Anne Quillet-Mary

An improved double fluorescence flow cytometry method for the quantification of killer cell/target cell conjugate formation.

We have developed an improved method to analyse stable associations (conjugate formation) between effector and target cells. Hydroethidine (red) stained lymphoblastoid target cells were cocentrifuged with carboxyfluorescein diacetate acetoxymethylester (green) stained human IL-2 activated cytotoxic cells (LAK). In the present studies either enriched or purified CD3 negative large granular lymphocytes (LGL) were used as cytotoxic cells. These fluorescent vital dyes localize intracellularly and therefore do not modify the cell to cell contact which eventually leads to the lytic events. Both dyes can be excited at a common wavelength (488 nm) using a single argon laser. Effectors firmly bound to target(s) (stable conjugates) were detected as two color fluorescent events (red and green). This method has several features: (a) the number of conjugates is recorded with reference to a fixed number of target cells; (b) the composition of conjugates (number of effectors or targets per conjugate) can be studied by analysis of the fluorescence intensities (red or green); (c) conjugate formation can be studied at E:T ratios comparable to those used in the classical 51Cr release cytotoxic assay; (d) it gives reproducible results and permits the study of very weak differences in binding properties. This method was used to study conjugate formation between human IL-2-activated cytotoxic cells (or purified CD3 negative LGL) and various lymphoblastoid target cells. We were able to demonstrate that cell lines susceptible to lysis formed more conjugates and were surrounded by more LAK effectors than their resistant counterparts and that no conjugate contained more than one target.

Immune recovery after fludarabine–cyclophosphamide–rituximab treatment in B-chronic lymphocytic leukemia: implication for maintenance immunotherapy

Thirty B-cell chronic lymphocytic leukemia patients were treated with fludarabine–cyclophosphamide–rituximab (FCR) and immune cell counts (natural killer (NK) cells, CD4, CD8, Tγδ and monocytes) were monitored from the end of treatment (EOT) up to 36 months (M36). Moreover, nonleukemic peripheral blood lymphocyte cytotoxicity (PBL/CTC) as well as rituximab (RTX)-dependent PBL/CTC was also measured at the initiation of therapy, EOT and M12. These parameters were correlated with post-FCR monitoring of the minimal residual disease (MRD) level in blood using a four-color flow cytometry technique. FCR induced a profound and sustained depletion of all T-cell populations, Tγδ being the most affected, whereas NK cells were relatively preserved. Both basal and interleukin-2-stimulated nonleukemic PBL/CTC against MEC-2, a CLL cell line, increased during the post-FCR period. There was no correlation between immune recovery parameters and MRD progression profile, except that patients with high post-FCR CD4+ counts experienced rapid MRD progression. MRD at M12 predicts clinical relapse. The limited data show RTX-mediated LBL/CTC activity against autologous B-cell cells in individuals with <1% residual disease at M12, opening avenues for immunomodulation post-FCR with anti-CD20 antibodies. To conclude, our study suggests that MRD increase at M12 precedes disease evolution post-FCR, and should be assessed as a surrogate marker for proactive management of CLL relapse.

B-chronic lymphocytic leukemia chemoresistance involves innate and acquired leukemic side population cells.

B-cell chronic lymphocytic leukemia (B-CLL) therapy remains unsatisfactory due to repeated resurgences of the chemoresistant disease. In this study, we investigated the basis of this chemoresistance by applying the ‘side population’ (SP) analysis to blood samples from B-CLL patients. We report the existence of few natural SP cells, which harbors phenotypic and cytogenetic hallmarks of B-CLL in most patients with this disease (n=22). SP cells appeared resistant to conventional B-CLL treatments, such as Fludarabine, Bendamustin or Rituximab. Indeed, treatment with Fludarabine (16/18 cases) or Bendamustin (5/7 cases) resulted in complete elimination of non-SP, whereas cells displaying the SP phenotype were the only surviving. Although some B-CLL SP cells were innately chemoresistant, chemotherapy by Fludarabine selected not only innate SP cells but also induced some acquired SP cells, which arose from non-SP by drug-driven evolution. This SP selection by chemotherapeutic treatments is further supported by the overall increase of the SP percentage in patients who experienced chemotherapy in the preceding year. Functionally, proliferative stimulation of SP cells was able to partially replenish in vitro the non-SP cell compartment of the B-CLL disease. The chemoresistance of B-CLL relies, in our model, on the cellular heterogeneity of B-CLL SP cells and on their regenerating dynamics.

TNFα stimulates NKG2D-mediated lytic activity of acute myeloid leukemic cells

The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show here that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFα, they display cytolytic activity toward various cellular targets including CFU-GM. This mechanism is dependent on stimulation of the granzyme B/perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP 10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patients with acute myeloid leukemia (AML) (FAB M0–M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by a pool >15% of leukemic cells. Furthermore, CD34+ hematopoietic progenitors undergoing granulomonocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which, upon stimulation by TNFα, leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFα accumulation, such as AML or myelodisplasic syndromes.

Protein kinase C‐ζ overexpression induces erythroid phenotype in the monocytic leukaemia cell line U937

Summary. Previous studies have established that protein kinase C‐ζ (PKC‐ζ) is critical for neuronal cell differentiation. However, the role of PKC‐ζ in haematopoietic cell differentiation is less clear. In this study, we have investigated the influence of PKC‐ζ overexpression on the phenotype of the human monocytic U937 leukaemic cells. In two PKC‐ζ‐overexpressing clones (U937 ζJ and U937 ζB), PKC‐ζ expression levels and activity were three to fourfold higher, and the enzyme accumulated both in the cytoplasm and in the nucleus compared with U937 control cells. PKC‐ζ‐overexpressing U937 cells exhibited an erythroid phenotype characterized by high levels of glycophorin A, cell haemoglobinization, increased GATA‐1 transcripts and protein expression, compared with controls. Immunoprecipitation studies revealed that GATA‐1 protein was constitutively phosphorylated in PKC‐ζ‐overexpressing cells. Moreover, GATA‐1 did not interact with PKC‐ζ but interacted with ERK1, which was constitutively activated and accumulated in the nucleus of U937 ζJ. However, ERK1 phosphorylation inhibition by PD098059 did not influence either GATA‐1 phosphorylation or GATA−1/ERK1 interaction. Collectively, these results suggest a model in which PKC‐ζ induces MEK‐dependent ERK1 activation, ERK1 translocation to the nucleus, GATA−1/ERK1 interaction and ERK1‐independent GATA‐1 phosphorylation resulting in GATA‐1 accumulation. To conclude, this study provides evidence for the role of PKC‐ζ in erythroid gene regulation.

Mithramycin A activates Fas death pathway in leukemic cell lines

Mithramycin A (MMA, trade name Plicamycin) can facilitate TNFα- (Tumor Necrosis Factor) and Fas ligand-induced apoptosis. Besides, several drugs play their anticancer effect through Fas apoptotic pathway. So we investigated the effect of MMA on Fas signaling. In this study we show that MMA induces apoptosis in Fas sensitive Jurkat cells and Fas resistant KG1a cells. This effect involves Fas apoptotic pathway: cell exposure to MMA leads to Fas clustering at the cell surface, DISC (Death Inducing Signaling Complex) formation and caspase cleavage. This phenomenon is independent of Fas ligand/Fas interaction and blockade of Fas death pathway partially inhibits MMA-induced apoptosis. Moreover the activation of Fas apoptotic pathway by MMA is correlated to the modulation of c-FlipL expression. Finally, pre-treatment with sub-lethal doses of MMA sensitizes KG1a cells to chemotherapeutic agents. Thus all these results may have important implications to improve clinical treatments.

Cancer stem cells of differentiated B-cell malignancies: models and consequences.

The concept of cancer stem cells has revolutionized our current vision of cancer development and was validated in solid tumors and cancers of the primitive hematopoietic compartment. Proof of the principle is still lacking, however, in malignancies of differentiated B-cells. We review here the current literature, which nevertheless suggests hierarchical organizations of the tumor clone for mostly incurable B-cell cancers such as multiple myeloma, lymphomas and B-chronic lymphocytic leukemia. We propose two models accounting for cancer stem cells in these contexts: a “top-to-bottom” clonal hierarchy from memory B-cells and a “bottom-to-top” model of clonal reprogramming. Selection pressure on the growing tumor can drive such reprogramming and increase its genetic diversity.

Abstract 2852: Obinutuzumab (GA101) efficacy in chronic lymphocytic leukemia in vitro is not diminished in high risk patients.

Introduction : we and others have already demonstrated the superiority of obinutuzumab (GA101) over rituximab (RTX) in in vitro depletion assays against chronic lymphocytic leukemia (CLL) cells. It has been recently suggested that complex karyotype (3 or more chromosomal abnormalities) and recurrent somatic mutations of the TP53, NOTCH1 and SF3B1 genes, influenced treatment-free and overall survivals in CLL patients. We address the question whether these factors may also influence the efficacy of anti-CD20 directed immunotherapy. Aim: To assess in vitro pre-clinical activity of RTX and GA101 against CLL cells in freshly isolated PBMCs, and correlate depletion efficacy with modern and classical (FISH, IgVH mutational status, age, gender, Binet stage, b2-microglobulin, bulky adenopathies>5cm) prognostic parameters in 95 CLL patients. Moreover, CD20 antigen density was assessed in 24 patients. Methods: PBMCs were isolated from blood samples by Ficoll gradient centrifugation. Antibody-mediated B cell depletions were determined by enumerating trypan blue negative, flow cytometrically CD19-positive B lymphocytes after treatment with 10 μg/ml of anti-CD20 antibodies. The Quantibrite® kit was used to determine CD20 density, measured as quantity of antibodies bound per cell (CD20-ABC). The Mann-Whitney test was used to compare median depletion according to relevant clinical and biological data. Results: patients characteristics were as follows: 69% males, median age 66y (36% >70y), advanced Binet stage B/C in 39% (31.7% had NCI2008 criteria for treatment), 22.6% had lymph nodes>5cm (56% had b2-microglobulin>3.5mg/l). Molecular and cytogenetics studies were as follows: unmutated IgVH in 42.7%, del13q/tri12/del11q/del17p=25.6%/22%/20%/5.8% respectively, complex karyotype (CK) was found in 20.8% of cases, and somatic mutation of TP53/NOTCH1/SF3B1 in 11.1%/12.6/4.8% of patients. The median B-CLL depletion in freshly isolated PBMC fraction due to treatment with were 61% for GA101 vs 21.5% for RTX (n=95, p 50% in 64% of cases. As shown in Figure 1, in all categories of patients, GA101 was superior to RTX. This was particularly noticed in the patients with TP53 deletion and/or mutation (n=7). Median CD20-ABC was 8100 in 24 patients, who were categorized as high or low CD20 expression based on that median. Low CD20-ABC patients had significantly less antibody-induced B-cell depletion, 12.8% (vs 25%, p=0.37) for RTX, and 42.5% (vs 67.5%, p=0.048) for GA101. CD20-ABC was not found to be correlated with clinicobiological parameters described above. Conclusions: according to modern prognostic parameters, GA101 confirms its higher efficacy than RTX (three-fold), and deserves further combination with chemotherapy or non-chemotherapy entities in CLL management. Citation Format: Loic Ysebaert, Emilie Laprevotte, Christian Klein, Jean-Jacques Fournie, Anne Quillet-Mary. Obinutuzumab (GA101) efficacy in chronic lymphocytic leukemia in vitro is not diminished in high risk patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2013-2852