B-CLL Patients Research Articles

Bendamustine with Rituximab Is Safe and Effective As FRONT LINE Therapy in Elderly B-CLL Patients. an ITALIAN RETROSPECTIVE MULTICENTER Experience

The front-line therapy for CLL young and fit patients is chemo-immunotherapy with Fludarabine-Cyclophosphamide and Rituximab (FCR). However, around three quarter of the patients with a newly diagnosed CLL are 65 years or older, with approximately 42% being older than 75 years and with age-related comorbidities. FCR regimen results in a significant myelosuppression and high rates of early and late infections. Hence, other less toxic regimens are under evaluation. Recently the German CLL study group reported an interim analysis of the CLL10 trial, who compared FCR vs Bendamustine-Rituximab (BR). The response rates to the 1st-line treatment with BR or FCR were comparable, and BR could be an alternative 1st-line treatment for medically fit pts. Notably, this study showed that pts treated with BR were often older (median 71 vs. 65 yrs; p<0.0001) and frequently with comorbidities (67% vs. 62%), suggesting that it may be a suitable option for this wide subpopulation. Here we report retrospective data from 12 Italian centres focused on the evaluation of efficacy and safety of BR in elderly SLL/CLL patients.Data on 70 untreated SLL/CLL patients with age ≥ 65 years assigned to receive front-line therapy with 6 monthly courses of Bendamustine (90 mg/sm at days 1-2) and RTX (375 mg/sm at day 1 for the first course, then 500 mg/sm for subsequent cycles) were collected. The primary end points were the Overall Response (CR/PR) and toxicity rates. Responses, evaluated at 2 months after the end of therapy, were defined according to the updated National Cancer Institute-Working Group guidelines.Seventy patients (47 males and 23 females) with a median age of 72 years (range, 65-87 years) were included in the study. Eight patients (11.4%) were unfit (CIRS score =/> 7). All patients had an ECOG performance status ranging from 0 to 2. Twenty-nine of 70 patients had Binet stage C, 12 patients were Binet A, 29 Binet B.Thirty-nine patients showed karyotype abnormalities at FISH analysis (data available in 54/70 patients). High risk FISH karyotype according to Döhner´s hierarchical model was detected in 17 patients (14 with del 11q, 3 with del 17p). Ten of them had del(13q14), 12 had trisomy 12 and 15 had normal karyotype. The analysis of the IGHV status, available in 50 patients, showed 25 patients with somatic mutation and 25 patients with germ-line sequences. Zap-70 data were available for 37/70 pts, 19 of them (51.3%) were Zap-70 positive. CD38 was available for 52 patients: it was positive in 27/52 pts (51.9%). (Table 1)A mean number of 5.46 courses of BR were given and the Bendamustine dose was reduced by more than 10% in 39 patients (55,7%). The main reason for dose reduction was haematological toxicities. The ORR rate was 88,6%,with 22 patients (31.4%) obtaining a CR and 40 patients (57,2%) obtaining a PR. Progression Free Survival, Time To Retreatment and Overall Survival rates at 2-years were 79%, 90.3% and 89.6%, respectively. Only the presence of del17 resulted to affect the response rate (p= 0.023) and PFS (p<0.001). Grade 3-4 hematological toxicity was recorded in 25 patients (35,7%). Extra-hematological toxicity grade I-III (skin reactions, gastrointestinal and cardiovascular symptoms, infusion related reactions, etc). was noticed in 35 patients (50,0%). Eleven patients (15.7%) were admitted to the hospital.Retrospective data from this group of elderly CLL patients indicate that Benda-R front-line at standard dose provides a high response rate with a good safety profile, even if more than 50% of patients experienced a Bendamustine dose reduction until 70 mg/sm. Even in this cohort of patients we confirmed del17 as a bad prognostic parameter.Table 1:Patients’ clinical-biological characteristics.Median age at diagnosis72 years (range 65-87)M:F47/23CIRS score =/>78/70 pts (11.4%)PB lymphocytes33.203/mmcBinet stage A12/70 pts (17.2%)Binet stage B29/70 pts (41.4%)Binet stage C29/70 pts (41.4%)ZAP70>20%19/37 pts (51.3 %)CD38> 30%27/52 pts (51.9%)Beta2microglobulin increased51/59 pts (86.4%)IgVH homology < 98%25/50 pts (50%)Normal FISH15/54 pts (27.8%)del 13q10/54 pts (18.5%)+1212/54 pts (22.2%)del 11q14/54 pts (25.9%)del 17p3/54 pts (5.6%)Bulky syndrome16/70 pts (22.9%)PB, peripheral blood; IgVH, immunoglobulin heavy chain variable genes;FISH, Fluorescent In Situ Hybridization. DisclosuresNo relevant conflicts of interest to declare.

Clinical and phenotypic features of CD5-negative B cell chronic lymphoproliferative disease resembling chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) B cells are phenotypically identified by surface expression of CD5 and CD23 antigens. Infrequently, patients with a monoclonal B cell lymphocytosis clinically resembling classic B-CLL have been found to harbor leukemic B cells lacking expression of the CD5 antigen. Little information is available concerning such CLL-like lymphoproliferative syndromes. Here, we provide phenotypic and clinical characteristics of 13 patients with CD5-negative chronic lymphoproliferative disorders selected from among 400 B-CLL patients followed up at a single academic center. Phenotypic analysis was carried out by flow cytometry using a broad panel of monoclonal antibodies including activation, costimulatory, adhesion, and growth factor receptor molecules. Moreover, intracellular staining and stimulation experiments were performed to investigate whether CD5 antigen was either retained in the cytoplasm of clonal B cells or not expressed due to defective cellular activation, respectively. Overall, CD5-negative leukemic cells were found to express significantly different levels of several membrane molecules, including CD95, CD69, CD23, CD25, CD80, and CD20, compared to "classic" CLL B cells. CD5 antigen was not detected in the cytoplasm of CD5-negative clonal B cells, nor could it be induced following in vitro activation. CD3+ T cell proportions were found to be less affected in CD5-negative patients than in classic B-CLL. Although these data suggest that CD5-negative clonal B cells are phenotypically different from classic B-CLL, clinical outcomes were similar to those shown by B-CLL patients, with most of the patients experiencing a long-lasting disease requiring chemotherapeutic intervention at some time during the disease course.

Loss of donor chimerism in remission after allogeneic stem cell transplantation of T-prolymphocytic leukemia patients following alemtuzumab induction therapy.

T-cell prolymphocytic leukemia is a rare aggressive malignancy with low susceptibility to conventional chemotherapy. The anti-CD52 antibody alemtuzumab induces remission, which requires consolidation by stem cell transplantation in eligible patients. In this case series, three chemotherapy-naïve T-PLL patients received alemtuzumab for remission induction and allogeneic SCT after reduced-intensity conditioning. While primary hematopoietic engraftment occurred in a timely fashion, donor chimerism declined in all patients between day 28 and day 290 post-transplantation. Loss of chimerism was not associated with disease recurrence, and full chimerisms were regained on donor leukocyte infusion. In six B-CLL patients, treated with identical regimens of alemtuzumab and SCT, a similar pattern of failing chimerism was not observed. We surmise that an accumulation of uncleared alemtuzumab in the plasma may impede the incoming graft after allogeneic SCT, which would indicate the need for close monitoring and management of engraftment to secure complete donor chimerism and putative cure in T-PLL patients.

Expression of subtypes of interleukin-17 ligands and receptors in patients with B-cell chronic lymphocytic leukemia.

The diversity of biological effects of cytokines from the IL-17 family, as well as the wide range of cells sensitive to their influence, suggests that these molecules might play a significant role in the malignant pro- cess. After the cells were initially isolated with Polymorphprep™, they were sorted with a MACS® magnetic separator, CD16 for neutrophils and CD19 for B lymphocytes. The presence of proteins: IL-17A, IL-17E, IL-17F, IL-17D, as well as receptors: IL-17R, IL-17BR, IL-17RC in cell lysates was confirmed by Western blot. The levels of IL-17A, IL-17E, and IL-17F in blood serum were determined with ELISA. The results indicate a lower expression of IL-17A, IL-17E, and IL-17F in PMNs and B lymphocytes of pa- tients with B-cell chronic lymphocytic leukemia compared to cells of healthy subjects. Elevated expression of IL- 17D was observed in the PMNs of patients, with a simultaneous decrease in the expression of this cytokine in leukemic B cells, in comparison to the control group. In patients with B-CLL, there also were observations of decreased expression of IL-17R, IL-17BR, and IL-17RC in leukemic B lymphocytes, compared to their expressions in the cells of healthy subjects. The blood serum of B-CLL patients demonstrated a significantly increased level of IL-17A at stage 0/I, II, and IV of disease; IL-17E at stage 0/I and II; IL-17F at stage 0/I, III, and IV of disease advancement, compared to the data obtained from the control group. The results clearly support the involvement of the studied proteins from the IL-17 family in the course of B-CLL and indicate that IL-17D may play a significant role in the course of this disease.

Efficacy and Safety Of Bendamustine In Combination With Rituximab For Elderly Patients With Previously Untreated B-Cell Chronic Lymphocytic Leukemia. A Retrospective Multicenter Study

Chronic Lymphocytic Leukemia (B-CLL) is the most prevalent adult leukemia in western countries, with a median age of onset of 65 years. Front-line therapy for B-CLL young patients is chemo-immunotherapy with Fludarabine-Cyclophosphamide and Rituximab (FCR). However, many B-CLL patients are elderly and with comorbidities. FCR regimen can result in a significant myelosuppression and a high rate of early and late infections, suggesting that it may be too toxic and therefore unsuitable for this large subpopulation of patients. Data from the CLL 5 phase III trial of the German CLL study group (GCLLSG) comparing Fludarabine vs Chlorambucil (Chl) in patients older than 65 years showed no differences in overall survival and progression free survival (PFS) between Fludarabine and Chl. Recently, Bendamustine as single agent showed superiority in comparison to Chl in terms of overall response rate (ORR) and PFS, with a good safety profile. Later, the addition of Rituximab (RTX) to Bendamustine (Benda-R) was shown to be efficacious and safe in the same treatment-naïve setting. Insufficient data are available in patients older than 70 years regarding the efficacy and safety, nevertheless increased incidence of extra-hematological toxicity was noted in this subgroup. Here we report our multicentre retrospective study focusing on responses and toxicities rate in elderly patients with B-CLL.We report data on 24 elderly patients with previously untreated B-CLL observed in 7 Italian Centers from November 2000 to June 2012. All patients were treated with a median of 6 cycles of Bendamustine (range, 3-6) at the median dose of 90 mg/m2 (range, 70-90 mg/m2) for 2 consecutive days every 28 days plus RTX (375 mg/m2 for the first course and 500 mg/m2 for subsequent cycles every 28 days). The median number of RTX cycles was 6 (range, 3-6). The mean dose of RTX was 4500 mg (range, 1500-6200 mg). The primary end points were the ORR (complete response CR and partial response PR) and hematological-extrahematological toxicities rate.Twenty male and four female with a median age of 72 years (range, 65-87 years) were included in the study. Only one patient was unfit with a CIRS score of 7. All patients had ECOG less than 2. Two B-CLL patients had A/I progressive stage according Binet and Rai, 10 patients had B/II and 12 patients had C/III or C/IV. The median lymphocytes count at diagnosis was 37.040/mmc (range, 2.200-140.000). FISH analysis was performed in 19/25 patients: 12 patients were classified as standard risk (normal karyotype, del13q14 or +12) and 7 patients as high risk (del11q and del17p). The analysis of the IgVH, available in 12 patients, showed 7 patients with somatic mutation and 5 patients with germ-line sequences. Only one patient was admitted to the hospital and one received reduced bendamustine dose for neutropenia. Fifteen patients received bendamustine at the dosage of 90 mg/m2 while 9 were treated with 70 mg/m2. The ORR rate was 87.5%: ten patients (41.7%) obtained a complete response and eleven patients (45.8%) obtained a partial response. Among biological features the presence of standard risk FISH karyotype showed a statistical significance in terms of better response to therapy (p= 0.013) and progression (p=0.034). Hematological toxicity was recorded in 7 patients (29%) (neutropenia grade III/IV), 5 of them required G-CSF. Extra-hematological toxicity grade I-III was noticed in 8 patients (3 skin reactions, 3 infusion related reactions, 2 nausea and vomiting). At the present only four patients showed a progressive disease with a PFS of 92% at 12 months. Only one unresponsive patient died from Richter disease 6 months after the end of therapy. When we stratified patients in two groups according to the age, we found that patients younger than 75 years (15 patients) showed a better response (p=0.004) and a delayed time to progression (p=0.027) in comparison to patients more than 75 years.Retrospective data from this group of elderly B-CLL patients indicate Benda-R front-line provide a high response rate and a good safety profile. Also in a subgroup of very elderly patients (age > 75 years) the association of bendamustine 90 mg/m2 and rituximab at standard dose is recommended because of a low rate of dose delay/reduction and acceptable hematological/extrahematological toxicities. Disclosures:No relevant conflicts of interest to declare.

Prospective, Observational Study To Assess The Safety Of Rituximab In Combination With Chemotherapy In Patients With Previously-Untreated Or Relapsed Or Refractory B Cell-Lineage Chronic Lymphocytic Leukaemia (B-CLL): Preliminary Results

Aim and BackgroundThe efficacy and safety of rituximab plus chemo (R+chemo) for first-line and relapsed B-CLL patients(pts) have been widely investigated in clinical trials and large patient cohorts, but much less is known about whether such regimens can be effectively and safely administered to unselected pts in the community setting to reflect the routine care of B-CLL pts. Therefore, this observational study was designed to characterize the type, severity and frequency of all adverse events occurring on-treatment and in the year following rituximab infusion. A secondary objective was to assess response rate (CR/nPR/PR), duration of response, disease-free survival, overall survival, and to evaluate the relationship between various baseline markers and clinical outcome parameters in a subset of pts in each study group. MethodsData were collected prospectively from B-CLL pts, in 47 Spanish hospitals, commencing a new treatment with prescribed R+chemo in accordance with normal clinical practice at the time of enrollment. Data cut-off for this interim analysis was May 31, 2013. ResultsA total of 218 pts have been enrolled, 108 and 110 pts in the first-line and relapsed arm, respectively. The median age (range) of the whole series was 69 (r: 29–87) years. ECOG status varied across the two groups, with a 1 ECOG status score of 23%, and 55% of first-line and relapsed pts, respectively. In the first-line arm, 26%, 47%, and 27%, had a Binet stage of A, B and C, respectively. In the relapsed arm, 11%, 42%, and 46% had a Binet stage of A, B and C, respectively. By first-line / relapsed arm, the proportion of pts with del(11q), del(17p), and unmutated IgVH) was: 14%/15%; 17%/10%; and 10%/9%, respectively. Treated pts were categorized as receiving fludarabine/cyclophosphamide/rituximab (FCR) 60%/20%, bendamustine/rituximbab (BR) 15%/42%, chlorambucil/rituximbab (ClbR) 12%/19%, or other R-based therapy 13%/20%, in the first-line and relapsed arm, respectively.The overall response rate (ORR) in the first-line arm was as follows: BR cohort (81%; 13/16), ClbR (69%; 9/13) and FCR cohort (68%; 44/65), while in the relapsed group the ORR was higher in the ClbR cohort (55%; 11/20), followed by other R-based (48%; 10/21) and FCR cohort (43%; 9/21).Adverse events were most frequently hematologic and infusion-related and included nuetropenia (34% first-line; 28% relapsed); fever (19%; 12%); nausea (14%; 11%); lymphopenia (12%; 9%), and vomiting (11%; 8%). CTC grade 3-5 adverse events occurred in 55% of all 218 pts. Grade3 and 4 neutropenia occurred in 23% (FCR), 25%(BR) and 15% (ClbR) of first-line pts. In the relapsed group this incidence was of 14% (FCR), 16% (BR), and 25% (ClbR). The reported incidence of CTC grade 3 or 4 febtrile neutropenia (FN) in the first-line FCR, and BR cohort was of 9%, and 6%, respectively, while pts in the relapsed group the incidence of grade 3-4 FN was of 19% for FCR and 16% for BR. No FN was reported in the ClbR cohort, on both arms. Infection was reported in 61% and 17% of pts in the first-line/relapsed CFR cohort; 13% and 50% in the first-line/relapsed BR cohort, and 16% and 12% in the first-line/relapsed ClbR cohort, respectively.Treatment discontinuations due to AEs in the first-line and relapsed arm was of 12% and 9%, while treatment related mortality occurred in 3% and 4%, respectively. ConclusionIn this unselected group of B-CLL patients taken from normal clinical practice, in first-line, or relapsed approach, rituximab seems an effective and relatively safe option when added to any chemotherapy regimen. Additional analysis of the whole study will give us further information on late toxicity and outcome. Disclosures:Garcia Bernaldez:Roche Pharma: Employment. Gonzalez-Grande:Roche Pharma: Employment.

Glycoengineered CD20 Antibody Obinutuzumab Activates Neutrophils and Mediates Phagocytosis Through CD16B More Efficiently Than Rituximab

Background Obinutuzumab (GA101) is a third generation, glycoengineered defucosylated anti-CD20 antibody which shows higher binding affinity for CD16A than fully glycosylated rituximab (RTX). This higher binding leads to stronger NK mediated antibody dependent cellular cytotoxicity activity (ADCC) by obinutuzumab compared to that induced by RTX. The GPI-anchored CD16B molecule is highly homologous to CD16A and is the major FcγR on polymorphonuclear neutrophils (PMN). We have therefore investigated the binding of obinutuzumab to CD16B and its functional activity on human PMN compared to parent rituximab (RTX) or to defucosylated rituximab (G2 antibody). Methods Binding to CD16B of glycoengineered or fully glycosylated anti-CD20 antibodies was measured by surface plasmon resonance (Biacore). For PMN activation and phagocytosis, we have used either purified PMN or analyzed PMN function in unmanipulated whole blood assays from normal donors or CLL patients. PMN activation was measured as CD11b upregulation and CD62L downmodulation by flow cytometry. Phagocytosis by PMN of chronic lymphocytic leukemia (CLL) cells was measured by triple fluorescence (PKH26, CD15-FITC and CD19-APC) and flow cytometry. Results Obinutuzumab or glycoengineered defucosylated rituximab (called G2) bound CD16B with about 7 fold higher affinity, compared to non-glycoengineered wild type parental antibodies. This was true either using surface plasmon resonance or measuring antibody binding to live PMN. Furthermore obinutuzumab activated PMN, either purified or in whole blood, more efficiently than RTX. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on PMN and in release of TNFα, IL-6 and IL-8. Activation was not accompanied by generation of reactive oxygen species or ADCC, but led to phagocytosis of anti-CD20 antibody opsonized CLL targets by purified PMN. Indeed up to 50% phagocytic PMN could be observed in presence of obinutuzumab or G2 antibodies after 6-24 hours incubation of purified PMN with CLL targets. Significant phagocytosis (15%) was also observed in whole blood, but only in presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using blocking F(ab) and F(ab’)2 fragments specific for CD16B and CD32A, that both these receptors are involved in PMN activation, phagocytosis and cell death induced by glycoengineered anti-CD20 antibodies. The possible effect of NA1 and NA2 polymorphisms of CD16B on obinutuzumab binding and phagocytosis is under further investigation. Conclusions We conclude that phagocytosis by PMN is an additional mechanism of action of obinutuzumab, mediated through its higher binding affinity for CD16B compared to RTX. Phagocytosis takes place in whole blood and is followed by PMN death. This effect may in part explain the neutropenia observed after treatment of B-CLL patients with GA101. Disclosures: Golay: Roche Glycart AG: Research Funding. Ferrara Koller:Roche Glycart AG: Employment. Rambaldi:Roche Italia: Consultancy, Honoraria. Klein:Roche Glycart AG: Employment. Introna:Roche Glycart AG: Research Funding.

Donor Vδ1+ γδ T cells expand after allogeneic hematopoietic stem cell transplantation and show reactivity against CMV-infected cells but not against progressing B-CLL

γδ T lymphocytes play an important role in immune reactions towards infections and malignancies. In particular, Vγ9–Vδ1+ T lymphocytes are thought to play protective antiviral roles in human CMV infection. Recently, Vδ1+ T lymphocytes were proposed to also have anti- B-CLL reactivity. Here we report a case of 48-year-old man who received allogeneic stem cell transplantation for progressive B-CLL. Within one year after transplantation, lymphoma relapsed despite a dramatic increase of Vδ1+ T cells in the patient’s blood. In vitro killing assays revealed activity of patient’s γδ cells against CMV target cells, but not against the relapsing lymphoma-cells. This argues for a contribution of Vδ1+ cells in the immune reaction against CMV reactivation, but does not support a strong correlation of expanded Vδ1+ T cells and favorable disease outcome in B-CLL patients.

Abstract 932: Predictive markers of the in vitro anticancer effect of the pan class I PI3K inhibitor BKM120 in primary B-CLL lymphocytes.

Abstract B-chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B-lymphocytes that are long-lived in-vivo but die quickly by apoptosis when cultured in-vitro. The phosphatidylinositol-3 kinase (PI3K) cascade which is a critical component of survival signaling has increased activity in CLL lymphocytes as compared to normal B lymphocytes. There are three classes of PI3Ks of which class I is the most clearly implicated in human cancer. The PI3K-δ inhibitor, CAL-101, promotes apoptosis and abrogated protection from spontaneous apoptosis induced by CD40 in primary CLL lymphocytes in-vitro. Thus the PI3K pathway appears to play a critical role in B-CLL cell survival. BKM120 is a pan class I PI3K inhibitor developed by Novartis. Phase I trials demonstrated that plasma concentrations (Cmax) of 5 μM can be obtained. In view of the critical role of PI3K in CLL homeostasis, the activity of BKM120 was examined in CLL lymphocytes in-vitro. BKM120 was cytotoxic in the 3 CLL cell lines and in 78% of the primary B-CLL lymphocytes samples isolated from 65 B-CLL patients, including patients with del11 and del17. Interestingly, 60% of the B-CLL samples tested in our study have an IC50 below the Cmax. Furthermore, BKM120 cytotoxicity correlated with the basal expression of proteins involved in the PI3K/Akt pathway (Akt, rictor, raptor, p70S6K and 4E-BP1) but not with PTEN, mTor, IgVH or CD38 expression in these primary B-CLL lymphocytes. Further analysis of these different markers demonstrated that only patients very sensitive to BKM120 (IC50 ≤3 μM) expressed low basal levels of mTor, raptor and p70S6K simultaneously. Inhibition of PI3K was demonstrated by detection of phosphorylated Akt (S473) after BKM120 treatment in the cell lines. Molecular biomarkers such as p70S6K and 4E-BP1 have been used as indicators of PI3K pathway inhibition in-vivo. We also have demonstrated that BKM120 decreased the phosphorylation status of 4E-BP1 and p70S6K in the primary B-CLL lymphocytes tested. As detected by AnnexinV/propidium iodine staining and analysis of caspase-3 cleavage, BKM120 induced apoptosis in primary B-CLL cells culture in the presence and absence of stromal cell support. Taken together, our results demonstrated that the class I PI3K inhibitor BKM120 may be useful as a single agent in CLL patients independently of their IgVh mutational status, CD38 expression or genomic deletions (del11 and del17). Furthermore, a combination of different biomarkers (expression of mTor, raptor, and p70S6K) may predict the response to BKM120 treatment. Also, BKM120 abolished the protection against apoptosis and drug resistance conferred by the microenvironment to the primary B-CLL lymphocytes in-vitro. Our preclinical study demonstrated that BKM120, a molecule with acceptable clinical toxicity, may be useful in the treatment for CLL. Citation Format: Lilian Amrein, May Shawi, Jeremy Grenier, Raquel Aloys, Lawrence Panasci. Predictive markers of the in vitro anticancer effect of the pan class I PI3K inhibitor BKM120 in primary B-CLL lymphocytes. [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 932. doi:10.1158/1538-7445.AM2013-932

Rai stage-related changes within T/NK cell populations from B-CLL patients

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Prognostic significance of flow cytometric quantification of circulating endothelial cells in chronic lymphocytic leukemia

Background Accumulating evidences have supported the role of angiogenesis in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). Detection of peripheral blood circulating endothelial cells (CECs) by flow cytometry is proposed to be a noninvasive indirect marker of angiogenesis. This work aimed to quantify CECs and endothelial progenitor cells (EPCs) by flow cytometry in patients with newly diagnosed CLL compared with healthy individuals, study their relationship with established risk predictors of the disease, and assess their prognostic significance. Materials and methods Flow cytometric quantification of CECs and EPCs was carried out for 50 newly diagnosed B-CLL patients and 20 healthy controls. Patients were followed up for assessment of the time to first treatment, response to therapy, and disease outcome. Results Patients with CLL had higher counts of CECs (median, 24.6×10 6 /l) and EPCs (median, 22.7×10 6 /l) compared with the controls (median, 2.8 and 1.9×10 6 /l for CECs and EPCs, respectively; P P >0.05), they were significantly related to higher white blood cell counts ( P P Conclusion This study shows that flow cytometric detection of peripheral blood CECs is a feasible indicator of abnormal angiogenesis in CLL that might be utilized as a biologic prognostic marker of a more aggressive disease course with a poor clinical outcome.

Advocating the need of a systems biology approach for personalised prognosis and treatment of B-CLL patients

The clinical course of B-CLL is heterogeneous. This heterogeneity leads to a clinical dilemma: can we identify those patients who will benefit from early treatment and predict the survival? In recent years, mathematical modelling has contributed significantly in understanding the complexity of diseases. In order to build a mathematical model for determining prognosis of B-CLL one has to identify, characterise and quantify key molecules involved in the disease. Here we discuss the need and role of mathematical modelling in predicting B-CLL disease pathogenesis and suggest a new systems biology approach for a personalised therapy of B-CLL patients.

S1P1 expression is controlled by the pro-oxidant activity of p66Shc and is impaired in B-CLL patients with unfavorable prognosis

AbstractAlthough intrinsic apoptosis defects are causal to the extended survival of chronic lymphocytic leukemia (CLL) B cells, several lines of evidence support a contribution of the peripheral lymphoid organs and BM microenvironment to the extended lifespan of leukemic B cells. Lymphocyte trafficking is controlled by homing signals provided by stromal cell–derived chemokines and egress signals provided by sphingosine-1-phosphate (S1P). In the present study, we show that expression of S1P1, the S1P receptor responsible for lymphocyte egress, is selectively reduced in CLL B cells with unmutated IGHV. Expression of S1P2, which controls B-cell homeostasis, is also impaired in CLL B cells but independently of the IGHV mutational status. We provide evidence herein that p66Shc, a Shc adaptor family member the deficiency of which is implicated in the apoptosis defects of CLL B cells, controls S1P1 expression through its pro-oxidant activity. p66Shc also controls the expression of the homing receptor CCR7, which opposes S1P1 by promoting lymphocyte retention in peripheral lymphoid organs. The results of the present study provide insights into the regulation of S1P1 expression in B cells and suggest that defective egress caused by impaired S1P1 expression contributes to the extended survival of CLL B cells by prolonging their residency in the prosurvival niche of peripheral lymphoid organs.

Overexpression of Deleted Lymphocytic Leukemia 1 (DLEU1) Inhibits Proliferation of Burkitt Lymphoma (BL).

AbstractAbstract 2654 Background:Burkitt Lymphoma (BL) represents approximately 40% of all childhood and adolescents Non Hodgkin Lymphoma (Cairo et al., Blood, 2007; Miles/Cairo, BJHaem., 2012), and pediatric BL patients with chromosome del13q14.3 region had significantly poorer outcome and inferior overall survival despite aggressive short, intensive multiagent chemotherapy in subset analysis (Poirel/Cairo et al., Leukemia, 2009; Nelson/Cairo/Sanger et al., BJHaem., 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is often deleted in B-CLL patients (Liu et al., Oncogene, 1997; Wolf et al., Hum. Mol. Genet., 2001) with the BL classifier genes (Dave et al., NEJM. 2006) on chromosome 13q14.3 region, is recognized to interact with Tubulin beta 2C (TUBB2C), RASSF1A, and E3 ubiquitin-protein ligase (UBR1), and p53 and their mRNA expression levels were significantly higher in BL than in diffuse large B-cell lymphoma (DLBCL) (Day/Cairo et al., AACR, 2008). DLEU1 may have function as a tumor suppressor gene, however, functions and mechanisms underlying DLEU1 expression in the cells are poorly understood. OBJECTIVE:We propose that DLEU1 may act as a tumor suppressor gene and sought out to examine whether gains of overexpression of DLEU1 in embryonic kidney cells and Burkitt Lymphoma cells result in inhibition of tumor proliferation METHODS:In order to get a full length of DLEU1 cDNA, total RNA was prepared from embryonic kidney epithelial 293T and Raji Burkitt lymphoma cells using Trizol (Invitrogen), and cDNA was synthesized by qScript™ cDNA Synthesis Kit (Quantas). The full length of cDNA encoding DLEU1 was fused into pEGFP-N3 vector. pEGFP-DLEU1fusion construct was transfected into 293T and Raji cells using lipofectamin or Amaxa Nucleofector™ kit V, respectively, followed by the manufacturer’s instruction. Twenty-four (293T) or Forty-eight hours (Raji) post transiently transfection, total RNA was extracted and 1ug of total RNA was used for cDNA synthesized as above. For comparison of mRNA expression of network genes, quantitative RT-PCR was performed using SsoFast™ EvaGreen® Supermix reagent by CFX96 Real-time system (Bio-rad) and statistical significance was performed by one-tailed Student t-test. For observation of expression of DLEU1 fusion protein, GFP only as control and GFP-DLEU1 transiently overexpressed 293T and Raji cells were observed under fluorescent microscope, and total lysates were prepared from both cells at 24 hours (293T) and 48hours (Raji) post-transfection and were hybridized with Rabbit polyclonal DLEU1 anitbody (Proteintech) on immune blotting. For cell proliferation assay, GFP-DLEU1 transiently overexpressed 293T and Raji cells were re-plated at 24hours post-transfection, and then counted every 24hrs. RESULTS:DLEU1 full length cDNA (234nt, 78amino acid) successfully fused into pEGFP expression vector and observed GFP fluorescence under fluorescent microscope in both 293T and Raji cells, and the predicted size of the fusion protein was confirmed by western blot (approximately 36kDa) [Figure 1]. The level of DLEU1 mRNA expression in GFP-DLEU1 transiently transfected 293T and Raji cells was significantly higher than GFP only transfected Raji cell as control [50% increase (p<0.05) and 25% increase (p<0.005), respectively]. In comparison of mRNA expression of DLEU1 network genes in DLEU1 overexpressed 293T cells, there were no significant differences in mRNA expression of c-myc compared to control, however, there was significantly increased expression of RASSF1 and UBR1 mRNA (>1.5-fold, p<0.03 and >1.2-fold, p<0.02), respectively, and significantly reduced expression of TUBB2C mRNA (<1.2-fold, p<0.03) in DLEU1 overexpressed 293T cells. In addition, in DLEU1 overexpressed Raji cells, there was no significant differences in c-myc mRNA similar to 293T cells, however, there was significantly increased expression of UBR1 mRNA (>1.1-fold, p<0.05), and significantly reduced expression of RASSF1 and TUBB2C mRNA (<1.4-fold, p<0.02 and <1.5-fold, p<0.03) in DLEU1 overexpressed Raji cells. DLEU1 transiently overexpressed cells showed an inhibition of cell proliferation in both 293T and Raji cells [60% reduction (p<0.05) at day 4 and 20% reduction (p<0.01) at day 5, respectively]. CONCLUSION:These results suggest that DLEU1 may act as a tumor suppressor gene and that following overexpression of DLEU1 resulted in inhibition of cell proliferation in Burkitt Lymphoma. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Single Cell Network Profiling (SCNP) Identifies Altered Signaling Between Patient Risk Groups in B-Cell Chronic Lymphocytic Leukemia (B-CLL).

AbstractAbstract 2876 Background:B-CLL follows a variable clinical course, with a subset of patients progressing quickly. The reasons for this outcome disparity are not fully understood; however, evidence suggests that B-cell receptor (BCR) signaling is a driving event in disease onset and progression. B-CLL cells also receive survival signals through additional receptors. SCNP is a multiparametric flow cytometry-based assay that measures, quantitatively at the single cell level, changes in intracellular signaling proteins in response to extracellular modulators. This provides a functional measure of pathway activity and intraclonal signaling differences within the larger B-CLL cell population. In prior studies, we observed higher αIgM-induced (→) p-ERK signaling in B-CLL samples from patients who had a shorter time to first treatment (TTFT) (Cesano et al. ASH 2011 Abstract 2834). Herein we examined the feasibility for SCNP to further define patient risk stratification. Objectives:The current study was designed to 1) map signaling profiles in early-stage B-CLL and to 2) identify signaling associations with clinical subgroups defining B-CLL prognosis (IGHV mutational status, cytogenetic risk, CD38 / ZAP-70 expression). Methods:Between 2006 and 2007, peripheral blood mononuclear cells (PBMCs) were collected and cryopreserved from a cohort of 39 untreated B-CLL patients, Rai stage 0 - II, at different time points during their clinical course. Of the 39 samples evaluated; 15 and 20 expressed CD38 (≥30% of cells) and ZAP-70 (≥20% of cells), respectively; 19 were IGHV unmutated (98% cutoff); and cytogenetic risk groups were evenly represented. SCNP analysis quantitatively measured 22 intracellular signaling proteins within CD19+CD5+ B-CLL cells, using a panel of 14 disease-relevant modulators. The Wilcoxon rank-sum test was used to identify signaling associations with CD38 and ZAP-70 expression, cytogenetic risk categories, del17p (includes p53 gene deletion) and IGHV mutational status. Results:Significant associations between patient risk categories and signaling are summarized in Table 1. IGHV unmutated and ZAP-70+ samples showed (1) elevated α-IgM or α-IgD-induced BCR signaling, either alone or in combination, (2) decreased CpGβ → p-ERK induction and (3) increased thapsigargin-induced (Ca2+ signaling) signaling. Of note, CD38+ samples did not show the same associations, but shared with unmutated IGHV samples a higher responsiveness to IFNα and weaker induction of p21 in response to bendamustine. The unfavorable cytogenetic group samples showed increased αIgM→p-ERK and had higher basal p-S6 that further increased with IgD crosslinking. Lack of p21 induction was also associated with unfavorable cytogenetics, which includes deletion of p53 (del17p), a regulator of p21 expression.Table 1Signaling Associations to Patient Risk Groups (Wilcoxon p-value)Signaling NodeCD38 ≥ 30%IGHV UnmutatedZAP-70 ≥ 20%Unfavorable CGX (del11q22.3 and/or del17p13)Favorable CGX (13q14.3)↑ αIgM → p-ERK0.0130.0059↑ αIgM → p-LYN0.0030.0060↑ αIgM → p-PLCγ20.0140.0244↑ αIgM → p-SYK0.024↑ αIgM → p-ERK*0.0180.04850.013↑ αIgM+αIgD → p-AKT0.043↑ αIgM+αIgD → p-ERK0.00390.0018↑ αIgM+SDF1α → p-ERK0.00500.0074↑ αIgD → p-S60.01190.034↓ CpGβ → IkB0.030↓ CpGβ → p-ERK0.0170.0355↑ R848 → IkB0.0055↓ R848 → IkB0.031↑ IFNα → p-STAT10.0026↑ IFNα → p-STAT30.0159↑ IFNα → p-STAT50.00240.0047↑ IL2 → p-STAT50.008↑ Thapsigargin → p-AKT0.0470.0102↑ Thapsigargin → p-ERK0.00920.0018↓ Thapsigargin → p-ERK0.0448↓ Bendamustine → p210.00210.0250.0003↑ Fludarabine → p-H2AX0.0073*Measured at 60 minutes; all other αIgM modulations measured at 10 minutes. Conclusions:This is the second, independent SCNP analysis of B-CLL signaling showing decreased bendamustine→p21 and increased αIgM→p-ERK signaling in samples with unfavorable cytogenetics. Further associations with IGHV unmutated status included increased BCR signaling in multiple nodes, altered TLR9 responsiveness, and decreased drug-induced p21. These data support the potential utility of SCNP in: (1) identifying in one assay those patients with a more aggressive form of B-CLL, including both unmutated IGHV and p53 pathway alteration, and (2) identification of patients with signaling profiles that may be more likely to respond to targeted therapies. Disclosures:Ptacek:Nodality, Inc.: Employment, Equity Ownership. Evensen:Nodality, Inc.: Employment, Equity Ownership. Friedland:Nodality, Inc.: Employment, Equity Ownership. Cordeiro:Nodality, Inc.: Employment, Equity Ownership. Ware:Nodality, Inc.: Employment, Equity Ownership. Cesano:Nodality, Inc: Employment, Equity Ownership. Hawtin:Nodality, Inc.: Employment, Equity Ownership.

HLA-G5 in the blood of leukemia patients and healthy individuals

In this work we focused on analysis of HLA-G5 molecules in the blood of patients with B-CLL leukemia and healthy individuals. Using sandwich ELISA, we found total soluble HLA-G, represented by sHLA-G1 and HLA-G5 in most of B-CLL patients while HLA-G5 alone was present only in few cases in both groups. These results lead us to assume that the majority of soluble HLA-G in blood is generated by proteolytic cleavage and shedding of membrane-bound HLA-G1. There is no correlation between the presence of HLA-G5 in blood of B-CLL patients and the stage of disease, age, and gender.

A particular expression pattern of CD13 epitope 7H5 in chronic lymphocytic leukaemia – a possible new therapeutic target

A total of 50 patients with chronic lymphocytic leukaemia (CLL), as well as the B-cell leukaemia cell lines MEC-1, JVM-3, and BV-173 were studied in order to assess the incidence of CD13/aminopeptidase N (APN) immunolabelling with a monoclonal antibody 7H5 compared to LeuM7 and to CD13 mRNA levels, and to correlate these data with the cytotoxic and apoptosis-induction activity of the natural phenolic APN inhibitor curcumin. CD13/APN was detected in a significant proportion of B-CLL patients (42/50, 84%), immunolabelled by 7H5 (42/50) ± LeuM7 (10/50). Molecular analysis for CD13 transcripts confirmed these data, resulting in a specific RT-PCR product in CD13 positive cases. Curcumin showed concentration-dependent cytoreductive efficacy and apoptosis-induction activity in all tested cell lines and primary cultures from CLL mononuclear cells. There was a clear tendency for a better response in CD13 positive cases. The incidence of CD13/APN in CLL suggests that the inhibition of APN/CD13 by curcumin may be an effective new molecular target for a more efficient therapy for these patients and warrants further investigations.

Prognostic significance of del 17p, ZAP-70 and CD38 as independent indicators for B-CLL: correlation to response to treatment and disease outcome

Prognostic markers as CD38 and ZAP-70 and specific chromosomal abnormalities as del 17p have now been developed to refine the risk of progressive disease in chronic lymphocytic leukemia (CLL). This study analyzed 40 recently diagnosed, untreated B-CLL patients for CD38 and ZAP-70 expression by flow cytometry and for del 17p by conventional cytogenetics (CCG) and by fluorescence in situ hybridization (FISH) technique to evaluate their effect on the clinical course of CLL and as risk factors for disease progression in addition to their impact on response to treatment and disease outcome. Twenty healthy age- and sex-matched subjects were included as a control. The results revealed that CD38 and ZAP-70 expression were detected in 42.5% and 47.5% of cases, respectively. They were associated with an unfavorable clinical course. Higher levels were significantly associated with increased risk of unfavorable response to treatment (P=0.003), with poor clinical outcome (P=0.0001). Del 17p was detected in 35% of cases by FISH technique and in 7.5% by CCG. The deletion was significantly associated with progressive clinical course; poor response to treatment (P=0.007) but not with disease outcome (P=0.103).Combined analysis of ZAP-70 and CD38 yielded concordantly negative results in 50% of patients and concordantly positive results in 40% of patients, while 10% were discordant. CD38+/ZAP-70+ patients were significantly associated with progressive disease (P<0.05) and with del 17p than CD38−/ZAP-70- patients (P=0.008).Time to disease progression (TDP) was 6months among CD38+/ZAP-70+ patients as compared to 16months in CD38−/ZAP-70− patients. In patients with discordant results, the TDP was 9months. Over-representation of the three parameters (CD38, ZAP-70 and del 17p) was detected in 22.5% of cases, and pointed towards even shorter TDP (4.5months), more aggressive disease; more resistance to chemotherapy and poor outcome thus providing a precise tool for identifying high-risk patients.In conclusion, the combined expression of CD38 and ZAP-70 together with del 17p in CLL is a precise diagnostic tool for identifying high-risk patients and convey rapid progression; they are accurate predictors of clinical outcome thus could be used to indicate when more novel chemotherapeutic approaches are needed and provided help in guiding individual patient treatment.

Alemtuzumab induction of intracellular signaling and apoptosis in malignant B lymphocytes

The molecular changes induced by alemtuzumab following binding of CD52 on B tumor cells were investigated. Alemtuzumab alone had no detectable impact on cell signaling but cross-linking of alemtuzumab on the surface of B tumor lines with anti-human Fc antibodies induced a transient Ca2 + flux followed by phosphorylation of several kinases involved in stress and survival pathways, and expression of associated proteins including TNF-α. Cross-linking of alemtuzumab also induced capping and caspase-dependent apoptosis of the tumor lines. When using primary cells from B-CLL patients, alemtuzumab alone was capable of inducing protein phosphorylation and apoptosis through the cross-linking of alemtuzumab by FcγRIIb receptors on B-CLL cells. Apoptosis was prevented by blocking of FcγRIIb receptors with anti-CD32 antibody. Overall, our results indicate that cross-linking of alemtuzumab on B tumor cells can occur naturally through Fc receptor interaction and leads to the activation of specific cellular pathways and induction of apoptosis.

Erratum to: Post-transcriptional silencing of Notch2 mRNA in chronic lymphocytic leukemic cells of B-CLL patients

Erratum to: Post-transcriptional silencing of Notch2 mRNA in chronic lymphocytic leukemic cells of B-CLL patients