimized on samples from patients with head and neck squamous cell carcinoma enabled identification of various immune cells including rare subpopulations.

In mass cytometry, the isolation of pure lymphocytes is very important to obtain reproducible results and to shorten the time spent on data acquisition. To prepare highly purified cell suspensions of peripheral blood lymphocytes for further analysis on mass cytometer, we used the new CD81+ immune affinity chromatography cell isolation approach. Using 21 metal conjugated antibodies in a single tube we were able to identify all basic cell subsets and compare their relative abundance in final products obtained by density gradient (Ficoll‐Paque) and immune affinity chromatography (CD81+ T‐catch™) isolation approach. We show that T‐catch isolation approach results in purer final product than Ficoll‐Paque (P values 0.0156), with fewer platelets bound to target cells. As a result acquisition time of 105 nucleated cells was 3.5 shorter. We then applied unsupervised high dimensional analysis viSNE algorithm to compare the two isolation protocols, which allowed us to evaluate the contribution of unsupervised analysis over supervised manual gating. ViSNE algorithm effectively characterized almost all supervised cell subsets. Moreover, viSNE uncovered previously overseen cell subsets and showed inaccuracies in Maxpar™ Human peripheral blood phenotyping panel kit recommended gating strategy. These findings emphasize the use of unsupervised analysis tools in parallel with conventional gating strategy to mine the complete information from a set of samples. They also stress the importance of the impurity removal to sensitively detect rare cell populations in unsupervised analysis. © 2016 International Society for Advancement of Cytometry