Improved Recognition of Hematogones from B Lymphoblastic Leukemia By 5-Color Flow Cytometric Analysis

Background: Hematogones (HG) are benign precursor B-cells that are seen in increased numbers in the bone marrow during childhood, following chemotherapy or bone marrow transplant, in certain immune deficiencies, and in autoimmune disorders. Flow cytometry typically shows expression of TdT, CD10, CD19, variable CD20, dim CD22, CD34 (early HG), and dim CD45. As this phenotype is also seen in patients with B lymphoblastic leukemia (B-ALL), it causes a significant problem in distinguishing leukemic blasts from HG, particularly in a regenerating marrow. Furthermore, hematogones are usually more numerous at baseline in younger patient populations, the same age group with a relatively higher incidence of B-ALL. Despite guidance by earlier studies using the above markers for differentiating HG from B-ALL, these markers are not always sufficient and may hinder correct interpretation. Previous studies demonstrate CD58 is commonly expressed in B-ALL but not in hematogones; however, CD58 as a single marker is somewhat limited when expressed at lower levels. In order to improve diagnostic accuracy, we generated a five color antibody panel including CD10, CD19, CD45, CD38, and CD58 to assess the utility of a single tube panel in distinguishing B-ALL from HG. Design: A total of 35 cases with immature B-cell populations, 16 B-ALL (diagnostic and residual/relapsed cases) and 19 HG, were analyzed by 5-color flow cytometry. 32/35 cases were bone marrow aspirates and 3/35 cases were peripheral blood. A single tube containing CD10 FITC/CD58 PE/CD19 ECD/CD38 PC5/CD45 PC7 was analyzed together with the standard acute leukemia panels. To eliminate technical and fluorochrome variability in expression level analysis, relative antigenic expression was determined through comparison with appropriate internal controls. Antigen expression, as measured by the geometric mean fluorescent intensity (MFI), was then compared between B-ALL and HG using the Mann-Whitney U-test to assess for significant difference. Results were correlated with the morphologic, immunohistochemical, cytogenetic, and molecular findings for precise diagnostic classification as HG or leukemia. Results: HG demonstrated significantly brighter expression of CD38 (p<0.01) than that seen in B-ALL. In contrast, B-ALL expressed significantly brighter CD58 (p <0.01) than HG, which showed dim to no expression of the antigen. HG also showed significantly bright expression of CD10 relative to internal control granulocytes; however, this level of expression was similar to that seen in B-ALL. Median antigen expression. Hematogones show bright CD38, but dim to no CD58. Conversely, B-ALL expresses very dim CD38 and variable CD58. CD10 expression, though, demonstrates overlap between the two populations. B-ALL = B lymphoblastic leukemia, MFI = mean fluorescent intensity Comparative antigenic expression levels in hematogones and B-ALL. Select representative histograms showing HG and B-ALL blasts for the antigens CD38, CD58, and CD10 were selected from various patients studied based on those with the closest relative MFI to the overall median detected for that population in the study. The far right column shows the distribution in MFI of relative antigen expression exhibited the populations studied. HG show significantly brighter CD38 expression than B-ALL does (p<0.01). While B-ALL generally expresses brighter CD58 relative to internal controls, expression levels are variable. HG, though, show significantly dimmer CD58 to essentially no CD58 expression, compared to B-ALL (p<0.01). Similar to CD38, HG demonstrate significantly bright CD10, while B-ALL shows overall bright CD10 but variable expression levels amongst studied cases. These expression levels for CD10 overlap between HG and B-ALL and show no real statistical difference. Conclusions: The combination of CD38 and CD58 in a single tube increases the diagnostic accuracy in differentiation of HG from B-ALL. Without utilizing both antigens together, certain cases would have been difficult to interpret. Based on this analysis, we recommend that these markers be utilized in the routine evaluation for acute lymphoblastic leukemia. This is especially critical in post treatment cases in order to avoid misdiagnosis. Furthermore, the use this single tube panel would cut costs while at the same time improve patient care. Table Table. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Vascular Endothelial Growth Factor (VEGF) Increases B-CLL CELL Migration Through Regulation of CXCR4/SDF1 AXIS

Abstract 3917 INTRODUCTION VEGF is an important pro-angiogenic factor involved in survival and dissemination of chronic lymphocytic leukemia cells. At high concentrations such as those present in bone marrow niches, VEGF reduces MMP9 production and favours bone marrow retention. But on the other hand, it is well known that autologous VEGF and CD49d are required for B-CLL cell migration. In other cancer cell lines, this effect seems to be mediated by CXCR4/SDF-1 axis. In this work we studied the role of exogenous VEGF on B-CLL migration as well its relationship with the G-coupled proteins CXCR4, CXCR7 and CD49d. MATERIALS AND METHODS We obtained peripheral blood mononuclear cells from 33 patients diagnosed of CLL according to established clinical and laboratory protocols at our institution after informed consent. We also used the CLL cell line Mec-1. B-lymphocytes were purified by Ficoll-Hypaque density gradient centrifugation anti-CD19 conjugated Dynabeads and stored in liquid nitrogen. Then we analysed by flow cytometry the expression of several chemokines and adhesion molecules, CXCR4, CXCR7 and CD49d before and after exogenous VEGF exposure (50 ng/mL), VEGF-R2 inhibitor (70 nM) or both. In order to evaluate the role of VEGF in the motility of these cells, we performed an in vitro 24-hours transmigration assay towards a media containing (or not) SDF-1. B-CLL (5 × 105 cells) cells were incubated on the upper chamber of transwell filters coated with human umbilical vein endothelial cells in the presence or absence (control) of vascular endothelial growth factor (VEGF) ([50 ng/ml], 24 hours), VEGFR2/KDR inhibitor [70nM] or both simultaneously. We quantified the migration ratio between those stimulated with VEGF compared with non-stimulated controls, inhibitor-treated cells versus control and both (R2-inh plus VEGF) versus inhibitor-treated cells. Migration ratio was given as mean ± SD. Statistical analysis was performed by non-parametric Wilcoxon test using SPSS statistical software (version 19.0). RESULTS Basal CXCR4, CXCR7 and CD49d expression levels of B-CLL cells were highly variable among the 33 patients analyzed. Mean fluorescent intensity (MFI) of CXCR4 expression was significantly higher on cells treated with VEGF versus untreated cells (average increase, 9.64 ± 95, p=0.028). However, we did not detect a significant difference in the percentage of cells expressing this receptor. On the other hand, VEGF treatment did not influence either the mean fluorescent intensity or the number of CXCR7 and CD49d expressing cells. Exposure to a VEGFR2 inhibitor reduced the percentage of cells expressing CXCR4 and CXCR7, suggesting a potential regulatory role of this receptor in the expression of these chemokines. Concerning B-CLL migration, we observed a significant increase in cell migration of cells treated with exogenous VEGF versus the control ones in both the Mec-1 cell line and the primary cells (27.66 ± 69.97 p= 0.03). Furthermore, the treatment with VEGFR2 inhibitor reduced significantly the migration index (−23.18 ± 33.5, p= 0.001) and the motility was restored by the addition of VEGF to the R2-inhibitor treated cells (36.04 ± 39.33, p=0.001). DISCUSSION This preliminary data suggest that VEGF seems to be involved in B-CLL migration through the regulation of CXCR4 expression levels. Detailed molecular mechanisms implicated in this process should be further studied. New therapeutic strategies focussed in blocking both the SDF1-CXCR4 axis and/or VEGF pathway could have a potential therapeutic implication by decreasing B-CLL cell migration into lymph nodes and bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Correlation of CD33 expression level with disease characteristics and response to gemtuzumab ozogamicin containing chemotherapy in childhood AML

CD33 is expressed on the majority of acute myeloid leukemia (AML) leukemic blasts and is the target for gemtuzumab ozogamicin (GO), a toxin-conjugated anti-CD33 mAb. In the present study, we quantified the CD33 mean fluorescent intensity of leukemic blasts prospectively in 619 de novo pediatric AML patients enrolled in Children's Oncology Group GO-containing clinical trials and determined its correlation with disease characteristics and clinical outcome. CD33 expression varied more than 2-log fold; a median mean fluorescent intensity of 129 (range, 3-1550.07) was observed. Patients were divided into 4 quartiles, quartiles 1-4 (Q1-4) based on expression and disease characteristics and clinical response defined across quartiles. High CD33 expression was associated with high-risk FLT3/ITD mutations (P < .001) and was inversely associated with low-risk disease (P < .001). Complete remission (CR) rates were similar, but patients in Q4 had significantly lower overall survival (57% ± 16% vs 77% ± 7%, P = .002) and disease-free survival from CR (44% ± 16% vs 62% ± 8%, P = .022). In a multivariate model, high CD33 expression remained a significant predictor of overall survival (P = .011) and disease-free survival (P = .038) from CR. Our findings suggest that CD33 expression is heterogeneous within de novo pediatric AML. High expression is associated with adverse disease features and is an independent predictor of inferior outcome. The correlation between CD33 expression and GO response is under investigation. These studies are registered at www.clinicaltrials.gov as NCT00070174 and NCT00372593.

Antihuman Leukocyte Antigen–Specific Antibody Strength Determined by Complement-Dependent or Solid-Phase Assays Can Predict Positive Donor-Specific Crossmatches

Context.—The association of circulating donor-specific antibody (DSA) strength with crossmatch results is of potential interest to predict allograft outcome. Objectives.—To systematically investigate the aforementioned association and to attempt to define a cutoff value for DSA strength that can predict a positive crossmatch result. Design.—We analyzed DSA strength and crossmatch results from the 2006 to 2008 proficiency testing samples of the American Society of Histocompatibility and Immunogenetics (n = 50). We further validated our findings in candidates for potential kidney transplant (n = 19). Results.—Proficiency test samples with positive antihuman globulin T-cell crossmatch results had significantly higher DSA strength, as assessed by Luminex (Austin, Texas) mean fluorescent intensity (MFI; MFI [SD], 7860 [4770]), compared with samples with negative crossmatch results (MFI [SD], 2900 [1820]; P = .001). Similarly, higher Luminex values were observed in samples from candidates for transplant with positive antihuman globulin T-cell crossmatch results (MFI [SD], 7910 [2370] versus 2840 [1960]; P &lt; .001). The MFI value of 6540 had 61% and 75% sensitivity and 92% and 94% specificity for predicting positive antihuman globulin T-cell crossmatches in proficiency test samples and in candidates for transplant, respectively. Conclusions.—The DSA strength correlates well with crossmatch results. An MFI of 6540 predicted a positive antihuman globulin T-cell crossmatch.

Abstract 2899: Ultrasound Exposed Microbubbles Cause Local Hyperpolarization of the Cell Membrane.

Background Ultrasound (US) and microbubbles have gained wide interest as a tool for local delivery of drugs and genes. However, the exact mechanisms underlying increased cellular uptake of drugs and genes are still not clear. Recently, we showed an increase in cell membrane permeability for Ca 2+ in rat cardiomyoblast (H9c2) cells exposed to US and microbubbles. In this study, we hypothesized that the Ca 2+ influx will have an effect on the membrane potential, and investigated whether Ca 2+ dependent potassium channels (BK Ca ) are involved. We particularly focused on local events where the microbubble was in contact with the cell membrane. Material and Methods H9c2 cells were cultured on US transparent membranes. US exposure consisted of bursts of 1 MHz with a peak negative pressure of 50 or 250 kPa. Pulse repetition frequency was 20 Hz, duty cycle 0.2%. Cells were exposed during 30s in the presence of Sonovue microbubbles. The membrane potential was monitored during US exposure using the fluorescent dye di-4-ANEPPS. The experiments were repeated in the presence of iberiotoxin (100 nM), a specific inhibitor of BK Ca channels. Results Surprisingly, despite the previously reported Ca 2+ influx, we found patches of hyperpolarization of the cell membrane, as reflected by local increases in di-4-ANEPPS mean fluorescent intensity (MIF), shown in table I as percentage of control (%MIF/control). This hyperpolarization was caused by the activation of BK Ca channels, as iberiotoxin completely prevented hyperpolarization. Conclusion US and microbubbles elicit a Ca 2+ influx, which leads to activation of BK Ca channels and a subsequent, local hyperpolarization of the cell membrane. This local hyperpolarization of the cell membrane may facilitate uptake of macromolecules through endocytosis and macropinocytosis. Table I

Development of a Unique siRNA and Intrabody Combinatorial HIV-1 Vector to Knockdown CXCR4 and Protect Cells from HIV-1 Challenge.

We overcome the problem of knocking down highly expressed gene function by combining intracellular single chain antibodies (intrabodies) and short interfering RNA (siRNA) to the same gene product. The chemokine receptor CXCR4 is expressed at high levels on T cells and is the co-receptor for pathogenic HIV-1s (termed X4-tropic). Furthermore, it has been shown that HIV-1 requires low amounts of CXCR4 for X4-tropic viral entry (Konopka and Düzgüne AIDS Res Hum Retro, 2002). In contrast to CCR5, due to the high levels of CXCR4, it has been more difficult to provide protection from HIV-1 challenge using conventional siRNA strategies. To address this issue a CXCR4 siRNA and intrabody gene combination HIV-1 vector has been generated. The CXCR4 intrabody is specific for the C-terminal domain of CXCR4 and is coupled to the KDEL endoplasmic retention signal and CXCR4 siRNA targets mRNA nucleotides 331–349 (Martinez, et al. AIDS, 2002). Using the myelomonocytic THP-1 cell line, a line that highly expresses cell surface CXCR4, it was found that CXCR4 intrabody or siRNA vectors mediated a 50% reduction in cells expressing CXCR4 as assessed by flow cytometry. In contrast, the level of CXCR4 cell knockdown increased to 82% using the CXCR4 siRNA and intrabody combinatorial vector. Moreover, the CXCR4 mean fluorescent intensity in cells containing the combination vector resembled the mean fluorescent intensity of cells analyzed with isotype control antibodies, i.e., little detectable CXCR4 expression as judged by flow cytometry. Similar CXCR4 knockdown trends were seen in primary CD4+ T-cells expressing the combinatorial vector as compared to vectors containing the intrabody gene or siRNA alone. HIV-1 X4-tropic challenge studies using a multiplicity of infection of 1 have shown that THP-1 cells expressing the combinatorial vector are resistant to infection (HIV-1 p24 production < 3 ng/ml), whereas THP-1 cells expressing the siRNA or intrabody gene vector were susceptible to infection (HIV-1 p24 > 15–35 ng/ml). These findings demonstrate that targeting both the message and protein product from the same gene is vastly superior to the targeting of either message or protein. The combinatorial vector is currently being tested in humanized mice for efficacy. Lastly, targeting a highly expressed cellular product at the message and protein level may prove beneficial for efficient gene knockdown outside the HIV-1 research and therapy field, such as for metastatic cancers associated with CXCR4/SDF-1 upregulation.