Quantification of CD38 expression in B-cell chronic lymphocytic leukemia (B-CLL): a comparison between antibody binding capacity (ABC) and relative median fluorescence (RMF)

The aim of this study was to investigate the efficacy of a combination of fludarabine (F) and cyclophosphamide (C) in the treatment of patients with refractory/recurrent B-cell chronic lymphocytic leukemia (B-CLL). Between November 1999 and December 2001, 63 patients with B-CLL (median age 60 years) received a regimen that consisted of F 25 mg/m² and C 250 mg/m², Days 1 - 3, intravenously, every 4 weeks , for a maximum of 6 courses. Response and toxicity were assessed according to current criteria (NCI-WG and WHO). Complete and partial remissions were achieved in 17.5 and 55.6% of patients, respectively; 19% of patients had stable disease and 7.9% of patients showed disease progression. The median follow-up was 16.5 (range 1.5 - 32) months. The median duration of progression-free survival (PFS) has not been reached among patients treated with FC regimen as second-line therapy. The median PFS was 13 (range 8 - 26) months in the 19 responding patients treated with FC regimen as third-line therapy. The most frequent side-effects were neutropenia (45%), thrombocytopenia (42%) and infections (57%). We conclude that the combination of fludarabine and cyclophosphamide demonstrated significant efficacy in pretreated, advanced B-CLL patients, with tolerable toxicity.     

The prognostic value of CD38 expression and its quantification in B cell chronic lymphocytic leukemia (B-CLL)

A large number of prognostic factors are available to help predict the outcome of patients who present with B-cell chronic lymphocytic leukemia (B-CLL). These include clinical stage, leukemic cell morphology, lymphocyte doubling time, the pattern of infiltration in bone marrow trephine biopsies, cytogenetic abnormalities, p53 function and serum factors such as beta-2 microglobulin. Two recently described major prognostic factors are immunoglobulin heavy chain variable region (IgVH) mutation status and cell membrane expression of CD38. These are both highly significant independent prognostic factors, but are not closely correlated.

Whereas IgVH mutational status is a time consuming and demanding technique, only available in a limited number of centres, CD38 expression by flow cytometry is relatively simple and rapidly obtained in most diagnostic laboratories. The predictive value of CD38 expression is enhanced by measurement of antigen density in terms of antibody binding capacity (ABC) rather than as the percentage of cells expressing the antigen. ABC correlates closely with relative median fluorescence (RMF), a parameter which is even more simply and cheaply obtained by flow cytometry. One of these methods of determining CD38 expression should be employed routinely.

Recent work suggests that membrane ZAP-70 expression determined by flow cytometry will prove to be an accurate proxy for IgVH mutational status and this assay will be within the reach of any laboratory skilled in flow cytometry. The combination of ZAP-70 expression, CD38 antigen density, p53 function and the concentration of serum factors such as soluble CD23, is likely to provide extremely accurate prognostic information in future studies. This will assist in identifying Stage A patients who may benfit from early and/or more intensive treatment, as well as Stage B and C patients who may require alternative treatment strategies at the outset.     

Intracellular cytokine profiles by peripheral blood CD3+ T-cells in patients with classical Hodgkin lymphoma

The intracellular profiles of T helper type 1 (Th1) and T helper type 2 (Th2) T-cell cytokines by peripheral blood (PB) CD3+ T-cells in patients with classical Hodgkin lymphoma (HL) has not been investigated before. The present study examines the cytoplasmic production of interleukin (IL) 2, 4, 10, tumour necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma) by activated PB CD3+ T-cells and compares them with the profiles observed with normal individuals. We report a significantly lower mean level of intracellular IL2, TNFalpha and IFNgamma at any time post-cell activation in cells isolated from patients with HL compared with the normal control group. In contrast, the mean level of cytoplasmic IL4 was significantly higher in the HL compared with the control group. No significant difference between the two groups was observed with IL10. In the HL patient group, there was a significantly higher percentage of CD3+CD8+ T-cells that synthesised IL4 compared with the CD3+CD4+ subpopulation, no such difference was observed in normal controls. The intensity of IL4 (expressed as relative median fluorescence) was significantly higher in the CD3+CD8+ cells of the patients with HL compared with the CD3+CD4+ sub-population, or with normal CD3+CD8+ cells. In conclusion, there is reduced intracellular IL2, TNFalpha and IFNgamma and increased cytoplasmic IL4 production by activated PB T-cells in patients with HL. The CD3+CD8+ sub-population is responsible for the increased levels of IL4.     

Evaluation of treatment outcome in 175 patients with Hodgkin lymphoma aged 60 years or over: the SHIELD study

The SHIELD program for Hodgkin lymphoma in patients 60 years of age or older, prospectively evaluated clinical features and outcome in a large patient cohort (n = 175). The central element was a phase 2 study of VEPEMB chemotherapy (n = 103, median age 73 years) incorporating comorbidity assessment. A total of 72 other patients were treated off-study but registered prospectively and treated concurrently with: ABVD (n = 35); CLVPP (n = 19), or other (n = 18). Of VEPEMB patients, 31 had early-stage disease (stage 1A/2A) and received VEPEMB 3 times plus radiotherapy. Median follow-up was 36 months. Complete remission (CR) rate (intention-to-treat) was 74% and 3-year overall survival (OS) and progression-free survival (PFS) were 81% and 74%, respectively. A total of 72 patients had advanced-stage disease (stage 1B/2B/3 or 4) and received VEPEMB 6 times. CR rate was 61% with 3-year OS and PFS of 66% and 58%, respectively. Of patients achieving CR, 13% with early-stage and 5% with advanced-stage disease progressed. Overall treatment-related mortality was 7%. In patients treated with curative intent with VEPEMB, ABVD, and CLVPP (n = 157), CR linked to several factors in univariate analysis. In a Cox regression model only, obtaining CR remained significant for OS and CR plus comorbidity and age for PFS. RS-EBV status had no significant effect on outcome.     

Expression and Production of Interleukin 4 in B-Cell Chronic Lymphocytic Leukaemia

Interleukin (IL) 4 is a T-cell derived pleiotropic cytokine whose properties include alterations of B-cell function. In B-cell chronic lymphocytic leukaemia (B-CLL), IL4 is involved in the mechanism of survival of the leukaemic B-cells. The present study examines the expression and production of IL4 by B- and T-lymphocytes derived from patients with B-CLL and provides evidence that IL4 is not an autocrine factor in B-CLL. Freshly isolated B-CLL cells enriched for B- and T-cells did not express mRNA for IL4 but expressed mRNA for IL4 receptor (IL4R). Activation of B-cells with phorbol ester and calcium ionophore and of T-cells with phytohaemaglutinin (PHA) upregulated IL4 mRNA expression. However phorbol ester and calcium ionophore did not affect the mean level of IL4 production by either B-CLL or normal B-cells. Furthermore, in the presence or absence of activation, the amount of IL4 synthesised by B-CLL B-cells was not significantly different than that observed with peripheral blood B-cells isolated from normal individuals (with activation: P=0.239; without activation: P=0.565). Also, there was no significant difference between normal and B-CLL B-cells in the level of cytoplasmic IL4 (P=0.47).

PHA-activated enriched B-CLL T-cells produced significantly higher levels of IL4 compared to normal control T-cells (P=0.0136). In addition, in 47% of cases with B-CLL T-cells, a significant higher level of intracellular IL4 was observed (P=0.0027). The levels of production of IL4 by the T-cell-enriched preparations correlated positively with the intensity of cytoplasmic IL4 in CD4⁺ and CD8⁺ cells in tested samples (r=0.49 and r=0.76, respectively).

The significant differences observed in the production of IL4 by B-CLL B- and T-lymphocytes may suggest a paracrine function of IL4 in B-CLL.     

γ-Linolenic Acid Induces Apoptosis in B-Chronic Lymphocytic Leukaemia Cells in Vitro

Gamma linolenic acid (GLA) is cytotoxic to many types of human cancer cells. Most chemotherapeutic agents are cytotoxic by inducing apoptosis. We examined the apoptotic activity of GLA on purified B-cells isolated from patients with B-cell chronic lymphocytic leukaemia (B-CLL) and from normal individuals. GLA significantly increased the degree of apoptosis in B-CLL B-cells after 24 hours of culture. The mean percentage of cells undergoing apoptosis when cultured in medium alone (spontaneous apoptosis) was 20% (range: 7 to 31%) (n=25) and in the presence of GLA (5μg-60μg) was: 42%-95%. In the presence of GLA 5μg/ml and dexamethasone the degree of apoptosis was 86% (range: 72 to 100%). GLA induced apoptosis in B-CLL B-cells at a higher level than that observed with normal B-cells at all lower concentrations tested 5, 10 and 15μg/ml: P=0.045; 0.027 and 0.022, respectively. At 30μg/ml of GLA, no significant difference in the percentage of cells displaying apoptosis between B-CLL and normal B-cells was observed (P=0.075). GLA induced apoptosis in B-CLL T-cells at both 10 and 30μg/ml. The degree of apoptosis in normal T-cells with GLA was also significant at the higher concentration of 30μg/ml. Interleukin 4 (IL4), a viability factor in B-CLL, and vitamin E, an anti-oxidant, protected B-CLL B-cells against GLA (20μg/ml)-induced apoptosis. These results demonstrate that GLA induces apoptosis in B-CLL B-and T-cells cells in-vitro and that they are more susceptible to GLA-induced apoptosis than normal peripheral blood B-and T-cells.     

Mitoxantrone in combination with an inhibitor of DNA-dependent protein kinase: a potential therapy for high risk B-cell chronic lymphocytic leukaemia

Defects in the DNA damage response pathway [e.g. del(17p)] are associated with drug‐resistant B‐cell chronic lymphocytic leukaemia (CLL). We previously demonstrated that over‐expression of DNA‐dependent protein kinase (DNA‐PK) correlates with chemo‐resistance and that inhibition of DNA‐PK sensitizes CLL cells to chemotherapeutics. Here, we investigated expression of DNA‐PK and other proteins that impact on drug resistance, and evaluated the effects of a DNA‐PK inhibitor (NU7441) on mitoxantrone‐induced cytotoxicity in CLL cells. NU7441 sensitized cells from 42/49 CLL samples to mitoxantrone, with sensitization ranging from 2‐ to 200‐fold Co‐culture of CLL cells in conditioned stromal medium increased chemoresistance but did not reduce sensitization by NU7441. Mitoxantrone treatment induced γH2AX foci and NU7441 increased their longevity (24 h). NU7441 prevented mitoxantrone‐induced autophosphorylation of the DNA‐PK catalytic subunit (DNA‐PKcs) at Ser 2056, confirming that DNA‐PK participates in repair of mitoxantrone‐induced DNA damage. del(17p) cases were more resistant to mitoxantrone than del(13q) cases, but were resensitized (7–16 fold) by co‐incubation with NU7441. Expression of DNA‐PKcs, Ku80, P‐glycoprotein and topoisomerase IIβ were significantly higher in del(17p) cases. PRKDC mRNA levels correlated with DNA‐PKcs protein expression, which predicted shorter survival. These data confirm the potential of DNA‐PK as a therapeutic target in poor prognosis CLL.     

Quantification improves the prognostic value of CD38 expression in B-cell chronic lymphocytic leukaemia

Recent studies have shown that CD38 expressed as a percentage of the antigen positivity can predict prognosis and disease progression in patients with B-cell chronic lymphocytic leukaemia (B-CLL). The present study showed that quantification of CD38 expressed as antibody-binding capacity (ABC) improves the prognostic value of the percentage of CD38 positivity in B-CLL. In a cohort of 81 patients with B-CLL, a level of CD38 expression of > or = 30% and an ABC value of 250 proved statistically valid cut-off points to predict disease progression (% CD38: P=0.0027; ABC: P < 0.0001). There was a positive and significant correlation between the percentage of CD38 expression and ABC (r=0.7; P < 0.0001). There was a better discrimination of survival using ABC rather than percentage CD38 positivity (P < 0.0001 compared with P=0.0027). Only ABC predicted for survival in patients under 60 years of age (P=0.0076) or with stage A disease (P=0.0195). Both percentage CD38 and ABC discriminated between time to first treatment for all patients but only ABC predicted time to treatment for stage A patients (P=0.0004). In conclusion, CD38 positivity is an important prognostic factor in B-CLL. However, quantification of CD38 is superior to the percentage positivity and should be used clinically in conjunction with other variables of predictive value to identify B-CLL patients that are likely to progress.     

CD31 density is a novel risk factor for patients with B-cell chronic lymphocytic leukaemia

CD31 is the physiological ligand for CD38. CD38 expression in a high percentage of malignant cells is a risk factor for patients with B-cell chronic lymphocytic leukaemia (B-CLL). A previous investigation demonstrated that quantification of CD38 improves upon the prognostic value of the percentage expression. A recent study states that the percentage of CD31 expression is not predictive in B-CLL. We reassessed the predictive power of CD31 in a cohort of 120 patients with B-CLL. Peripheral blood cells were stained with PCP-labelled anti (alpha)-CD19, FITC-alpha-CD5 and PE-alpha-CD31 antibodies. CD31 expression was quantified using beads of specific antibody binding capacity and the density was correlated with clinical outcome. End points were disease-specific survival and time to treatment (TTT). We report that CD31 density was significantly lower in the group of patients with Binet stage B and C of disease progression (P=0.0003). There was an inverse, significant correlation between CD31 and CD38 densities (R= -0.281, P=0.002). All CLL-related deaths occurred in patients with low CD31 density. Low CD31 predicted for poor disease outcome (survival, P=0.0087; TTT, P=0.0064) and identified Binet stage A patients (survival, P=0.0350; TTT, P=0.0716) and those with low CD38 (survival: all patients, P<0.0001; stage A, P=0.003) who followed a more aggressive clinical course. Disease-specific survival of patients with low CD31 and high CD38 densities was significantly shorter than all other groups. In addition, low CD31 density was a poor risk factor irrespective of patient age (survival: all patients, P=0.045; stage A, P=0.021) and identified patients with Binet stage B/C as the highest risk group (P<0.0001). In conclusion, low CD31 density is an adverse prognostic indicator in B-CLL. Also, low CD31 density enhances the prognostic power of CD38 density. The interaction between CD31 and CD38 and its clinical significance in B-CLL requires further investigation.     

Micro-vessel density and the expression of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PdEGF) in classical Hodgkin lymphoma (HL)

There is little information to date regarding the role of angiogenesis in Hodgkin lymphoma (HL). The present study examines micro-vessel density and the expression of vascular endothelial growth factor (VEGF) and platelet-derived endothelial growth factor (PdEGF) in lymph node biopsies of patients with HL at presentation and relapse. Using immunohistochemistry, the degree of new blood vessel formation and the expression of VEGF and PdEGF was assessed in Hodgkin-rich tissue. The micro-vessel density (MVD) increased with disease progression in seven out of 11 cases. Expression of VEGF was observed in endothelial cells (EC) of some micro-vessels and also in follicular dendritic cells. The Hodgkin/Reed-Sternberg (H-RS) cells as well as the inflammatory lymphocytes were negative for VEGF. Cytoplasmic or cytoplasmic and nuclear expression of PdEGF by the H-RS cells was observed in five of the 11 presentation cases. The expression of PdEGF increased with disease progression in seven cases. In conclusion, Hodgkin tissue shows prominent vascularization. The increased MVD and PdEGF expression with disease progression merits further investigation.