Using digital RNA counting and flow cytometry to compare mRNA with protein expression in acute leukemias.

BACKGROUND: The diagnosis of malignant hematologic diseases has become increasingly complex during the last decade. It is based on the interpretation of results from different laboratory analyses, which range from microscopy to gene expression profiling. Recently, a method for the analysis of RNA phenotypes has been developed, the nCounter technology (Nanostring® Technologies), which allows for simultaneous quantification of hundreds of RNA molecules in biological samples. We evaluated this technique in a Swiss multi-center study on eighty-six samples from acute leukemia patients. METHODS: mRNA and protein profiles were established for normal peripheral blood and bone marrow samples. Signal intensities of the various tested antigens with surface expression were similar to those found in previously performed Affymetrix microarray analyses. Acute leukemia samples were analyzed for a set of twenty-two validated antigens and the Pearson Correlation Coefficient for nCounter and flow cytometry results was calculated. RESULTS: Highly significant values between 0.40 and 0.97 were found for the twenty-two antigens tested. A second correlation analysis performed on a per sample basis resulted in concordant results between flow cytometry and nCounter in 44-100% of the antigens tested (mean = 76%), depending on the number of blasts present in a sample, the homogeneity of the blast population, and the type of leukemia (AML or ALL). CONCLUSIONS: The nCounter technology allows for fast and easy depiction of a mRNA profile from hematologic samples. This technology has the potential to become a valuable tool for the diagnosis of acute leukemias, in addition to multi-color flow cytometry.

L'immunoscintigraphie par les anticorps monoclonaux radiomarqués. Evolution d'une méthode de diagnostic du cancer et espoir d'une nouvelle forme de thérapie [Immunoscintigraphy using radio-labeled monoclonal antibodies. Development of a method of cancer diagnosis and hopes for a new form of therapy].

Personal results are presented to illustrate the development of immunoscintigraphy for the detection of cancer over the last 12 years, from the early experimental results in nude mice grafted with human colon carcinoma to the most modern form of immunoscintigraphy applied to patients, using I123 labeled Fab fragments from monoclonal anti-CEA antibodies detected by single photon emission computerized tomography (SPECT). The first generation of immunoscintigraphy used I131 labeled, immunoadsorbent purified, polyclonal anti-CEA antibodies and planar scintigraphy, as the detection system. The second generation used I131 labeled monoclonal anti-CEA antibodies and SPECT, while the third generation employed I123 labeled fragments of monoclonal antibodies and SPECT. The improvement in the precision of tumor images with the most recent forms of immunoscintigraphy is obvious. However, we think the usefulness of immunoscintigraphy for routine cancer management has not yet been entirely demonstrated. Further prospective trials are still necessary to determine the precise clinical role of immunoscintigraphy. A case report is presented on a patient with two liver metastases from a sigmoid carcinoma, who received through the hepatic artery a therapeutic dose (100 mCi) of I131 coupled to 40 mg of a mixture of two high affinity anti-CEA monoclonal antibodies. Excellent localisation in the metastases of the I131 labeled antibodies was demonstrated by SPECT and the treatment was well tolerated. The irradiation dose to the tumor, however, was too low at 4300 rads (with 1075 rads to the normal liver and 88 rads to the bone marrow), and no evidence of tumor regression was obtained. Different approaches for increasing the irradiation dose delivered to the tumor by the antibodies are considered.

Dysmegakaryopoiesis predicting response to therapy in acute myeloid leukaemia. A histologic and clinical study

With standard induction therapy between 50 to 85% of patients with Acute Myeloid Leukaemia (AML) achieve Complete Remission (CR). We investigated whether any morphological feature of bone marrow (BM) plastic embedded biopsies could predict failure of therapy. We reviewed BM plastic embedded biopsies from 54 adult patients presenting with untreated AML. The main histologic parameters analysed were cellularity, dysmegakaryopoiesis (DysM), percentage of marrow blasts and fibrosis. CR was obtained in 34 of 49 treated patients (69%). The rate of CR was significantly lower in the group of patients presenting with DysM: CR was achieved in 54% of the 28 treated patients with DysM and in 90% of the 21 treated patients without DysM (p less than 0.02). Patients with DysM had a significantly lower blood count and bone marrow blasts at presentation. Median age was not significantly different in the 2 groups. Cellularity and fibrosis were not predictive. DysM may be the hallmark of an AML subgroup with distinct clinical behaviour and lower rate of CR with conventional therapy. DysM should be carefully looked for on BM marrow biopsies and aspirate from AML patients at diagnosis.

Exposure to Leishmania braziliensis triggers neutrophil activation and apoptosis.

BACKGROUND: Neutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate. METHODS AND FINDINGS: Inoculation of serum-opsonized L. braziliensis promastigotes in mice induced neutrophil accumulation in vivo, peaking at 24 h. In vitro, exposure of thyoglycollate-elicited inflammatory or bone marrow neutrophils to L. braziliensis modulated the expression of surface molecules such as CD18 and CD62L, and induced the oxidative burst. Using mCherry-expressing L. braziliensis, we determined that such effects were mainly observed in infected and not in bystander cells. Neutrophil activation following contact with L. braziliensis was also confirmed by the release of TNF-α and neutrophil elastase. Lastly, neutrophils infected with L. braziliensis but not with L. major displayed markers of early apoptosis. CONCLUSIONS: We show that L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection.

CD103 marks a subset of human CD34(+)-derived langerin(+) dendritic cells that induce T-regulatory cells via indoleamine 2,3-dioxygenase-1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive molecule expressed in some subsets of normal and neoplastic cells. Mature human dendritic cells (DCs) have been shown to express IDO1, but little is known about its expression and function during DC differentiation from bone marrow hematopoietic stem/progenitor cells (HSPCs). Here, we show that during in vitro differentiation along the myeloid DC lineage, CD34(+) HSPCs acquire IDO1 expression, which acts in a tolerogenic manner by inducing a population of fully functional CD4(+)CD25(+) FOXP3(+) T-regulatory cells. Phenotypically, CD1a(+)CD14(-) HPSC-derived DCs expressed IDO1, langerin, CD11b, and CD1c. Cell-sorting experiments demonstrated that IDO1 expression is found in a subset of CD1a(+)CD14(-)langerin(+) cells, expressing CD103, which is capable of inducing T-regulatory cells in an IDO1-dependent manner. In conclusion, DC differentiation from CD34(+) HSPCs results in the expression of a functionally active IDO1 protein in CD1a(+)langerin(+), CD103-expressing DCs. These data point toward IDO1 expression as part of a tolerogenic signature during DC development.

ErbB Ligands in Angiogenesis

The formation of new blood vessels, i.e. angiogenesis, is an important phenomenon during normal development and wound repair, as well as during various pathological processes, such as tumor growth and metastasis. Specific growth factors regulate angiogenesis by modulating the different cellular functions of endothelial cells (EC), and periendothelial cells, such as pericytes (PC) and smooth muscle cells (SMC), which interact with ECs in a paracrine manner. ErbB receptors form a subgroup of transmembrane receptor tyrosine kinases that interact with growth factors of the epidermal growth factor (EGF) family. ErbB receptors regulate behaviour of a variety of normal as well as tumor cell types. Cancer drugs that target epidermal growth factor receptor (EGFR, ErbB1) or ErbB2 receptor have been approved for clinical use. It has been speculated that part of the antitumor activity of ErbB inhibitor compounds result from an antiangiogenic mechanism. The results presented here indicate a role for endothelial-derived EGF-like growth factors heparin binding EGF-like growth factor (HB-EGF) and neuregulin-1 (NRG-1) in the paracrine regulation of angiogenesis. HB-EGF, EGFR and ErbB2 are shown to mediate interaction between ECs and SMCs in vitro, and gefitinib, an inhibitor of EGFR kinase activity, suppresses recruitment of PCs/SMCs in vivo. NRG-1 is shown to regulate EC functions in vitro and angiogenesis in vivo by indirect mechanisms involving vascular endothelial growth factor-A (VEGF-A) and VEGF receptor-2 (VEGFR-2). Furthermore, EGFR activity is demonstrated to regulate recruitment of bone marrow-derived perivascular cells during tumor neovascularization in vivo. These results indicate that ErbB signaling is involved in the cellular processes of new blood vessel formation. This study gives new information about the role of ErbB ligands and receptors in angiogenesis and vasculogenesis and about the mechanisms by which ErbB inhibitor drugs such as gefitinib affect tumor growth.

The CXCR4/CXCR7/CXCL12 Axis Is Involved in a Secondary but Complex Control of Neuroblastoma Metastatic Cell Homing.

Neuroblastoma (NB) is one of the most deadly solid tumors of the young child, for which new efficient and targeted therapies are strongly needed. The CXCR4/CXCR7/CXCL12 chemokine axis has been involved in the progression and organ-specific dissemination of various cancers. In NB, CXCR4 expression was shown to be associated to highly aggressive undifferentiated tumors, while CXCR7 expression was detected in more differentiated and mature neuroblastic tumors. As investigated in vivo, using an orthotopic model of tumor cell implantation of chemokine receptor-overexpressing NB cells (IGR-NB8), the CXCR4/CXCR7/CXCL12 axis was shown to regulate NB primary and secondary growth, although without any apparent influence on organ selective metastasis. In the present study, we addressed the selective role of CXCR4 and CXCR7 receptors in the homing phase of metastatic dissemination using an intravenous model of tumor cell implantation. Tail vein injection into NOD-scid-gamma mice of transduced IGR-NB8 cells overexpressing CXCR4, CXCR7, or both receptors revealed that all transduced cell variants preferentially invaded the adrenal gland and typical NB metastatic target organs, such as the liver and the bone marrow. However, CXCR4 expression favored NB cell dissemination to the liver and the lungs, while CXCR7 was able to strongly promote NB cell homing to the adrenal gland and the liver. Finally, coexpression of CXCR4 and CXCR7 receptors significantly and selectively increased NB dissemination toward the bone marrow. In conclusion, CXCR4 and CXCR7 receptors may be involved in a complex and organ-dependent control of NB growth and selective homing, making these receptors and their inhibitors potential new therapeutic targets.

Cell-Cell interaction in erythropoiesis: role of human monocytes

Erythroid burst forming units (BFU-E) are proliferative cells present in peripheral blood and bone marrow which may be precursors of the erythroid colony forming cell found in the bone marrow. To examine the possible role of monocyte-macrophages in the modulation of erythropoiesis, the effect of monocytes on peripheral blood BFU-E proliferation in response to erythropoietin was investigated in the plasma clot culture system. Peripheral blood mononuclear cells from normal human donors were separated into four fractions. Fraction-I cells were obtained from the interface of Ficoll-Hypaque gradients (20-30% monocytes; 60-80% lymphocytes); fraction-II cells were fraction-I cells that were nonadherent to plastic (2-10% monocytes; 90-98% lymphocytes); fraction-III cells were obtained by incubation of fraction-II cells with carbonyl iron followed by Ficoll-Hypaque centrifugation (>99% lymphocytes); and fraction-IV cells represented the adherent population of fraction-II cells released from the plastic by lidocaine (>95% monocytes). When cells from these fractions were cultured in the presence of erythropoietin, the number of BFU-E-derived colonies was inversely proportional to the number of monocytes present (r = ¿0.96, P < 0.001). The suppressive effect of monocytes on BFU-E proliferation was confirmed by admixing autologous purified monocytes (fraction-IV cells) with fraction-III cells. Monocyte concentrations of ¿20% completely suppressed BFU-E activity. Reduction in the number of plated BFU-E by monocyte dilution could not account for these findings: a 15% reduction in the number of fraction-III cells plated resulted in only a 15% reduction in colony formation. These results indicate that monocyte-macrophages may play a significant role in the regulation of erythropoiesis and be involved in the pathogenesis of the hypoproliferative anemias associated with infection and certain neoplasia in which increased monocyte activity and monopoiesis also occur.