Françoise Solly

Comparable flow cytometry data can be obtained with two types of instruments, Canto II, and Navios. A GEIL study.

Flow cytometry (FC) instruments settings classically rely on local establishment of photomultipliers (PMT) voltages adapted to the measurements expected to be performed. In the era of multiparameter FC (MFC), it appears more and more desirable that comparable patterns of fluorescence are obtained in different settings. This relies on a harmonization of settings between instruments. Although this has been shown to be feasible within a given brand of flow cytometers, little information is available about broader comparisons in a given center or in a multicenter fashion. Here, we report a two‐phase series of experiments first performed between a Canto II (BD Biosciences) and a Navios (Beckman Coulter) instruments in the same center. PMT values adjusted on the reference instrument (RI) Canto II were used to establish target values for PMT settings on the paired Navios practice instrument (PI). This allowed to show the good correlation of all but peaks 1 and 2 of Rainbow® beads between RI and PI. Using 4‐ or 8‐color stained leukocytes, the similitude of the settings was further confirmed. A complex set of matrices was then established between five centers all equipped with both instruments. Using Bland & Altman difference comparisons for median fluorescence values, it was shown that using either Rainbow beads or CD16 stained polymorphonuclears to set‐up target values on the RI CantoII, highly superimposable results could be obtained on all 9 PI. The latter were obtained using Rainbow beads or Compbeads® for comparisons. In summary, this two‐phase study demonstrates the feasibility of different methods allowing for a robust harmonization of settings for MFC.

Oncogene- and drug resistance-associated alternative exon usage in acute myeloid leukemia (AML)

In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro, 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents.

One tube with eight antibodies for 14-part bone marrow leukocyte differential using flow cytometry

Bone marrow analysis by flow cytometry is part of the routine diagnosis of hematological disorders in medical laboratories. Differential leukocyte count and identification of abnormal cell subsets is currently performed through morphological examination on bone marrow smears by skilled cytologists. In this work, we propose a single 8‐color tube for providing equivalent information, using flow cytometry.

CD180 expression in B-cell lymphomas: A multicenter GEIL study

CD180, a related member of the Toll‐like receptor family, is lost or underexpressed at the plasma membrane in circulating cells of various B‐cell lymphomas except marginal zone lymphomas (MZL). In order to confirm its clinical relevance in routine analysis, we evaluated prospectively the expression of CD180 in 236 patients from 5 French University Hospital laboratories on behalf of the GEIL. Highly comparable results were obtained in all centers using the EuroFlow standardization protocol. We observed that CD180 median fluorescence (MdFI) was significantly higher in MZL and hairy cell leukaemia (HCL) compared to all other B‐cell proliferations (P < 0.05). CD180 intensity could distinguish lymphomas with numerous villous lymphocytes from other MZL. ROC curve analysis identified a CD180 MdFI threshold for which the diagnosis of MZL could be assessed with 77% sensitivity and 92% specificity. This study showed that CD180 can be considered as a single positive robust marker of MZL and should be therefore included in flow cytometry panels for the diagnosis of mature B‐cell neoplasms. Harmonization process is of great interest in order to evaluate new markers in multicentric studies and to set up decisional thresholds.

TET2 exon 2 skipping is an independent favorable prognostic factor for cytogenetically normal acute myelogenous leukemia (AML)

In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2Shigh patients were found to be younger than TET2low patients without a difference in the rate of complete remission. However, TET2E2Shigh patients exhibited a significantly lower CIR (p<10-4). TET2E2S and FLT3-ITD, but not age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both the intermediate-1 and favorable ELN genetic categories, TET2E2S remained significantly associated with prolonged survival. There was no correlation between TET2E2S status and outcomes in 34 additional AML patients who were unfit for IC. Therefore our results suggest that assessments of TET2 exon 2 splicing status might improve risk stratification in CN-AML patients treated with IC.

Abstract 2470: Adhesion-mediated dysfunctions in myelodysplastic syndromes microenvironment

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objectives: The recent evidences demonstrate that in myelodysplastic syndromes (MDS) a particular role is played by stromal microenvironment dysfunctions, which mediate the direct contact with hematopoietic precursor cells (HPC). The aims of our study were to assess the putative growth deficiencies of mesenchymal stromal cells (MSC) selected from MDS individuals, with regard to their ability to generate a microenvironment suitable to HPC development. Then, we intended to decode the focal adhesion (FA) signalling pathways and to understand whether adhesion-mediated processes contribute to transduction of intrinsic proliferative signals, as well as their impact on HPC-to-MSC interactions. Methods: To this end, we imagined a selection procedure using MSC specific markers expression (STRO-1 and CD73), we performed their phenotypic evaluation, and we conducted the functional assays on MSC and HPC selected from MDS patients vs. healthy volunteers. Finally, we have used the immunofluorescence microscopy to characterize the FA proteins (paxillin and focal adhesion kinase [FAK]), and of their regulators, p130CAS and HSP90. Results: The MSC production in STRO-1+ and CD73+ cell cultures from refractory cytopenia (RC) marrows was deficient, and, in addition, the clonogenic ability of these fractions was strongly diminished. The relative proliferation in MSC cultures from RC is the result of a continuous division process occurring at a low rate and lacking the ability to generate the normal functional progenitors required to form colonies. By contrast, in refractory anemia with excess of blasts (RAEB) settings, the proliferation rate is moderately improved due to the reduced doubling time of STRO-1 cells. However, this was not accompanied, at the end point, by complete functional maturity as reflected in the CFU-F number. Likewise, we have to point out the diminution of CFU-F capacity of CD73+ fractions in MDS that directly correlates with the CD44 mitigate on their surface. In addition, the doubling time of MSC from MDS inversely correlate with their expression for CD49e (Δ5-integrin). The proliferation differences occur in MDS cultures compared to normal settings can be attributed equally to the qualitative defects of FA proteins (FAK, and paxillin). The MSC from RAEB cultures highlight a strong complexation of FA proteins to HSP90 in nuclear area, which support a proliferative behaviour of these cells. Moreover, this high colocalisation to HSP90 indicates the cessation of proteasome-mediated recycling of these proteins. Furthermore, the preliminary results indicate the fact that the clonogenic potential of HPC is controlled by adhesion mechanisms dependent on stroma, and FAK is one of the molecules involved in this process. Conclusions: These data prove that MSC selected from MDS patients are intrinsically pathological and they could influence HSC behaviour by their direct interactions via FA proteins signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2470. doi:1538-7445.AM2012-2470