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In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 2723-2723

Abstract: Abstract 2723 With the advent of highly effective chemo-immunotherapy regimens, the treatment of chronic lymphocytic leukemia (CLL) is no longer simple palliation but aims to achieve a minimal residual disease (MRD)-negative remission. Historically, CD5 and CD19 with demonstration of clonality was the mainstay of MRD evaluation in CLL. However, this approach is limited by the presence of normal B1-cells (CD5+CD19+) and the inability to demonstrate light-chain restriction with very low B-cell numbers. More recently, an international standardized approach (ISA) to the assessment of MRD in CLL was published with a detection accuracy of 95.7% for the detection CLL above 0.01%, involving 8 monoclonal antibodies (mAbs) in a 5 test system.1 CD160 is an activatory NK cell receptor, but is not expressed on normal B-lymphocytes, B1-cells, naive B-cells or activated B-lymphocytes. CD160 is expressed on malignant B-cells in 〉 98% of cases of CLL, but in only 15% of non-CLL cases. This makes CD160 a unique marker for the assessment of MRD. Methods: Utilizing the anti-CD160 monoclonal antibody (BY55, Coulter Immunotech, Marseille, France), we have developed a highly sensitive CD160 Flow Cytometric Assay (CD160FCA) incorporating CD2, CD5, CD19, CD23 and CD160. Whole blood (1×106 leukocytes) were labeled with mAbs, followed by an ammonium chloride-based lysing method. Data acquisition was captured using a sequential gating strategy focusing only on the malignant population. The CD160FCA was evaluated in two centers and the results compared with the ISA methodology in two different centers. Results: As expected, light-chain restriction could not be reliably measured in patients with very low B-cell numbers. 23 cases were analyzed at four centers. Concordant results were found in 22/23 cases with a sensitivity of 1.00 (P=0.0023, OR 91.00). Of the 23 patients 87% were MRD positive by the CD160FCA compared to 82% by the ISA. There were no cases that were MRD positive by the ISA and negative by the CD160FCA. There was a close correlation between the two methodologies for disease levels above 0.01% (Spearman Rank R=0.9913, P 〈 0.0001). When studying the median fluorescence intensity (MFI) of CD160, the MRD positive cases had a mean MFI of 374 (288-461 95%CI) and the negative cases 47 (2-93 95%CI) (Figure 2). The CD160 FCA offers a single tube analysis for MRD, which is highly sensitive, independent of the therapy and can be employed throughout treatment. Importantly, not only is this approach simpler, cheaper and faster than current MRD approaches, it is still informative in cases where no light chain clonal population is found, or where restriction studies have failed. With increasing evidence that MRD detection in CLL can predict patient outcome, CD160FCA represents a simple tool for MRD assessment. Reference: Rawstron AC, Villamor N, Ritgen M et al. International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia. Leukemia. 2007;21(5):956-964. Disclosure: No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V116.21.2723.2723

Language: English

Publisher: American Society of Hematology

Publication Date: 2010

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 2170-2170

Abstract: Biosimilar filgrastim is now widely used for haematopoietic stem cell mobilisation in Europe. Previous studies have reported differences in mobilisation efficacy between originator filgrastim and lenograstim, although others have reported comparable efficacy. This is the first study to compare biosimilar filgrastim with lenograstim for autologous haematopoietic stem cell transplant (HSCT). We also report our use of biosimilar filgrastim for mobilisation in sibling allogeneic transplant, for which there is limited previous data. Methods Data from patients with lymphoma or multiple myeloma (MM) who underwent autologous HSCT mobilised with biosimilar filgrastim (HX575) between October 2011 and April 2013 at St Bartholomew's Hospital, London, were compared with a historical control group of patients who underwent HCST using a similar mobilisation protocol with lenograstim from January 2009 to September 2011. Peripheral blood (PB) cells counts (white blood cell [WBC] and CD34+ cells) were monitored after 7–8 consecutive days of G-CSF injection (approximately 5 μg/kg) and apheresis was performed on day 8 if PB CD34+ cell count was ≥10 cells/µl. G-CSF administration and apheresis were then performed daily until a PB CD34+ cell dose of ≥2.0 x106/kg (lymphoma), ≥ 4.0 x106/kg (MM ≥60 years old) or ≥ 8.0 x106/kg (MM 〈 60 years old) was achieved. Data from a separate group of sibling donors and recipients with haematological malignancies who underwent allogeneic HSCT between October 2010 and April 2013 are also reported. Results A total of 259 patients were included in the autologous HSCT comparison (biosimilar filgrastim, n=104; lenograstim, n=155). Both groups had similar characteristics (overall, 66% male, median age 56 years) although the biosimilar group had a lower percentage of patients with lymphoma (19% vs 35%). In patients with lymphoma and older MM patients (≥60 years old), no significant differences were observed between groups with regard to stem cell mobilisation parameters. However, in MM patients 〈 60 years old, all parameters were significantly superior in the biosimilar filgrastim group compared with lenograstim, including the need for one rather the two aphereses (Table 1). Among patients who proceeded to transplant, no significant differences were observed between biosimilar filgrastim and lenograstim in median number of days to ANC recovery 〉 0.5 x109/l (lymphoma: 13 [9, 35] vs 13 days [9, 36] ; MM: 14 [9, 34] vs 12 [10, 33] days) or platelet recovery 〉 20 x109/l (lymphoma: 21 [9, 35] vs 23 days [10, 35] ; MM: 19 [9, 38] vs 18 [9, 39] days). In the allogeneic setting, 48 sibling donors received biosimilar filgrastim. Mean CD34+ count at the first apheresis was 6.1 x 106/kg. Thirteen donors needed a second apheresis, four of whom required a third. Among the recipients, median days to ANC recovery was 16 (10–28) and to platelet recovery was 13 (9–54). Conclusions Biosimilar filgrastim is as effective as lenograstim for autologous HSCT in patients with lymphoma or MM patients ≥60 years old. However, mobilisation with biosimilar filgrastim appeared to be superior to that with lenograstim in younger MM patients. Biosimilar filgrastim was also successfully used to mobilise sibling donors for allogeneic transplantation. Disclosures: Agrawal: Sandoz Biopharmaceuticals: Consultancy, Honoraria, Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V122.21.2170.2170

Language: English

Publisher: American Society of Hematology

Publication Date: 2013

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detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 5305-5305

Abstract: IL-6 in the cancer microenvironment plays a crucial role in the constitutive activation of STAT3, which contributes to cancer cell progression. IL-6 binds to a heterodimer of membrane-bound receptors, IL-6R (CD126) and gp130, that triggers phosphorylation of JAK2 and downstream STAT3. The phosphorylated STAT3 acts as a transcription factor, binding target DNA and promoting tumorigenesis, cell proliferation and survival. IL-6 signaling pathways have been extensively studied as therapeutic targets in both cancer and autoimmune conditions. The anti-IL6R antibody Tocilizumab is a humanized anti CD126 antibody, approved by the FDA (Food and Drug Administration) for treatment of rheumatoid arthritis, that can block IL-6 classic and trans-signalling pathways. We investigated IL-6R expression in CLL cells and analysed the correlation of IL-6R expression with chemo-sensitization and the potential of targeting IL-6R to overcome drug resistance in vitro. 41 CLL patients were studied. Membrane-bound and soluble IL-6 receptors, CD126 and gp130, were measured by flow cytometry or ELISA. Constitutive activation of STAT3 and the expression of Bcl-2 family proteins were detected by western blotting. Cell cycle distribution was assessed with Ki67 and cell viability indicated by propidium iodide (PI) negative staining. CLL cells were found to express variable levels of membrane-bound receptors of IL-6, CD126 (average 22%, range 3-60%) and gp130 (average 12%, range 3-25%), which was significantly higher than normal B-cells (CD126 average 1.7%, range 0-6.3%; gp130 average 1.3%, rage 0-7.9%). Although in vitro Chlorambucil-induced cell death was variable among the cases, there was a strong correlation with CD126 expression (Figure 1): higher CD126 expression was associated with increased resistance to Chlorambucil. Treatment of CLL cells with Tocilizumab led to inhibition of constitutive STAT3 activation decreased Mcl-1, Bcl-xl expression and cells exited from G0 phase of the cell cycle. Tocilizumab did not induce cell death in CLL cells. Combined treatment with both Chlorambucil and Tocilizumab significantly increased cell death (p 〈 0.01). Interestingly, higher CD126 expression was highly significantly correlated with the increased cell death seen with combined Chlorambucil and Tocilizumab (p 〈 0.0001, Figure 2) These results suggest that inhibiting IL6 signal pathways via IL-6R blockade with Tocilizumab is a new approach in CLL, leading to increased chemo-sensitisation to standard chemotherapeutic agents. Although CLL cells with higher CD126 expression exhibited more drug resistance in vitro, they also showed better response to Tocilizumab. Targeting the combination of Tocilizumab with anti-cancer therapy to CLL patients with high IL-6R expression might achieve clinical therapeutic efficacy. Disclosures: No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V122.21.5305.5305

Language: English

Publisher: American Society of Hematology

Publication Date: 2013

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 3646-3646

Abstract: Background: Adult patients undergoing remission-induction (RI) chemotherapy for newly diagnosed acute lymphoblastic leukaemia (ALL) are at high risk of invasive fungal disease (IFD), particularly invasive aspergillosis. The prevalence of IFD in this population has not been widely studied. There is currently no approved standard for antifungal prophylaxis and it is recommended by the European Working Group for Adult ALL that azole antifungal agents be avoided because of drug interactions with vincristine, a standard component of induction chemotherapy regimens. Methods: This was a double-blind, multicentre, placebo-controlled study in patients aged ≥18 years receiving RI chemotherapy for newly diagnosed ALL. Study subjects received either liposomal amphotericin B (L-AMB) 5mg/kg or placebo by IV infusion twice weekly in a 2:1 random allocation. The primary endpoint of the study was the proportion of subjects with proven or probable invasive fungal infection (IFI) assigned in a blinded review by an independent data review board (IDRB) using EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) definitions (1). Subjects were closely monitored for signs and symptoms of IFI, including biweekly galactomannan and β-D-glucan assessments. Clinical, laboratory or radiographic findings suggestive of IFI prompted further investigation according to a specified protocol. Subjects with defined clinical and mycological criteria or a halo sign on chest CT had prophylaxis interrupted and pre-emptive antifungal therapy initiated while further diagnostic procedures were undertaken. Prophylaxis was stopped when subjects met protocol-specified criteria for proven or probable IFI and appropriate antifungal therapy started. Secondary objectives included safety and tolerability of prophylactic L-AMB and the impact of IFI prevention on the efficacy of RI chemotherapy. Results: A total of 355 subjects from 83 centres in 13 countries in Europe (including Turkey and Israel) and South America were randomised and received at least 1 dose of either L-AMB (n=237) or placebo (n=118). Sixteen subjects were excluded from the efficacy analysis, 1 due to misdiagnosis (AML) and 15 due to protocol-prohibited antifungal treatment. 7.9% (18/228) subjects in the L-AMB group and 11.7% (13/111) subjects in the placebo group were considered by the IDRB to have experienced a proven or probable IFI (p=0.24, RR 0.33 [CI -0.32 to 0.66]). Of these, 1 case (0.4%) in the L-AMB group and 3 cases (2.7%) in the placebo group were considered proven. In subjects who were neutropenic (absolute neutrophil count 〈 0.5x109/L) ≥10 days, the incidence was 6.9% (12/174) in the L-AMB group versus 13.1% (11/84) in the placebo group (p=0.10). Overall mortality was similar: 7.2% (17/237) in the L-AMB group vs 6.8% (8/118) in the placebo group (p=1.00) as was the incidence of serious adverse events (SAEs; 33.3% (79/237) vs 32.2% (38/118), [p=0.90]). SAEs of any grade related to the study drug were reported for 8.4% subjects (20/237) in the L-AMB group compared with 1.7% subjects (2/118) (p=0.02) in the placebo group. The most common ( 〉 5%) study drug-related adverse events (AEs) were hypokalaemia (10.5% [25/237] vs 3.4% [4/118] p=0.02) and increased creatinine (6.3% [15/237] vs. 0% [p=0.003] ). Complete remission at the end of RI chemotherapy was achieved by 72.6% (172/237) subjects in the L-AMB group versus 78.8% (93/118) subjects in the placebo group (p=0.24). Conclusions: In this study, the largest of its kind to date, the rate of IFI among adult patients undergoing RI chemotherapy for newly diagnosed ALL was 11.7% in the placebo group. Study subjects who received L-AMB at 5mg/kg twice weekly for prophylaxis showed a reduction in IFIs relative to placebo (RR 0.33 [CI -0.32 to 0.66]), although this did not reach statistical significance (p=0.24). L-AMB prophylaxis was generally well tolerated. The incidence of IFI in patients with ALL receiving RI therapy indicates that antifungal prophylaxis is merited in this patient population. Further analysis is needed to determine which patients might benefit the most. clinicaltrials.gov number: NCT01259713 (1) De Pauw et al 2008 Clin Infect Dis 46: 1813–1821 Disclosures Cornely: Gilead Sciences: Consultancy, Research Funding, Speakers Bureau. Off Label Use: Liposomal Amphotericin B (AmBisome®) is a broad spectrum antifungal agent indicated for the treatment of severe systemic and/or deep mycoses, empiric treatment of presumed fungal infections in febrile neutropenic patients and treatment of visceral leishmaniasis. In this study the efficacy, safety and tolerability of prophylactic liposomal amphotericin B (AmBisome®) was explored for the prevention of invasive fungal infections in subjects receiving remission-induction chemotherapy for Acute Lymphoblastic Leukaemia (the AmBiGuard trial).. Leguay:Gilead Sciences: Research Funding. Maertens:Gilead Sciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Castagnola:Gilead Sciences: Research Funding. Anagnostopoulos:Gilead Sciences: Research Funding. Allione:Gilead Sciences: Research Funding. Heussel:Gilead Sciences: Consultancy, Speakers Bureau. Donnelly:Astellas, Gilead, Merck, Pfizer: Consultancy, Honoraria, Research Funding. Agrawal:Gilead, Pfizer, Astellas, MSD, Bio-Rad: Honoraria, Research Funding. Garner:Gilead Sciences: Employment, Equity Ownership. Simcock:Gilead Sciences: Employment, Equity Ownership. Hawkins:Gilead Sciences: Employment, Equity Ownership. Goekbuget:Gilead Sciences: Consultancy, Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V124.21.3646.3646

Language: English

Publisher: American Society of Hematology

Publication Date: 2014

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detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 4124-4124

Abstract: Expression of CD160 on normal immune cells is primarily observed on human and murine circulating cytotoxic lymphocytes (NK cells and a small subset of T-cells). CD160 exhibits a broad specificity for major histocompatibility complex molecules. While normal B-cells do not express CD160, malignant B-cells of CLL (and HCL) are CD160+.1 Triggering of CD160 on CLL cells mediates increased viability, PI3K dependent growth signals and marked IL-6 and IL-8 secretion.2 In this study, the role of the different PI3K subunits in CD160 effector functions in CLL have been investigated in terms of cytokine/chemokine secretion and cell viability. Unstimulated CLL cells underwent apoptosis over 5 days in culture, which was significantly inhibited by the anti-CD160 antibody, CL1-R2 (p 〈 0.01). Similarly, the viability of purified CLL cells was enhanced by addition of a microenvironment milieu (CD19 negative fraction from BM samples) and further improved by CL1-R2 triggering of CD160 (p=0.03). Without stimulation, CLL cells secreted minimal levels of IL-2, IL-4, IL-10, TNF-alpha, INF-gamma, CCL2 and CCL5 (as assessed by cytokine bead array (BD Biosciences). At baseline, low levels of IL-6 (median: 23 pg/ml) and IL-8 (median: 114 pg/ml) were detected. Incubating CL1-R2 with CLL cells led to a significant increase in IL-6 (median: 1431 pg/ml, p 〈 0.01), IL-8 (median: 7945 pg/ml, p 〈 0.01), CCL2 (monocyte chemotactic protein-1, median: 813.3 pg/ml, p=0.01) and CCL5 (median: 53.61 pg/ml, p=0.03). These effects of CD160 triggering were suppressed, in a dose-dependent manner, by the pan PI3K inhibitors LY294002 (p=0.01) and Wortmannin (p 〈 0.01, Table 1).Table 1(above): n= 12CD160 (10μg/ml) Median pg/ml (range)CD160 (10μg/ml) and LY294002 10μM Median pg/ml (range)CD160 (10μg/ml) and LY294002 20μM Median pg/ml (range)IL-61431 (147 - 8972)108 (4.0 - 5512) p=0.003929 (3.38-5250) p=0.0019IL-87945 (950 - 9665)4394 (71 - 7744) p=0.00171958 (28 - 7024) p=0.0075CCL2813 (84 - 879)7.0 (1.3 - 81) p=0.00631.8 (0.0 - 20) p=0.0045CCL554 (42 - 106)36 (24 - 72) p=0.001821 (6.5 -27) p=0.0024 The roles of the different p110-catalytic subunits of PI3K in CD160 mediated activation were investigated. CLL cells from 5 different patients were incubated with the specific isoform-selective inhibitors PI103 (p110α), TGX-115 (p110β) and IC87114 (p110δ) at concentrations of 0.5, 1 and 10μM, in the presence of CL1-R2. CD160 induced cell viability was significantly reduced in a dose dependent manor by both PI103 (p=0.004) and IC87114 (p=0.002). Although the p110β inhibitor demonstrated a reduction in CD160 mediated cell viability it was not significant. Combining the p110α and p110δ inhibitors, a further decrease in CD160 mediated cell viability was observed (p 〈 0.01), but not in a synergistic manner. Upon performing cytokine and chemokine bead array assays on the same patients, the CD160 induced cytokine and chemokine production of IL-6, IL-8, CCL2 and CCL5 were suppressed by all the inhibitors at a concentration of 10 μM (Table 2). However, only p110α and p110δ subunit inhibition resulted in significant reductions, with a further decrease in both CD160 mediated cytokine and chemokine secretion when both were inhibited together (IL-6: p 〈 0.01, IL-8: p=0.03, CCL2: p=0.02, CCL5: p=0.03), but not with a synergistic or additive effect (Table 2).Table 2(Above): n=5CD160 (10μg/ml)CD160 (10μg/ml) and PI103 (p110a)CD160 (10μg/ml) and TGX-221 (p110b)CD160 (10μg/ml) and IC87114 (p110d)CD160 (10μg/ml) and PI103+IC87114IL-6457198 p=0.05365 p=NS189 p=0.02101 p 〈 0.01IL-848613405 p=0.044066 p=NS3143 p=0.032625 p=0.03CCL2696130 p=0.04583 p=NS111 p=0.0246 p=0.02CCL59159 p=0.0573 p=NS61 p=0.0543 p=0.03 These results demonstrate that CL1-R2 engagement of CD160 leads to: (1) a direct survival signal in CLL cells; and (2) production of IL-6, IL-8, CCL2 and CCL5. These functions are PI3K-dependent. These PI3K dependent responses signal largely through the p110α and p110δ isoform-selective catalytic subunits. From a clinical perspective, this data lends further support to targeting the p110α subunit, in addition to p110δ, as a therapeutic strategy. However, none of the inhibitors, alone or in combination, reduced cytokine secretion to basal levels, suggesting that CD160 is also signalling via other undefined pathways. References: 1. Farren TW et al. Blood. 2011; 118 (8): 2174-2183. 2. Liu FT et al. Blood. 2010; 115 (15): 3079-3088 Disclosures: No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V122.21.4124.4124

Language: English

Publisher: American Society of Hematology

Publication Date: 2013

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 2832-2832

Abstract: Abstract 2832 Introduction: CLL is a heterogenous disease. At diagnosis, prognostication is possible using a number of clinical and biological factors, which can inform the choice of treatment. In terms of response to treatment, minimal residual disease (MRD) detection is becoming increasingly important in CLL. Utilizing the CD160FCA assay, we have evaluated the time to MRD detection for a number of established prognostic markers. Methods: Utilizing the anti-CD160 monoclonal antibody (BY55, Coulter Immunotech, Marseille, France), we have developed a highly sensitive CD160 Flow Cytometric Assay (CD160FCA) incorporating a sequential gating strategy. Between July 2010 and July 2011, 63 patients were investigated for MRD by the routine clinical diagnostic service. 47 (73%) had completed treatment and were evaluable for long-term outcome. Response rates and event-free survival (EFS) were determined. The time to MRD detection post therapy was determined using CD160FCA for the prognostic indicators, ZAP-70, CD38, beta-2 microglobulin (B2M) and cytogenetic abnormalities. Results: As expected, establishing clonality by light-chain restriction could not be reliably measured in patients with very low B-cell numbers, and hence could not reliably be used to determine MRD. The CD160FCA could reliably monitor patients for MRD, including immunotherapy with Rituximab and Campath. 28 Patients (44%) were in complete remission (CR) following therapy by current NCI guidelines. Of these patients, 9/28 (32%) were CR-MRD positive and had significantly shortened EFS compared with CR-MRD negative patients (61 days vs 957 days P 〈 0.0001). Those patients with adverse cytogenetic markers had shorter time to disease detection (TTD): p53 mutation (30.5 days), 17pdel (61 days) and 11qdel (122 days) compared to +12 (427d), 13qdel (957d) and 13qdel with additional abnormalities (760 d). Of interest, patients with bi-allelic 13qdel had significantly shorter TTD compared to those with monoallelic 13qdel (46d vs 957d). Likewise CD38 status (CD38+: 92d vs CD38-: 669d; P=0.03) and B2M prior to treatment (B2M high: 91d vs B2M normal: 822d; P=0.004) were predictors of TTD based on residual disease assessment by the CD160FCA. 25 patients (40%) were assessed for ZAP-70 prior to commencing therapy. Those patients who were ZAP-70 positive had shorter TTD compared to those who were negative (ZAP+: 61d vs ZAP- 822d; P=0.01). Conclusion: Many clinical studies base their entry criteria on clinical and biological prognostication, as this provides insights into the biology of CLL and its response to therapy. The CD160FCA is a single tube tumor specific assay for MRD detection, which is highly sensitive, independent of the therapy and can be employed throughout treatment. Patients in CR had significantly different EFS based on their MRD status following treatment using the CD160FCA. For those patients with adverse prognostic markers (including CD38, ZAP-70 and B2M), the detection of MRD or relapsing disease using CD160FCA, is significantly shorter than those with a normal or good prognosis. Disclosures: No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V118.21.2832.2832

Language: English

Publisher: American Society of Hematology

Publication Date: 2011

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detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 4131-4131

Abstract: Chronic lymphocytic leukemia (CLL) is associated with profound defects in immune function, resulting in failure of anti-tumor immunity and increased susceptibility to infection. We have previously demonstrated alterations in gene expression profiles of T cells from CLL patients, which translate into functional defects in T-cell immune synapse formation, motility and cytotoxicity (Gorgun et al. JCI 2005; Ramsay et al. JCI 2008, Blood 2013). However a comparison of the transcriptome of natural killer (NK) cells from CLL patients and controls has not been investigated. NK cells were isolated from the peripheral blood of patients with CLL and healthy donors, followed by gene expression profiling using the Affymetrix U133Plus2.0 platform. 117 probes showed a 〉 2-fold decrease in expression while only 18 probes showed a 〉 2-fold increase in expression (adjusted p-value 〈 0.05) in CLL NK cells compared to healthy donor NK cells. Strikingly, 52 out of the 117 significantly down-regulated probes (44.4%) were for interferon-inducible genes including STAT1 (Signal Transducers and Activator 1), SOCS1 (Suppressor of cytokine signaling 1), interferon regulatory factor genes IRF7 and IRF9, and oligoadenylate synthetase genes OAS1, OAS2, and OAS3. The majority of these genes were inducible by both type 1 and type 2 interferons. Many of these genes have been implicated in host immunity to viral infections, and so it is possible that decreased NK-cell responsiveness to interferon contributes to the increased susceptibility of CLL patients to viruses. Notably, there was also altered expression of signaling pathways in common with T cells from CLL patients, with dysregulation of the cytoskeleton genes RAB3GAP1, RAB38, and EPHA1 and down-regulation of JUN mirroring the dysregulated JNK-signaling and the altered actin cytoskeleton pathways we have found in T cells from CLL patients. These changes were not due to differences in the relative frequencies of CD56DIM and CD56BRIGHTNK cells. Lenalidomide has significant clinical activity in CLL. It is not directly toxic to tumor cells in vitro, but instead is thought to activate anti-tumor immunity and block pro-tumor micro-environmental factors. NK-cell proliferation has been shown to correlate with clinical response to lenalidomide in CLL (Chanan Khan et al. BJ Haem 2011). Therefore, we investigated the effect of lenalidomide treatment on the gene expression profiles of NK cells from CLL patients in comparison to healthy controls. PBMCs from CLL patients or healthy controls were cultured in the presence of 1μM lenalidomide or vehicle control for 48 hours, followed by NK-cell isolation, RNA extraction and gene expression profiling. There were striking differences in the effect of lenalidomide on NK cells from CLL patients compared with healthy NK cells. In CLL NK cells, lenalidomide repaired the down-regulation of interferon-inducible genes, by increasing the expression of genes such as OAS3, IFIT1, IFI44L, IFIT3, OAS1, PDK4, and ACTN1. Pathway analysis highlighted the effect of lenalidomide on inducing interferon signaling, showing significant activation of interferon α, γ, and λ as upstream regulators. While many of the interferon-inducible genes were up-regulated 〉 3-fold in CLL NK cells, only OAS3 was significantly up-regulated in healthy NK cells with lenalidomide. Furthermore, the gene for IFNγ, IFNG, was actually significantly down-regulated in healthy NK cells by this agent. Lenalidomide also significantly down-regulated the expression of 5 genes encoding killer-cell immunoglobulin-like receptors (KIRs): KIR2DL1, KIR2DL2, KIR2DS3, KIR2DS4, and KIR3DL2, in healthy NK cells, but did not significantly down-regulate KIR genes in the CLL NK-cell dataset. Lenalidomide treatment did have some overlapping effects on CLL and healthy NK cells, including up-regulation of genes ARL11, CYFIP, and CORO1B that regulate the actin cytoskeleton pathway. In conclusion, NK cells from CLL patients have down-regulation of interferon response genes and pathways known to regulate normal immune function in response to bacteria and viruses. Lenalidomide has a differential effect on CLL and healthy NK cells: in CLL NK cells it repairs defective interferon responses, whereas in healthy NK cells it down-regulates inhibitory pathways. Disclosures: Riches: Celgene: Research Funding. Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V122.21.4131.4131

Language: English

Publisher: American Society of Hematology

Publication Date: 2013

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 104, No. 11 ( 2004-11-16), p. 1478-1478

Abstract: Background: The most favourable outcomes for patients with MM are seen in those achieving a complete or near complete response following initial therapy. Therefore, it is logical to explore strategies aimed at improving initial response rates. Bortezomib has shown significant activity in patients with advanced MM and has been shown to be superior to pulsed dexamethasone in this setting. Additional efficacy is seen when dexamethasone is combined with bortezomib and in vitro synergy is observed with cytotoxic agents such as adriamycin. On this basis, the PAD regimen was investigated as front line therapy for patients with MM. Aims: The primary objective of this Phase I/II study was to assess the feasibility of harvesting PBSCs after PAD, with secondary objectives being assessments of safety, toxicity, response rate (RR), PFS and OS. Methods: Patients with previously untreated MM were eligible. Patients were treated with 4x21 day cycles of PAD comprising bortezomib 1.3 mg/m2 on days 1,4,8 & 11 along with 40 mg dexamethasone on days 1–4, 8–11 & 15–18 during cycle 1 and days 1–4 during cycles 2–4. During days 1–4 of each cycle, patients also received 0 mg/m2, 4.5 mg/m2 or 9 mg/m2 of adriamycin at levels 1,2 & 3 respectively. Following harvesting, patients received high dose melphalan (MEL200) with stem cell (PBSC) rescue. Gene expression profiling was performed on purified plasma cells from diagnostic samples for pharmacogenomic analysis. Results: Thus far, 21 patients have been enrolled (19 male 2 female, median age 55 years [range 36–66]). All 21 have completed PAD with a 95% PR/CR rate. 20/21 patients mobilised PBSC successfully (median collection 3.8 x 106 CD34+ cells/kg, range 1.6–10.4). 16 patients have received MEL200 with median neutrophil ( 〉 0.5 x109/L) and platelet engraftment ( 〉 20 x109/L) of 15 (1–24) and 13 (10–33) days respectively. Of patients who are assessable at 3 months post MEL200, 11/12 have achieved at least a PR (CR 4, nCR 1, VGPR 4 & PR 2). Toxicities have generally been acceptable with 15 grade 3 events: 6 infections, 4 episodes of shingles, 2 nausea and vomiting, 2 neuropathy and 1 postural hypotension. Overall, 48% of patients have experienced sensory or painful neuropathy which is of Grade 1 severity in 43% of cases. Of note, neuropathic symptoms are improving in all patients after completion of therapy. Summary: These preliminary data show that PAD is well tolerated in the majority of patients, is highly effective and does not prejudice subsequent PBSC collection. A further cohort of patients will receive PAD with bortezomib at 1.0 mg/m2 in order to determine whether the frequency of neuropathy can be reduced whilst preserving efficacy. PAD should be further evaluated as first line therapy in prospective randomised trials.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V104.11.1478.1478

Language: English

Publisher: American Society of Hematology

Publication Date: 2004

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 4678-4678

Abstract: Background: CD160 is a functional receptor on NK cells, T -cells and neoangiogenic blood vessels. CD160 is not expressed on normal B-lymphocytes or myeloid cells. We have previously reported that CD160 is aberrantly expressed on CLL (Agrawal S. et al. Blood, 100 [Abstract no. 1488] 2002, Agrawal S, et al. Blood, 94 [suppl 1] , p119a, 1999). CD23 is almost invariably expressed on CLL cells, but it is also found on a significant proportion of patients with Mantle Cell Lymphoma (MCL). Aims: To provide further data on CD160 expression in CLL, assess the pattern of CD23 expression using the CD5+23+ / CD5+19+ ratio and the utility of these markers in differentiating B-LPDs, particularly, atypical CLL and MCL. Methods: Samples from more than 500 consecutive patients were examined for CD160 expression. Flow cytometry was performed using fresh whole blood and red cell lysis (Pharmalyse solution, BD), with anti-CD160 monoclonal antibody (BY55, Coulter Immunotech, Marseille, France) added to the routine panels. The ‘CLL score’ (Moreau EJ et al. [1997]. Am J of Clin Pathol; 108: 378–382) is based on the markers CD5, CD23, CD79b, FMC7 and IgM. Atypical cases of CLL were confirmed by bone marrow, lymph node and cytogenetic studies. The CD5+23+ / CD5+19+ ratios were calculated for 271 patients with CLL and 7 patients with documented typical CD23+ MCL. Results: 423/425 ( 〉 99%) CLL cases were positive for CD160, including 41/42 (98%) cases with a low CLL score (3 or less).Using CD5, CD23 and CD160 - which are almost universally expressed in CLL - a new ‘mini CLL score’ has been developed with each marker scoring one point. The mini CLL scores for 564 cases of B-LPD including CLL, MCL, Hairy Cell Leukemia (HCL), Splenic Marginal Zone Lymphoma (SMZL), Lymphoplasmacytic Lymphoma(LPC) and Waldenstrom’s Macroglobulinaemia (WM), are shown below: Mini CLL Scores for 564 cases of B-LPD Mini CLL Score 0 1 2 3 CLL 0 0 5 420 MCL 0 11 7 4 HCL 0 9 19 1 SMZL/LPC/WM 3 18 9 0 Other B-LPD 22 22 14 0 The CD5+23+ / CD5+19+ ratio proved useful in further differentiating between CLL and MCL. Only 3 out of 271 (1.1%) patients with CLL had a ratio of 0.75 or less, whereas 255 patients (94%) had a ratio of 0.95 or higher. Out of the 7 patients with CD23+ MCL, 6 patients (86%) had a ratio of 0.75 or less. All 4 MCL patients with a mini CLL score of 3 had ratios below 0.75. CD5+23+ / CD5+19 Ratio (CD23r) for CLL and CD23+ MCL CD23r 〉 .94 .94–76 .75–51 〈 .51 CLL 255 13 3 MCL 1 3 3 Discussion: By using the 3 most robust markers expressed in CLL, the mini CLL score reduces diagnostic doubt and is particularly useful for difficult cases with atypical morphology and immunophenotyping. A mini CLL score of 3 has a positive predictive value for CLL of 99%; the negative predictive value of a score of 〈 3 is 96%. The specificity of the mini score can be further improved by calculating the CD5+23+ / CD5+19+ ratio which in CLL is significantly higher then in CD23+ cases of MCL. Incorporating this tool into diagnostic practice could allow for simpler and cheaper diagnostic antibody panels. In addition to its diagnostic utility, CD160 is a functional molecule in CLL (separate abstract) and has a role in CLL biology.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V110.11.4678.4678

Language: English

Publisher: American Society of Hematology

Publication Date: 2007

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 3480-3480

Abstract: Abstract 3480 Background: Outcomes for patients with refractory or relapsed acute myeloid leukaemia (AML) are extremely poor and allogeneic haemopoietic stem cell transplantation (HSCT) provides the best hope for prolonged disease free survival. Ablative HSCT combines high dose, anti-leukaemic chemoradiotherapy and the graft versus leukaemia (GVL) effect, but is associated with significant toxicity and mortality and these risks rise exponentially with advancing age. Several factors mitigate against an attempt to transplant patients after relapsed disease, including a low probability of achieving second remission, high re-induction mortality, prolonged cytopenias and opportunistic infections and prolonged hospitalization. Based on promising results [1], we undertook a phase II study of sequential chemotherapy immediately followed by reduced intensity conditioning (RIC)-Allo with the aim of increasing the safety and applicability of AlloHSCT, while maintaining its antileukaemic efficacy. Patients/Methods: All eligible patients received treatment with Daunorubicin 45mg/m2 OD IV D-15 to D-13 and AraC 1.5g/m2 BD IV D-15 to D-10, a three day rest period, and conditioning with Fludarabine 25mg/m2 D-6 to D-2 and Cyclophosphamide 1g/m2 D-3 and -2 before receiving HSCT. Graft versus Host Disease (GVHD) prophylaxis was with Cyclosporine and Methotrexate. To date 33 patients were enrolled (table 1), of whom 31 patients underwent transplant. Results: 28/31 (90.3%) patients engrafted (neutrophils ≥0.5 and platelets ≥ 20) at a median of 33.5 days (15-49), while 3 died between d21-51 due to sepsis. The median d30, d60 and d100 whole blood chimerism was 96% (range 4–100), 80% (range 5–100), and 76% (range 0–100) respectively. 6/31 (19.4%) developed acute GVHD; 5 grade 1–2, 1 grade 4. Chronic GVHD was documented in 8 patients, extensive GVHD in 4/8. 7 patients had CMV re-activation (no CMV disease) and 1 non-specified pneumonitis. Median time of hospitalization was 37 days (30-61). No patient has required DLI to date. 18 pts (56.25%) achieved complete remission (CR) as assessed by day 30 bone marrow (BM). 2 had 〈 5% blasts BM, 4 refractory disease, 4 died before evaluation, and 3 did not have a BM examination. 7 (6 previously documented CR in BM) of the 33 patients (21.2%) relapsed. Median time to relapse was 162 days (59–408). 18 /33 (54.5%) have died; 6 (33.4%) of sepsis, 5 (27.8%) of relapsed leukaemia, 4 (22.2%) of GVHD, and 3 (16.7%) of refractory leukaemia. D100 treatment related mortality (TRM) was 18.2% (n=6) and overall TRM is 30.3% (n=10). Median time of TRM was 84 days (range 21–519). 15 of the 33 patients (45.5 %) are alive, 12 of whom (36.4%) are disease free with a median follow up of 13 months (range 2–31 months). For these patients, the underlying diagnosis was relapsed AML in 7 (50 %), refractory AML in 5 (28.6 %), high risk MDS in 2 (14.3%) and other in 1 (7.1 %). 2 patients with relapsed and 1 with refractory AML subsequently relapsed after having achieved remission. Overall survival for the 31 patients is 45.2% with disease-free survival of 38.7%. Conclusions: Sequential treatment with cytoreductive therapy and immediate RIC Allo is associated with good engraftment rates with a TRM acceptable for this high risk group. Our preliminary data indicate a favourable survival outcome for these patients with a particularly poor prognosis. 1. Schmid C, Scheuning M, Schwerdtfeger et al. Long-term survival in refractory acute myeloid leukemia afetr sequential treatment with chemotherapy and reduced-intensity conditioning for allogeneic stem cell transplantation. Blood. 2006;108:1092-1099. Disclosures: Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V116.21.3480.3480

Language: English

Publisher: American Society of Hematology

Publication Date: 2010

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7