Nancy D. Bone

Chromosome anomalies detected by interphase fluorescence in situ hybridization: correlation with significant biological features of B-cell chronic lymphocytic leukaemia

Summary. Fluorescence in situ hybridization (FISH) was used to detect 6q–, 11q–, +12, 13q–, 17p– and translocations involving 14q32 in interphase nuclei from blood and/or bone marrow from 113 patients with B‐cell chronic lymphocytic leukaemia (B‐CLL). A total of 87 patients (77%) had a FISH anomaly: 13q– × 1 was most frequent (64%) followed by 13q– × 2 (28%), +12 (25%), 11q– (15%), 17p– (8%) and 6q– (0%). FISH results for blood and bone marrow cells in 38 patients were similar. Purified CD5+/CD19+ cells from blood were studied in eight patients and results indicate that in some patients not all B cells have FISH anomalies. We used a defined set of hierarchical FISH risk categories to compare FISH results by stable versus progressive disease, age, sex, Rai stage, CD38+ expression and IgVH mutational status. Significant differences in FISH risk distributions were associated with Rai stage, disease status and CD38+, but not by age, sex or IgVH mutational status. To look for baseline factors associated with high‐risk disease, multivariate analysis of age, sex, Rai stage, CD38+ and disease status versus FISH risk category was performed. Importantly, only CD38+ was significantly associated with high‐risk FISH categories (+12, 11q– and 17p–) after adjustment for the effects of other variables.

Analysis of clonal B‐cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B‐chronic lymphocytic leukaemia

Recent reports suggest that the expression of germline (GL) Ig variable region heavy‐chain genes (VH) is a negative prognostic factor for B‐cell chronic lymphocytic leukaemia (B‐CLL) patients and that CLL B‐cell CD38 expression may be a surrogate marker of Ig VH gene status. Currently, however, the usefulness of this surrogate marker is controversial. Therefore, our goal was to study the ability of CD38 to act as a surrogate marker for Ig VH somatic mutation (SM), and to identify differences in overall survival (OS), progression‐free survival (PFS) and response in B‐CLL patients based on these two markers. We first assessed the relationship between CD38 expression and Ig VH status on 131 B‐CLL patients, including 66 patients enrolled in three North Central Cancer Treatment Group Trials. Although the mean percentages of CD38+ clonal B cells were significantly higher for patients classified as GL versus SM, CD38 was not a reliable marker for clonal B‐cell SM. Overall, GL patients exhibited significantly shorter OS and PFS times than SM patients. Despite the inability of clonal B‐cell CD38 expression to predict Ig VH mutation status, patients with ≥ 30% CD38+ cells did have shorter PFS and OS times than did CLL patients with < 30% CD38+ cells. Thus, the relationship between CD38 expression and Ig VH mutation status in B‐CLL is not straightforward. Nevertheless, analysis in a co‐operative group clinical trial setting suggests that both B‐cell markers alone or in combination may have clinical usefulness. These data strongly encourage the study of these biological markers as they relate to disease heterogeneity in B‐CLL.

Potential therapeutic application of gold nanoparticles in B-chronic lymphocytic leukemia (BCLL): enhancing apoptosis

B-Chronic Lymphocytic Leukemia (CLL) is an incurable disease predominantly characterized by apoptosis resistance. We have previously described a VEGF signaling pathway that generates apoptosis resistance in CLL B cells. We found induction of significantly more apoptosis in CLL B cells by co-culture with an anti-VEGF antibody. To increase the efficacy of these agents in CLL therapy we have focused on the use of gold nanoparticles (GNP). Gold nanoparticles were chosen based on their biocompatibility, very high surface area, ease of characterization and surface functionalization. We attached VEGF antibody (AbVF) to the gold nanoparticles and determined their ability to kill CLL B cells. Gold nanoparticles and their nanoconjugates were characterized using UV-Visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). All the patient samples studied (N = 7) responded to the gold-AbVF treatment with a dose dependent apoptosis of CLL B cells. The induction of apoptosis with gold-AbVF was significantly higher than the CLL cells exposed to only AbVF or GNP. The gold-AbVF treated cells showed significant down regulation of anti-apoptotic proteins and exhibited PARP cleavage. Gold-AbVF treated and GNP treated cells showed internalization of the nanoparticles in early and late endosomes and in multivesicular bodies. Non-coated gold nanoparticles alone were able to induce some levels of apoptosis in CLL B cells. This paper opens up new opportunities in the treatment of CLL-B using gold nanoparticles and integrates nanoscience with therapy in CLL. In future, potential opportunities exist to harness the optoelectronic properties of gold nanoparticles in the treatment of CLL.

B-CLL cells are capable of synthesis and secretion of both pro- and anti-angiogenic molecules.

Initial work has shown that clonal B cells from B-chronic lymphocytic leukemia (B-CLL) are able to synthesize pro-angiogenic molecules. In this study, our goal was to study the spectrum of angiogenic factors and receptors expressed in the CLL B cell. We used ELISA assays to determine the levels of basic fibroblast growth factors (bFGF), vascular endothelial growth factor (VEGF), endostatin, interferon-α (IFN-α) and thrombospondin-1 (TSP-1) secreted into culture medium by purified CLL B cells. These data demonstrated that CLL B cells spontaneously secrete a variety of pro- and anti-angiogenic factors, including bFGF (23.9 pg/ml ± 7.9; mean ± s.e.m.), VEGF (12.5 pg/ml ± 2.3) and TSP-1 (1.9 ng/ml ± 0.3). Out of these three factors, CLL B cells consistently secreted bFGF and TSP-1, while VEGF was expressed in approximately two-thirds of CLL patients. Of interest, hypoxic conditions dramatically upregulated VEGF expression at both the mRNA and protein levels. We also employed ribonuclease protection assays to assay CLL B cell expression of a variety of other angiogenesis-related molecules. These analyses revealed that CLL B cells consistently express mRNA for VEGF receptor 1 (VEGFR1), thrombin receptor, endoglin, and angiopoietin. Further analysis of VEGFR expression by RT-PCR revealed that CLL B cells expressed both VEGFR1 mRNA and VEGFR2 mRNA. In summary, these data collectively indicate that CLL B cells express both pro- and anti-angiogenic molecules and several vascular factor receptors. Because of the co-expression of angiogenic molecules and receptors for some of these molecules, these data suggest that the biology of the leukemic cells may also be directly impacted by angiogenic factors as a result of autocrine pathways of stimulation.

CD49d expression is an independent predictor of overall survival in patients with chronic lymphocytic leukaemia: a prognostic parameter with therapeutic potential

In vitro studies have demonstrated that surface expression of CD49d on chronic lymphocytic leukaemia (CLL) B cells facilitates leukaemic cell–stromal interactions by binding to fibronectin. This interaction reduces both spontaneous and drug‐induced apoptosis. The present study measured CD49d expression by flow cytometry in a cohort of untreated CLL patients previously accrued to a prospective observational study and evaluated the relationship with overall survival (OS). Among the 158 CLL patients tested, the percentage of leukaemic B cells expressing CD49d ranged from 0 to 100%. When all risk factors were treated as continuous variables, CD49d expression showed moderate correlation with expression of ZAP‐70 (r = 0·54; P < 0·0001) and CD38 (r = 0·58; P < 0·0001) but not %IGHV mutation. As a continuous variable, CD49d expression strongly correlated with OS (P < 0·0001). Recursive partitioning analysis suggested the 45% threshold of CD49d expression best predicted OS. Multivariate analysis, controlling for disease stage, ZAP‐70, IGHV status and fluorescent in situ hybridization defects identified CD49d as an independent predictor of OS and was a better predictor of clinical outcome than ZAP‐70, IGHV, or cytogenetics. This observational cohort study suggests that CLL B‐cell expression of CD49d is an easily measurable and independent predictor of OS and CD49d expression in CLL. Importantly, anti‐CD49d antibodies are already approved for treatment of other human diseases. Clinical testing of anti‐CD49d therapy in CLL appears warranted.

VEGF receptors on chronic lymphocytic leukemia (CLL) B cells interact with STAT 1 and 3: implication for apoptosis resistance

We have previously shown that chronic lymphocytic leukemia (CLL) B cells secrete vascular endothelial growth factor (VEGF) in vitro, have constitutively active VEGF receptors R1 and R2, and respond to exogenous VEGF by specifically upregulating Mcl-1 and XIAP in association with decreased cell death. We found that epigallocatechin (EGCG) decreases VEGF receptor phosphorylation and induces apoptosis in CLL B cells. The mechanism(s) by which VEGF receptor activation increases Mcl-1 and XIAP and promotes survival remains unknown. To further define the signaling pathway mediating VEGF induction of antiapoptotic proteins in CLL B-cells, we investigated downstream effects of VEGF–VEGF receptor binding on the STAT signaling pathway. We find that CLL B cells abundantly express cytoplasmic serine phosphorylated (p)-STAT-1 and p-STAT-3, VEGF-R1/2 are physically associated with p-STAT-1 and p-STAT-3, and p-STAT-3 (but not p-STAT-1) is found in the CLL nucleus. VEGF receptor ligation selectively induces activation and perinuclear translocation of STAT 3 through receptor-mediated endocytosis. The inhibition of VEGF receptor activation with either tyrosine kinase inhibitors or VEGF neutralizing antibodies inhibit VEGF receptor phosphorylation, decrease p-STAT-3 (serine 727), Mcl-1, and induces cell death in CLL B cells. Thus, a VEGF–VEGF receptor pathway in CLL B cells can be linked to activation of STAT proteins that are able to enhance their apoptotic resistance.

Direct and complement dependent cytotoxicity in CLL cells from patients with high-risk early-intermediate stage chronic lymphocytic leukemia (CLL) treated with alemtuzumab and rituximab.

The mechanism of cytotoxicity of alemtuzumab and rituximab in chronic lymphocytic leukemia (CLL) is not well understood. We obtained fresh CLL cells from early-intermediate stage high-risk patients just prior to treatment with alemtuzumab and rituximab to study mechanisms of action and resistance. Alemtuzumab had minimal direct cytotoxicity but caused significant complement dependent cytotoxicity (CDC) although a subpopulation of CLL cells had intrinsic resistance. Rituximab had no direct cytotoxicity and caused minimal CDC in cells from most patients. These data suggest that CDC has a therapeutic role in patients treated with alemtuzumab and that measures to decrease resistance to CDC could increase efficacy.

Expression of the chemokine receptors CXCR4 and CCR7 and disease progression in B-cell chronic lymphocytic leukemia/ small lymphocytic lymphoma.

OBJECTIVE To assess the clinical relevance of chemokine receptor expression on the progression of B-cell chronic lymphocytic leukemia (B-CLL). PATIENTS AND METHODS Peripheral blood mononuclear cells from 45 patients with B-CLL were purified and compared with lymph node samples collected from 17 of these patients. Also compared were B cells obtained from peripheral blood samples from 5 healthy controls and B cells from reactive lymph nodes from 3 otherwise healthy persons. The patients were treated at the Mayo Clinic in Rochester, Minn, between January 15,1991, and February 7, 2003. Mononuclear cells were stained by a 2-color (fluorescein isothiocyanate/phycoerythrin) flow cytometric assay using antibodies to the chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR2, CCR4, CCR5, CCR6, and CCR7) and also to CD19. RESULTS Of the 45 patients in this study, 20 had Rai stage 0 disease, 12 had stage I disease, 3 had stage II disease, 2 had stage III disease, and 8 had stage IV disease. The mean fluorescent intensity (MFI) of the chemokine receptor expression on B-CLL cells was compared with normal controls and was not significantly different, except for an increase in the median expression of CXCR3 (P = .003) and CCR7 (P = .001) on B-CLL cells. We also found a significant increase in the expression of CXCR4 and CCR7 in B-CLL cells from patients with stage IV compared with stage 0 disease (P = .001 and P = .02, respectively). Furthermore, circulating B-CLL cells showed significantly higher expression of CXCR4 and CCR7 when compared with B lymphocytes in lymph nodes (P = .003 and P < .001, respectively). CONCLUSION The expression of CXCR4 and CCR7 on B-CLL cells correlates with Rai stage. Also, these chemokine receptors may be down-regulated once malignant B cells enter the lymph nodes. To our knowledge, this is the first published report that shows the strong association of Rai stage with CXCR4 and CCR7 expression levels in B-CLL cells.

Interleukin 4 content in chronic lymphocytic leukaemia (CLL) B cells and blood CD8+ T cells from B‐CLL patients: impact on clonal B‐cell apoptosis

B‐chronic lymphocytic leukaemia (CLL) clonal B cells are characterized by resistance to apoptosis. We evaluated clonal B cells and blood T cells for interleukin 4 (IL‐4) content as IL‐4 is able to increase CLL cell resistance to apoptosis. The content of IL‐4 in CD8+ T cells of CLL patients (n = 9) ranged from 37% to 63% of the total CD8+ T cells (mean level of 49% ± 3·4) compared with a range of 5–10% for control CD8+ T cells. Clonal B cells positive for cytoplasmic IL‐4 ranged from 1% to 97% (mean value 57·8 ± 6·9%). CD8+ T cells and clonal B cells secreted detectable levels of IL‐4, but only clonal CLL B cells (n = 4) secreted IL−4 in association with increasing cell numbers. Fludarabine (F‐ara‐AMP, 0·1–100 μmol/ml) was able to downregulate the IL‐4 content of CD8+ T cells, but not clonal B‐cell IL‐4. Culture supernatant from CLL CD8+ T cells decreased the spontaneous apoptotic rate of clonal B cells that was reversed with anti‐IL‐4 and soluble IL‐4 receptor. These findings show that IL‐4 is present in the microenvironment of B‐CLL. In addition, use of agents that can interfere with IL‐4 presentation to clonal B cells can be effective in increasing clonal B‐cell apoptosis.

Percentage of Smudge Cells on Routine Blood Smear Predicts Survival in Chronic Lymphocytic Leukemia

PURPOSE Smudge cells are ruptured chronic lymphocytic leukemia (CLL) cells appearing on the blood smears of CLL patients. Our recent findings suggest that the number of smudge cells may have important biologic correlations rather than being only an artifact of slide preparation. In this study, we evaluated whether the smudge cell percentage on a blood smear predicted survival of CLL patients. PATIENTS AND METHODS We calculated smudge cell percentages (ratio of smudged to intact cells plus smudged lymphocytes) on archived blood smears from a cohort of previously untreated patients with predominantly early-stage CLL enrolled onto a prospective observational study. The relationship between percentage of smudge cells, patient survival, and other prognostic factors was explored. RESULTS Between 1994 and 2002, 108 patients were enrolled onto the study and had archived blood smears available for review; 80% of patients had Rai stage 0 or I disease. The median smudge cell percentage was 28% (range, 1% to 75%). The percentage of smudge cells was lower in CD38(+) versus CD38(-) patients (P = .019) and in Zap70-positive versus Zap70-negative patients (P = .028). Smudge cell percentage as a continuous variable was associated with prolonged survival (P = .042). The 10-year survival rate was 50% for patients with 30% or less smudge cells compared with 80% for patients with more than 30% of smudge cells (P = .015). In multivariate analysis, the percentage of smudge cells was an independent predictor of overall survival. CONCLUSION Percentage of smudge cells on blood smear is readily available and an independent factor predicting overall survival in CLL.