Pascale Flandrin-Gresta

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.

Transmission of leukemic donor cells by allogeneic stem cell transplantation in a context of familial CLL: should we screen donors for MBL?

To the editor: A family history of chronic lymphocytic leukemia (CLL) is one of the best characterized risk factors for the development of CLL. First-degree relatives of individuals with CLL have a 3- to 8-fold increased risk for CLL.[1][1],[2][2] CLL often has an indolent behavior, but some

Recommendations for accreditation of laboratories in molecular biology of hematologic malignancies.

Over recent years, the development of molecular biology techniques has improved the hematological diseases diagnostic and follow-up. Consequently, these techniques are largely used in the biological screening of these diseases; therefore the Hemato-oncology molecular diagnostics laboratories must be actively involved in the accreditation process according the ISO 15189 standard. The French group of molecular biologists (GBMHM) provides requirements for the implementation of quality assurance for the medical molecular laboratories. This guideline states the recommendations for the pre-analytical, analytical (methods validation procedures, quality controls, reagents), and post-analytical conditions. In addition, herein we state a strategy for the internal quality control management. These recommendations will be regularly updated.

Congenital Acute Leukemia with Initial Indolent Presentation—A Case Report

Congenital acute leukemia is a rare event, presenting usually as an aggressive disease with a poor prognosis. A differential diagnosis is the transient myeloproliferative disorder observed in Down syndrome. We describe the case of an apparently healthy newborn male child presenting with normal peripheral blood (PB) counts but with a blast population on differentials. The childs condition and the blast population remained unchanged during the first year of life.

Expression of embryonic stem cell markers in acute myeloid leukemia

Acute myeloid leukemia is driven by leukemic stem cells which can be identified by cross lineage expression or arrest of differentiation compared to normal hematopoietic stem cells. Self-renewal and lack of differentiation are also features of stem cells and have been associated with the expression of embryonic genes. The aim of our study was to evaluate the expression of embryonic antigens (OCT4, NANOG, SOX2, SSEA1, SSEA3) in hematopoietic stem cell subsets (CD34+CD38− and CD34+CD38+) from normal bone marrows and in samples from acute myeloid leukemia patients. We observed an upregulation of the transcription factors OCT4 and SOX2 in leukemic cells as compared to normal cells. Conversely, SSEA1 protein was downregulated in leukemic cells. The expression of OCT4, SOX2, and SSEA3 was higher in CD34+CD38− than in CD34+CD38+ subsets in leukemic cells. There was no correlation with biological characteristics of the leukemia. We evaluated the prognostic value of marker expression in 69 patients who received an intensive treatment. The rate of complete remission was not influenced by the level of expression of markers. Overall survival was significantly better for patients with high SOX2 levels, which was unexpected because of the inverse correlation with favorable genetic subtypes. These results prompt us to evaluate the potential role of these markers in leukemogenesis and to test their relevance for better leukemic stem cell identification.

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 1961: Embryonic stem cells antigens: expression in acute myeloid leukemia cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Acute Myeloid Leukemia (AML) is characterized by the expansion and resistance to apoptosis of myeloid cells blocked in early stages of differentiation. A subset of stem or progenitor cells, termed leukemic stem cells (LSC), which retain or reacquire self-renewing properties, as well as the capacity to remain in a poorly differentiated stage, give rise to the leukemic clone. The identification and purification of the LSC can provide a powerful tool for diagnosis, prognosis and therapy. Several studies suggested that LSC belong to the CD34+ CD38- compartment. Self-renewal and lack of differentiation are properties of embryonic and induced pluripotent stem cell which specifically express a set of differentiation antigens (SSEA1 and SSEA3) and transcription factors (OCT¾, SOX2, and NANOG), designated as ESCA. We postulate that an epigenetic reprogramming could induce the ESCA expression on HSC. The aim of our study was to investigate the expression ESCA in the CD34+CD38- cells from normal bone marrow (NBM) and from 50 AML BM patients. Then compare ESCAs expression between CD34+CD38- and CD34+CD38+ cells. We studied the expression in 5 cell lines (NTERA-2 which is control cell line, KG1a, U937, THP-1 and HL60 leukemic cell lines) and their potential involvement in myeloid differentiation in 2 cell lines (HL60 and THP-1). Thereafter, we inhibited the expression of ESCA in 3 leukemic cell lines (HL60, KG1a and U937) to better understand their role in abnormal proliferation or differentiation. The preliminary experiments showed an important expression of ESCA on all 5 cell lines by Multicolor Flow Cytometry (MFC), confirmed by RT-PCR. Then, we compared the expression of ESCA in CD34+CD38- cells between normal and leukemic marrow. We observed an up-regulation of two transcription factors OCT3/4 and SOX2 and the protein SSEA3 with 2-fold higher expression in AML cells compared to normal HSC. In addition, we found the down regulation of SSEA1 protein involved in cell adhesion, migration and differentiation. We also compared the expression of ESCA between the CD34+CD38- and CD34+CD38+ AML population. We observed a higher expression of OCT3/4 and SSEA3 (1.3-fold) in CD34+CD38-. We found a higher expression of SSEA1 (1.9-fold), NANOG and SOX2 (1.2-fold) in AML CD34+CD38+ population compared to CD34+CD38- population. We investigated whether there was a relationship between the overexpression of ESCA and a cytogenetic subgroup of AML. We found that AML t(15;17) expressed OCT3/4 at higher level than the other ESCA. Finally, the inhibition experiments showed that Oct3/4 was strongly inhibited in the KG1a cell line. In conclusion, these results suggest that the deregulation of ESCA may have a potential role in leukemogenesis by maintaining the LSC properties. This prompts us to test the relevance of these markers for LSC identification and as therapeutic strategies. Citation Format: Lydia Campos, TIPHANIE PICOT, CARMEN AANEI, PASCALE FLANDRIN-GRESTA, EMMANUELLE TAVERNIER, DENIS GUYOTAT. Embryonic stem cells antigens: expression in acute myeloid leukemia cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1961. doi:10.1158/1538-7445.AM2014-1961

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