Chronic Lymphocytic Leukemia Group Research Articles

Platelet Function Prior to Ibrutinib Treatment Is More Severely Impaired in Chronic Lymphocytic Leukemia Than in Mantle Cell Lymphoma

INTRODUCTION. Ibrutinib is an oral small-molecule drug that irreversibly inhibits Bruton's tyrosine kinase and thus suppresses proliferation of B cells. Ibrutinib therapy in chronic lymphocytic leukemia (CLL) and in mantle cell lymphoma (MCL) is associated with frequent bleeding complications that were reported to be more severe in CLL. This difference was suggested to be related to differential adenosine diphosphate (ADP) disruption by CD39, expressed by tumor cells. Here we investigate platelet functional activity in CLL and MCL patients before initiation of ibrutinib.MATERIALS. Fifty one adult patients with relapsed and refractory CLL and MCL and 20 healthy donors were included in the study. Platelet functional activity was characterized by flow cytometry before and after activation with SFLLRN plus collagen-related peptide. Levels of CD42b, CD61, CD62P, PAC1, annexin V binding, and mepacrine release were determined. Aggregation with collagen, ADP and ristocetin were measured.RESULTS. Among 36 CLL patients 25 (69%) were men, the median age was 66 (range, 31 to 83 years) and 23 (64%) had a refractory disease. In 34 previously treated patients the median number of prior treatments was 2 (range, 1 - 6). Two CLL patients with del (17p) received ibrutinib as a first line. In a group of MCL patients (n=15) there were 12 men (80%), the median age was 63 years (range, 49-79). Seven (54%) patients had a high MIPI score. The median number of previous chemotherapy lines in MCL patients was 1 (range, 1 - 6). Lymphocyte count before initiation of ibrutinib was significantly higher in CLL group (median 17,5 х 109/L range 0,48 - 189,7) compared to MCL group (median 1,75 х 109/L, range 0,35 - 6,59, p=0,037). Initial platelets count was also different being lower in CLL than in MCL patients (127 х 109/L (15 - 248) versus 186 х 109/L (63 - 311), p=0,005). Platelets of untreated patients with CLL had statistically significant impairments in their responses compared to those with MCL. Specifically, they had decreased integrin activation in response to stimulation (46±19% versus 73±18%; for healthy donors, 100±17%), and relative mepacrine release (2.9±0.9 versus 3.5±0.8; healthy, 4.0±0.7). P-selectin release was impaired milder (77±17% versus 92±14%; healthy, 100±10%), and procogulant platelet formation impairment was not statistically significant (8±6% versus 12±7%; healthy, 12±3%). In control experiments, addition of MeS-AMP and MRS2179, selective inhibitors of ADP receptors, had comparable effects on the healthy donor platelets. Aggregation in response to ADP was dramatically impaired in CLL compared with MCL (24±18% versus 42±16%), as well as that in response to collagen (47±23% versus 72±13%). Upon ibrutinib treatment, further decrease of PAC1 binding and procoagulant platelets formation almost to the level of resting platelets were observed; activation with SFLLRN alone produced a similar result with donor platelets.CONCLUSIONS. Patients with CLL and MCL have initially impaired response of platelets. The defects of platelet function in CLL is much greater suggesting a possible explanation of the different clinical severity. DisclosuresVorobyev:Janssen: Speakers Bureau. Ptushkin:Janssen: Speakers Bureau.

High Aurora Kinase and Low Dido Levels Characterizes a Sub-Group of Chronic Lymphocytic Leukemia with Chromosomal Gains and High White Blood Cell Counts: Potential Inter-Regulatory Role of E2F1 and Mir-17-92 Cluster

During cell cycle division Aurora kinases (AURKA and AURKB) participate in the formation and control of mitotic spindle fibers, while, protein isoforms (DIDO1, DIDO2 and DIDO3), derived by alternative splicing of the DIDO gene, assist at the junction of microtubules to kinetochores. Thus, both are relevant to cell cycle maintenance. Interestingly, overexpression (or gain of function) of AURKs or low expression (or loss of function of DIDO) are both associated with centrosomal amplification and chromosomal instability (CIN), leading to aneuploidy. Among hematological diseases with CIN records, chronic lymphocytic leukemia (CLL) can display centrosome amplification and changes in AURKs expression levels leading to aneuploidy. The Despite this, there are no studies evaluating the potential association of these genes with CIN in CLL. By evaluating their gene expression levels in CLL samples from patients with or without chromosomal aberrations, we show that increased levels of AURKA and AURKB and, conversely, reduced levels of DIDO variants, are both significantly associated with chromosomal gains and with increased white blood cell (WBC) counts. Clearly, CLL samples without any cytogenetic abnormality had expression levels similar to samples mostly harboring non-numerical aberrations. The finding that the expression levels of AURKs and DIDO variants are completely opposed, showing a discrete inter-related pattern, led us to investigate the potential regulatory mechanism behind this. Given that other have previously shown that the oncogenic miR-17-92 cluster is significantly upregulated in purified CLL patient cells expressing unmutated IGHV genes (as compared to mutated patient cells), and that miR-17 is expressed at significantly higher levels in unmutated or ZAP-70 high cases (bad prognostic cases generally associated with chromosomal instability), we investigated the potential negative regulation of DIDO variants by microRNAs from this cluster. In addition, based on the already described regulatory mechanism by which AURKA overexpression induces the E2F1-mediated transcription upregulation of the miR-17-92 cluster (with an observed expression correlation of both proteins in cancer specimens); we decided to investigate this regulatory axis in CLL. Notably, we found that all DIDO variants are predicted to be heavily targeted by several miRs of this oncogenic cluster. We show that CLL samples with low DIDO expression, in addition to the already mentioned AURK high levels, displayed significant higher levels of the transcription factor E2F1 and of its transcriptional target, the miR-17-92 primary transcript (MIR17HG). Moreover, by using the NTERA-2 cell line as a model, we show that siRNA knockdown of AURKA (at the transcript and protein level, as confirmed by qPCR and western blot) is accompanied by a striking significant reduction of E2F1 and also of MIR17HG. Furthermore, transfection of NTERA-2 cells with synthetic mimics of the miR-17~92 cluster (namely, miR-19a, miR-20a and miR-92a) results in a clear and significant reduction in the transcript levels of all DIDO variants. Finally, specific siRNA inhibition of the DIDO3 variant (but not the others) led to a significant reduction in the transcript levels of all DIDO variants, indicating an additional mechanism contributing to the downregulation of DIDO transcripts. Altogether, our results demonstrate the existence of a potential interconnected regulatory mechanism between AURK and DIDO, associated with CIN and higher WBC counts in CLL. More importantly, the high expression levels of AURKs and the associated low levels of DIDO variants are specifically associated with cytogenetic abnormalities presenting chromosomal gains, highlighting the specific cellular mechanism underlying the CIN observed in this distinct CLL group. Given the central role of CIN in cancer genesis and progression, these findings will likely have an important impact on prognosis or treatment of CLL. Funded by: FAPESP, CNPq and CAPES. DisclosuresNo relevant conflicts of interest to declare.

Diagnosis and treatment of chronic lymphocytic leukemia: recommendations from the Brazilian Group of Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia is characterized by clonal proliferation and progressive accumulation of B-cell lymphocytes that typically express CD19+, CD5+ and CD23+. The lymphocytes usually infiltrate the bone marrow, peripheral blood, lymph nodes, and spleen. The diagnosis is established by immunophenotyping circulating B-lymphocytes, and prognosis is defined by two staging systems (Rai and Binet) established by physical examination and blood counts, as well as by several biological and genetic markers. In this update, we present the recommendations from the Brazilian Group of Chronic Lymphocytic Leukemia for the diagnosis and treatment of chronic lymphocytic leukemia. The following recommendations are based on an extensive literature review with the aim of contributing to more uniform patient care in Brazil and possibly in other countries with a similar social–economic profile.

Abstract 292: Integrated analysis of ibrutinib resistance in chronic lymphocytic leukemia

Abstract BACKGROUND: Ibrutinib covalently binds to Bruton's tyrosine kinase, inhibits B-cell receptor signaling, and is active in chronic lymphocytic leukemia (CLL). Progressive disease (PD) on ibrutinib has been reported due to histologic transformation or mutations of BTK or PLCG2. Here we report integrated analysis of the clinical and molecular characteristics of CLL pts who progressed on ibrutinib (PD group). METHODS: Under a phase II investigator-initiated trial (ClinicalTrials.gov, NCT01500733) 84 CLL pts with TP53 aberration (deletion 17p or TP53 mutation) or age ≥65 were treated with ibrutinib 420mg daily. Samples from the PD group were tested for mutations of BTK and PLCG2 by a high-sensitivity assay using branched and locked nucleic acids. In pts with mutations at PD, stored samples from earlier time-points were also sequenced. RESULTS: 13 (15.5%) of 84 pts progressed at a median follow up of 24 months. 3 of 4 early PDs (≤12 months) were due to histologic transformation, while 8 of 9 late PDs (median 34.9 months) were due to CLL. PFS was inferior in subgroups with TP53 aberration, unmutated IGHV, advanced Rai stage, high β-2 microglobulin and relapsed/refractory disease (log-rank p<0.05 for all tests). 8 pts with progressive CLL were subsequently treated with small molecules targeting PI3K or Bcl-2, and 6 were alive to date (longest follow-up 15 months). Two types of non-synonymous mutations at BTK exon 15 (C481S, C481R) and five types of non-synonymous mutations at PLCG2 exon 19, 20 and 24 (R665W, P664S, P664L, S707Y, L845F) were identified in 8 out of 9 pts having progressive CLL. No mutation was found in pts with transformation and in one pt with progressive CLL. Concomitant BTK and PLCG2 mutations were found in 5 out of 8 pts (62.5%). Mutations pre-dating clinical PD were identified in stored samples from 6 pts as early as 13 months before progression (range 1.8-13.0). The median time to the first detected mutation was 23.1 months (range 5.4-34.7). Mutational complexity increased over time as reflected by increasing types of mutations (n = 3) and allele frequencies (n = 3) at later time-points. Both PD with progressive CLL and non-PD groups showed equivalent depth of best response in peripheral blood (PB) and bone marrow during treatment (p>0.05). At PD, tumor burden increased by 2 to 32-fold from nadir based on PB flow cytometry. CONCLUSION: Most pts with progressive CLL on ibrutinib carry BTK and/or PLCG2 mutations. Concurrent mutations of BTK and PLCG2 are common at progression, and either or both of these mutations can be acquired many months before clinical progression. In cases with detectable mutations but without clinical progression, pts can benefit from prolonged treatment with ibrutinib until clinical progression occurs. Upon clinical progression with CLL, pts can still show responses to alternative targeted agents. Citation Format: Inhye E. Ahn, Adam Albitar, Chingiz Underbayev, Sarah Herman, Xin Tian, Susan Soto, Maryalice Stetler-Stevenson, Irina Maric, Mohammed Z. Farooqui, Maher Albitar, Adrian Wiestner. Integrated analysis of ibrutinib resistance in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 292.

Obinutuzumab (GA101) is highly effective against chronic lymphocytic leukemia cells in ex vivo B-cell depletion irrespective of high-risk prognostic markers

The anti-CD20 monoclonal antibody rituximab has revolutionized the management of B-cell non-Hodgkin's lymphoma and chronic lymphocytic leukemia (CLL). Addition of rituximab to the frontline standard chemotherapy (fludarabine–cyclophosphamide, FC) within the rituximab fludarabine cyclophosphamide regimen has recently shown to statistically increase both progression-free survival (PFS) and overall survival (OS) in the randomized CLL8 phase III trial of the German CLL Group.1 This was the first example of improved OS in first-line CLL, although restricted to those patients fit enough to receive FC chemotherapy. However, the translation of these results into the clinical setting is hampered by the fact that ca. one half of CLL patients are 72 years at the time of first therapy, 40% being aged more than 75 years2 with increased burden of comorbidities (for example, kidney function impairment) and thus not eligible for FC chemotherapy.

Patients with chronic lymphocytic leukaemia (CLL) differ in the pattern of CTLA-4 expression on CLL cells: the possible implications for immunotherapy with CTLA-4 blocking antibody.

Recently, systemic administration of a human monoclonal antibody directed against cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on circulating T cells in patients with chronic lymphocytic leukaemia (CLL) has been considered. Also, CLL cells have been shown to express CTLA-4, increased levels of which in the leukaemic compartment are a predictor of good clinical outcome. Since both CLL and Treg microenvironment cells can be targeted by the CTLA-4 blocking antibody in this immunotherapy approach, the investigation of the functional effect of CTLA-4 blockade on CLL cells might be of potential clinical relevance. The main aim of this study was to examine the effect of CTLA-4 blockade on proliferation activity and apoptosis of CLL cells in patients with low and high CTLA-4 expression. We found that in the high CTLA-4-expressing CLL group, CTLA-4 blockade on the CLL cell surface resulted in a significant increase in the median percentages of Ki67+ cells and a tendency to decrease in the proportion of apoptotic cells. In contrast, in the low CTLA-4 expressors, CTLA-4 blockade did not affect the proliferation activity or the frequency of apoptosis. This study reports for the first time the different effect of CTLA-4 blockade on CLL cells in CLL patients depending on the levels of CTLA-4 expression. CTLA-4 blockade seems to induce pro-survival signals in leukaemic cells from CLL patients exhibiting high CTLA-4 expression, suggesting that an immunotherapy approach based on the systemic use of monoclonal anti-CTLA-4 antibodies could be an unfavourable strategy for some CLL patients.

Molecular prediction of durable remission after first-line fludarabine-cyclophosphamide-rituximab in chronic lymphocytic leukemia

AbstractFludarabine, cyclophosphamide, and rituximab (FCR) has represented a significant treatment advancement in chronic lymphocytic leukemia (CLL). In the new scenario of targeted agents, there is an increasing interest in identifying patients who gain the maximum benefit from FCR. In this observational multicenter retrospective analysis of 404 CLL patients receiving frontline FCR, the combination of three biomarkers that are widely tested before treatment (IGHV mutation status, 11q deletion and 17p deletion; available in 80% of the study cohort) allowed to identify a very low-risk category of patients carrying mutated IGHV genes but neither 11q or 17p deletion that accounted for 28% of all cases. The majority of very low-risk patients (71%) remained free of progression after treatment and their hazard of relapse decreased after 4 years from FCR. The life expectancy of very low-risk patients (91% at 5 years) was superimposable to that observed in the matched normal general population, indicating that neither the disease nor complications of its treatment affected survival in this favorable CLL group. These findings need a prospective validation and may be helpful for the design of clinical trials aimed at comparing FCR to new targeted treatments of CLL, and, possibly, for optimized disease management.

Prognostic Significance of VEGF and bFGF in Egyptian CLL Patients

Though an impressive number of prognostic factors have been identified in chronic lymphocytic leukemia (CLL), which exhibits a high variability in its clinical presentation and course, many of them have not yet been applied in routine clinical practice. Among these factors are the angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), previously reported as prognostic markers for disease progression. The objective of the present study was to assess the impact of serum VEGF and bFGF levels at diagnosis in Egyptian CLL patients and their relationship with disease prognostic parameters and patients' outcome, aiming to apply them in routine clinical practice. For this purpose, 45 newly diagnosed CLL patients and 25 healthy controls were recruited. VEGF and bFGF were found to be significantly higher in the CLL group when compared to the control group (P 320 pg/ml for VEGF and >145 pg/ml for bFGF was able to predict poor prognosis (treatment failure) in CLL patients with prognostic accuracies of 96.7% and 89.10% respectively. In conclusion: VEGF and bFGF represent powerful prognostic markers in B-CLL, partially because of the simplicity of obtaining their samples and their assays procedure, the reliability of their assessment besides being optimal targets for potential novel therapies in conjunction with classical therapies.

Superior efficacy of rituximab-based chemoimmunotherapy as an initial therapy in newly diagnosed patients with B cell indolent lymphomas: long-term results from a single center in China

BackgroundRituximab has been confirmed to improve the survival of patients with B cell indolent non-Hodgkin lymphomas (B-iNHLs) in Western world as previously reported, however, it is rarely reported in Chinese cohort. This study is to investigate the efficacy and safety of rituximab-based chemoimmunotherapy and select subpopulations most sensitive to the regimen in Chinese B-iNHL patients.Methods334 B-iNHL patients from our center were retrospectively assessed.ResultsPatients received R-based chemoimmunotherapy showed significantly higher rates of overall response (OR) (93.0 % vs. 53.4 %, P < 0.001) and complete response (CR) (63.3 % vs. 16.0 %, P < 0.001) compared with the patients received other therapies. Survival analysis showed that rituximab-based chemoimmunotherapy could obviously improve the progression-free survival (PFS) (110 vs. 49 months, P = 0.001) and overall survival (OS) (120 vs. 72 months, P < 0.001) in patients with B-iNHLs. Interestingly, in chronic lymphocytic leukemia (CLL) patients, we found that the patients with β2-microglobulin (β2-MG) < 3.5 mg/L, lactate dehydrogenase (LDH) < 220 U/L, zeta-chain-associated protein kinase 70 (ZAP-70) negative, and non high-risk genetic abnormality could achieve more benefits from R-based regimens with higher CR rate (P = 0.003, 0.029, 0.013 and 0.038, respectively). Meanwhile, more CLL patients achieved minimal residual disease (MRD) negative after rituximab-based treatment (46.5 % vs. 10.3 %, P < 0.001). Moreover, CLL patients with MRD < 1 %, LDH < 220 U/L, complete remission (CR) or partial remission (PR), β2-MG < 3.5 mg/L and non high-risk cytogenetic abnormality showed superior outcome compared to the controls (P = 0.001, 0.000, 0.000, 0.001 and 0.013, respectively). No other side-effects increased in chemoimmunotherapy group except the elevation of grade 3–4 neutropenia.ConclusionsOur results demonstrate the superior efficacy of rituximab–based chemoimmunotherapy as an initial therapy in Chinese cohort with newly diagnosed B-iNHLs and further identify subpopulations that are more sensitive to R-based chemoimmunotherapy in CLL group.

CD22 signal abnormalities in the pathogenesis of immune related pancytopenia

To investigate the expression of CD22 and its downstream signal molecule spleen tyrosine kinase (SYK) and their phosphorylation of B lymphocytes in patients with immune related pancytopenia(IRP), and to explore the role of CD22 in pathogenesis of IRP. The expression of CD22, SYK and their phosphorylation, along with the expression of IgG and IgM, which obtained from B lymphocytes in peripheral blood of 46 patients with IRP(22 new diagnosed and 24 remitted patients returned to normal after treatment), 22 healthy controls and 12 chronic lymphocytic leukemia(CLL) patients from February to December 2014 were analyzed by flow cytometry. And the mRNA expression of CD22 in peripheral blood mononuclear cell was determined by real-time quantitative PCR. The ratios of CD22+ cells and phosphorylated CD22(pCD22)+ cells of B lymphocytes in new diagnosed group (60. 03% ± 20. 94% 71. 32% ± 11. 16%) were significantly higher than those in remission group (46. 92% ± 20. 04%, 55. 82% ± 14. 42%), normal control group (46. 86% ± 17. 78%, 53. 28% ± 14. 76%) and CLL group (39. 74% ± 18. 96%, 59. 07% ± 17.09%) (all P <0.05). The ratios of phosphorylated SYK( pSYK) + cells in the four groups had the same trend (all P <0. 05). The ratio of pCD22+ cells/pSYK+ cells in new diagnosed group was significantly lower than that in normal control group and CLL group (27. 39 (5. 06 - 102. 70) vs 55. 95 (15. 25 - 298. 53), 56. 92(5. 60 - 228. 96), both P <0. 05), and pCD22+ cells positively correlated to pSYK+ cells ( r = 0. 341, P < 0. 05). The expression of IgG in new diagnosed group and remission group was significantly higher than that in normal control group, and the expression of IgM in new diagnosed group was significantly higher than that in normal control group and CLL group (all P <0. 05). The expression levels of CD22 mRNA in new diagnosed group was significantly higher than that in remission group, normal control group and CLL group (all P <0. 05). The BCR signal pathway of B lymphocyte in IRP patients is enhanced, and the quantity and function of CD22 are increased, while which are still insufficient to inhibit B cell proliferation, and these may have some relationships with the pathogenesis of IRP. [Key words] Pancytopenia; Antigens, CD22; Immune related pancytopenia; Spleen tyrosine kinase; Phosphorylation

TCL1 targeting miR-3676 is codeleted with tumor protein p53 in chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia and dysregulation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease based on transgenic mouse studies. To determine a role of microRNAs on the pathogenesis of the aggressive form of CLL we studied regulation of TCL1 expression in CLL by microRNAs. We identified miR-3676 as a regulator of TCL1 expression. We demonstrated that miR-3676 targets three consecutive 28-bp repeats within 3'UTR of TCL1 and showed that miR-3676 is a powerful inhibitor of TCL1. We further showed that miR-3676 expression is significantly down-regulated in four groups of CLL carrying the 11q deletions, 13q deletions, 17p deletions, or a normal karyotype compared with normal CD19(+) cord blood and peripheral blood B cells. In addition, the sequencing of 539 CLL samples revealed five germ-line mutations in six samples (1%) in miR-3676. Two of these mutations were loss-of-function mutations. Because miR-3676 is located at 17p13, only 500-kb centromeric of tumor protein p53 (Tp53), and is codeleted with Tp53, we propose that loss of miR-3676 causes high levels of TCL1 expression contributing to CLL progression.

Minimal residual disease detection with tumor-specific CD160 correlates with event-free survival in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the detection of minimal residual disease (MRD) correlates with outcome in the trial setting. However, MRD assessment does not guide routine clinical management and its assessment remains complex. We incorporated detection of the B cell, tumor-specific antigen CD160 to develop a single-tube, flow cytometry assay (CD160FCA) for CLL MRD to a threshold of 10−4 to 10−5. One hundred and eighty-seven patients treated for CLL were enrolled. Utilizing the CD160FCA methodology, there was a high level of comparison between blood and bone marrow (R=0.87, P<0.001). In a validation cohort, CD160FCA and the international standardised approach of the European Research Initiative on CLL group demonstrated high concordance (R=0.91, P<0.01). Patients in complete remission (CR) and CD160FCA negative had longer event-free survival (EFS) (63 vs 16 months, P<0.01) and prolonged time to next treatment (60 vs 15 months, P<0.001) vs MRD positive patients; with a median time to MRD positivity of 36 months. In multivariate analysis, CD160FCA MRD detection was independently predictive of EFS in patients in CR and even predicted EFS in the good-risk cytogenetic subgroup. CD160FCA offers a simple assay for MRD detection in CLL and gives prognostic information across different CLL risk groups.

BI 836826, a Novel Fc-Engineered Antibody in Combination with Phosphoinositide-3-Kinase Inhibitor for Treatment of High Risk Chronic Lymphocytic Leukemia

Novel chronic lymphocytic leukemia (CLL) therapies target phosphoinositide 3-kinase (PI3K; idelalisib) and Bruton’s tyrosine kinase (BTK; ibrutinib). Despite their success, certain high-risk groups, notably those with dysfunctional P53, demonstrate lower response rates than other CLL groups. As such, combination therapies could potentially overcome this deficiency. As CD37-mediated signaling resulted in direct cytotoxicity that was enhanced with PI3K inhibition (Lapalombella 2012), we aimed to combine BI 836826, a novel IgG1 chimerized and Fc-engineered anti-CD37 mAb, with BCR-pathway inhibitors to possibly enhance clinical efficacy in high-risk CLL patients and overcome potential inhibition of ADCC.Consistent with previous report, BI 836826 displays both ADCC and direct pro-apoptotic activity (Heider 2011). We assessed the potential effect of idelalisib and ibrutinib on CD37 expression and NK-cell function. Following 24hr incubation of primary CLL cells (n=4) with idelalisib, ibrutinib, or DMSO, there was no significant difference in surface expression of CD37 (fold change ~ 1.01; p > 0.16 for all comparisons). To ensure that NK-cell Fc-receptor function was unimpaired by BCR-pathway inhibitors, we conducted CD107ab degranulation assays (n=5). Data revealed that Fc-receptor engagement of BI 836826 and NK-cell lytic degranulation was not abrogated by idelalisib or ibrutinib (mean CD107ab expression: DMSO = 10.14%; idelalisib = 5.67%; ibrutinib = 2.41%). In combination with idelalisib, BI 836826 was still capable of eliciting an effective NK-cell response superior to rituximab (difference = 4.51%; p < 0.0001). To further qualify NK-cell function, TNFα (n = 6) or INFγ (n = 12) release by NK cells after pretreatment with idelalisib or ibrutinib was evaluated. Similarly, cytokine release was not completely inhibited by idelalisib and ibrutinib; and BI 836826 was still capable of inducing cytokine release. To confirm the potential added benefit of combination therapy, we conducted 51Cr release ADCC assays with primary CLL cells and healthy allogeneic NK-cells (Fig1) or CLL patient autologous NK-cells (Fig2). Results confirmed that neither ibrutinib nor idelalisib could ablate BI 836826-induced NK-cell ADCC. However, there was a more pronounced inhibition of NK-cell ADCC with ibrutinib. As previously demonstrated (Kohrt 2014), NK-cell ADCC mediated by rituximab was almost completely abrogated by ibrutinib, likely secondary to off-target interleukin-2-inducible T-cell kinase inhibition (Dubovsky 2013).Our preliminary data show that signaling induced by BI 836826 may demonstrate the same enhanced direct cytotoxicity with PI3K inhibition as previously reported with other CD37 mediated signaling agents (Lapalombella 2012). Evaluation of direct pro-apoptotic activity of BI 836826 (0.1ug/mL) in combination with idelalisib (1uM) on direct cell death (24hr incubation) demonstrated enhanced activity in the combination setting with similar activity in primary patient CLL cells with dysfunctional P53 (n=6; Fig3A) and functional P53 (n=4; Fig3B).In conclusion, these results demonstrate that idelalisib could potentially be used in combination with BI 836826, which has the added benefit of directly killing inhibitor-resistant P53-null primary CLL cells and lacks complete inhibitor-induced ADCC ablation. [Display omitted] [Display omitted] [Display omitted] DisclosuresOff Label Use: Off-label use of idelalisib and BI 836826 in combination for CLL patients. Jones:Pharmacyclics: Consultancy, Research Funding. Heider:Boehringer Ingelheim: Employment.

A Molecular Model to Predict Durable Remission after First Line Fludarabine-Cyclophosphamide-Rituximab Treatment in Chronic Lymphocytic Leukemia

Background. Fludarabine, cyclophosphamide, and rituximab (FCR) has represented a significant advancement in the treatment of patients with chronic lymphocytic leukemia (CLL) and has the potential of inducing durable remissions of the disease. In the new scenario of targeted agents for CLL, there is an increasing interest in identifying patients who may gain the maximum benefit in terms of disease control by a single shot of FCR chemo-immunotherapy.Purpose. Here we aimed at identifying predictors of durable remission after first line FCR treatment.Methods. The study was based on 405 progressive and previously untreated CLL patients who consecutively received standard FCR (up to 6 cycles) as first line therapy in 19 hematologic centers between 2001 and 2010. According to an intention to treat approach, all patients who received at least one FCR cycle were registered in the study. This series was representative of a FCR treated CLL cohort with respect to baseline characteristics, including age (median: 61 years; >65 years in 33% of patients), gender (male in 68% of patients), stage (progressive Binet A in 11% of patients; Binet B in 59%; Binet C in 30%) and number of FCR courses (median: 6; <6 in 42% of patients). Most patients were evaluable for IGHV mutation status (unmutated in 192/296, 65%) and genomic aberrations at treatment requirement (17p deletion in 30/306, 9.8%; 11q deletion in 56/300, 19%; +12 in 70/298, 23%; 13q deletion in 108/301, 36%).Results. After a median follow-up of five years, 159 patients have progressed and 72 have died, accounting for a 5-year progression free survival (PFS) according to the IWCLL criteria of 47% (median: 58 months) and for a 5-year overall survival (OS) of 81% (median: not reached). When the demographic effects of age, gender and year of treatment were compensated, the 5-year and 10-year survival of the whole CLL cohort were 85% and 68%, respectively, of those expected in the matched normal general population (p<0.001). By multivariate analysis, unmutated IGHV genes, 11q deletion and 17p deletion maintained independent association with both PFS and OS, thus providing the rationale to utilize them in the development of a model to predict remission duration after FCR. By recursive partitioning analysis, 17p deletion was the most important variable in predicting PFS after FCR, followed by 11q deletion and IGHV mutation status (Fig. 1A). Based on the application of an amalgamation algorithm, cases harboring unmutated IGHV genes and 11q deletion were grouped into a single category because they showed an identical drop of the PFS curve (Fig. 1A). By this approach, three CLL subgroups were hierarchically classified. Clinical features and treatment indication were superimposable across the three risk groups. The low risk category comprised patients harboring mutated IGHV genes but neither 11q or 17p deletion, and accounted for 26% of all cases. Most of the low risk patients (67%) remained free of progression after FCR (Fig. 1B), and their hazard of relapse dropped to zero after 5 years of follow-up. Consistently, the PFS curve of low risk patients plateaued after 5-years from FCR (Fig 1B). The life expectancy of low risk patients (91% at 5 year) was superimposable to that observed in the matched normal general population (Fig. 1D), indicating that neither the disease, nor complications of its treatment affected survival in this favorable group of CLL. Conversely, patients belonging to the intermediate risk (IGHV unmutated and/or 11q deleted) and high risk (17p deleted) categories showed a constant increase of the hazard of progression over time and almost all were projected to relapse after FCR, although at a different rate: 10% per year of follow-up in the intermediate risk group and 17% per year of follow-up in the high risk group (Fig. 1B-C). The 5-year life expectancy of intermediate and high-risk patients was significantly impaired compared to that observed in the matched general population (85% and 60%, respectively) (Fig. 1D), indicating an excess of deaths related to the disease or treatment complications in these unfavorable groups of CLL.Conclusions. The combination of three biomarkers that are routinely tested at treatment allows to segregate a subgroup of CLL (IGHV mutated without 17p or 11q deletion) that may achieve a durable remission after first line FCR treatment and experience an expected survival similar to that of the general population. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

NOTCH1 Mutations Are Associated with High CD49d Expression in Chronic Lymphocytic Leukemia

Mutations of NOTCH1 have emerged as one of the most frequent somatic alterations in chronic lymphocytic leukemia (CLL), affecting up to 10–15% of patients. These mutations (~80% are 7544-7545delCT frameshift deletions) generate a truncated protein that accumulates in the cell and activates the downstream NOTCH1 signaling which is implicated in apoptosis resistance and increased survival of CLL cells.CD49d (α4 integrin chain) is one of the most relevant negative prognosticator in CLL, expressed by ~40% of CLL cases, and associated with aggressive/accelerated clinical courses, whose key role in CLL cell microenvironmental interactions has been thoroughly investigated.Literature data indicate that NOTCH1 has a role in activating the integrin signaling in several cell models. Given the higher CD49d expression characterizing trisomy 12 CLL, a CLL subset in which the NOTCH1 pathway is more often activated by NOTCH1 mutations, this study was aimed at investigating the contribution of NOTCH1 in the regulation of CD49d expression in CLL.The 7544-7545delCT NOTCH1 mutations were investigated by ARMS-PCR in 1027 CLL cases, all characterized for CD49d expression and for the cytogenetic profiles by FISH. NOTCH1 mutated cases were 158/1027 (15%), with a higher prevalence (36.7% of NOTCH1 mutated cases) in the trisomy 12 cytogenetic group.Analysis of CD49d expression highlighted a very strong association between the presence of NOTCH1 mutations and CD49d expression (p<0.0001). In particular, high CD49d expression (>30% of positive cells) was found in 102/158 (64.5%) NOTCH1 mutated cases as compared to 285/869 (32.8%) NOTCH1 wild-type cases. Of note, excluding trisomy 12 CLL, again NOTCH1 mutated CLL (100/865, 11.6%) displayed a significantly higher frequency of CD49d+ cases (52%) as compared to NOTCH1 wild-type CLL (25.7%) (p<0.0001).We next analyzed the percentage of mutated NOTCH1 DNA in the context of the CLL clone by a quantitative real-time PCR (QRT-PCR) approach set up by us to quantify the delCT NOTCH1 mutation. Using the 10% cut-off value to discriminate between cases with high (high NOTCH1) and low (low NOTCH1) mutation burden, 90/138 (67.7%) and 43/138 (32.3%) CLL cases were classified as low NOTCH1 and high NOTCH1, respectively. A higher prevalence of CD49d+ cases was found in the high NOTCH1 group (79%) as compared to the low NOTCH1 group (58%) (p=0.03). Moreover, a significant association between CD49d expression and a high NOTCH1 mutation burden was observed also excluding trisomy 12 CLL, with 69% of CD49d+ cases in the high NOTCH1 group, compared to 41% of CD49d+cases in the low NOTCH1 group (p=0.03).The association between NOTCH1 mutations and CD49d expression was next confirmed by next-generation sequencing results using the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 8 CLL cases characterized by both CD49d bimodal expression, and the presence of 7544-7545delCT NOTCH1 mutations at the subclonal level. In 7/8 cases, the CD49d+ component showed a higher NOTCH1 mutation burden compared to the CD49d- component, this difference reaching statistical significance in 4/7 cases. Of note, a similar clustering of mutations could not be observed in the CD49d- and CD49d+ components of other CLL cases characterized by bimodal CD49d expression and subclonal mutations of SF3B1 (n=1), BIRC3 (n=2) or TP53 (n=2).To verify whether NOTCH1 accumulation, as occurring in NOTCH1 mutated CLL, may influence CD49d expression, MEC-1 cells were transfected with a vector containing either a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or a NICD carrying a missense mutation (c.5304G>A) generating a stop codon at the beginning of the sequence, as control. The higher levels of both NOTCH1 transcript (fold increase over control=2.2) and protein (fold increase over control=1.3) characterizing mutated-NICD MEC-1 cells, was paralleled by higher levels of CD49d expression (mean fluorescence intensity=23.300 versus 12.400) in these cells.Altogether our data demonstrate a direct correlation between NOTCH1 mutations and CD49d expression also outside the trisomy 12 CLL group, and suggest that accumulation of NOTCH1 may be directly or indirectly responsible for the up-regulation of CD49d expression. DisclosuresNo relevant conflicts of interest to declare.

CD180 Expression in B-Cell Lymphomas: A Multicenter Geil Study

CD180 is a Toll-Like Receptor homolog strongly expressed on normal human B-cells and involved in innate immune responses. Previous proteomic analyses on microparticles derived from mature B-cell neoplasms allowed us to identify CD180 as a marker of marginal zone lymphomas (MZL)(Miguet, Leukemia, 2013). Using flow cytometry on blood samples we showed that this protein is lost or underexpressed at the plasma membrane for almost all B-cell lymphomas except MZL. In order to confirm its clinical relevance, we conducted a prospective multicenter flow cytometry study in 5 French University Hospital laboratories, on behalf of the GEIL. Blood or bone marrow samples from 236 patients were studied (20 normal controls ; 74 chronic lymphocytic leukemia (CLL); 21 mantle cell lymphoma (MCL); 42 lymphoplasmacytic lymphoma (LPL); 13 follicular lymphoma (FL) ; 58 MZL, 14 of which with numerous villous lymphocytes; 8 hairy cell leukemia (HCL)). Analyses were performed either on FACSCanto II (BD Biosciences, 3 centers) or on Navios (Beckman Coulter, 2 centers) instruments. Harmonization process was performed using Rainbow beads (Spherotech).For the CLL group, CD180 Median fluorescence (MdFI) in each center was not significantly different (Anova test, p>0.05). Instruments’ harmonization was therefore effective enough to obtain similar data from all centres. In the whole cohort, CD180 was significantly less expressed in the group of lymphomas -including CLL, MCL, LPL and FL- than in controls (Mann-Whitney test, p<0.05). Conversely, in the group of MZL and HCL, CD180 MdFI was not different from those of controls (Mann-Whitney test, p>0.05) but significantly higher than in CLL, MCL, LPL and FL (Mann-Whitney test, p<0.0001). Distinction between MZL and lymphomas with numerous villous lymphocytes was possible (Mann-Whitney test, p=0.0012) but not between MZL and HCL. ROC curve analysis determined a CD180 MdFI threshold of 1800 which allow the positive diagnosis of MZL with a sensitivity of 77% and specificity of 92%.These results underline the efficiency of CD180 as a single positive and robust marker for MZL diagnosis, and confirm that between centers and between instruments harmonization is largely feasible in routine practice as published recently (Solly F et al. Cytomery part A, 2013). It should be emphasized that among the group of lymphomas with intense expression of CD180, all interestingly originating from the spleen, those with numerous villous lymphocytes display the highest expression. We described for the first time in this study the strong positivity of CD180 in HCL. Anti-CD180 antibody may be included in diagnosis combination markers in order to improve the diagnosis of chronic B-cell malignancies DisclosuresNo relevant conflicts of interest to declare.

Clinical relevance of vascular endothelial growth factor type A (VEGFA) and VEGF receptor type 2 (VEGFR2) gene polymorphism in chronic lymphocytic leukemia.

Vascular endothelial growth factor type A (VEGFA) is a key regulator of angiogenesis and vascular permeability. Chronic lymphocytic leukemia (CLL) cells are able to secrete VEGFA and express VEGFA receptors, thus it can be hypothesized that VEGFA-mediated signaling influences CLL clone survival. In this case-control study we verified whether inherited differences in activities of VEGFA and its main receptor VEGFR2 impact predisposition to CLL or the course of the disease. Four functional single nucleotide polymorphisms (SNPs) including two SNPs in VEGFA gene, namely rs2010963 (+405G>C) and rs3025039 (+936C>T) and two SNPs in VEGFR2 gene including rs7667298 (-271G>A) and rs1870377 (+1719A>T) were genotyped using PCR-based assays in 223 Caucasian CLL patients and 150 matched controls. Regarding VEGF rs2010963 SNP, we observed an association between CLL and allele C distribution with an OR of 1.52 (95% CI, 1.002-2.312), p=0.04. The distribution of other genotypes and alleles was similar in CLL and control groups. No genotype or allele was significantly associated with important prognostic factors in CLL including clinical stage, IgVH mutational status, ZAP-70 expression and FISH cytogenetic abnormalities. In conclusion, the results of our study indicate that genetic polymorphisms in VEGFA mediated pathway may influence the susceptibility to CLL.

Association between polymorphism of GLI1 gene SNP rs2228226 and chronic lymphocytic leukemia in Chinese population.

A non-synonymous single-nucleotide polymorphism (SNP) (rs2228226C>G), in exon 12 of glioma-associated oncogene homolog 1 (GLI1) (Q1100E), encodes a change from glutamine to glutamic acid (Q1100E). The variant GLI1 protein exhibited reduced transactivation function in vivo, decreasing the ability of activating hedgehog signal, which has been proposed as an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL). The GLI1 Q1100E (NCBI SNP ID: rs2228226) genotypes in 155 CLL patients were detected by direct sequencing. The difference between frequencies of GLI1 Q1100E genotype among CLL patients and controls was statistically significant (p < 0.001). Logistic regression analysis revealed that in comparison with G/G, GLI1 SNP1100 C/C genotype was associated with a significantly increased risk of CLL (OR 3.787, 95% CI 1.814-7.907, p < 0.001). Compared with the SNP1100 C/G genotype, C/C genotype significantly increased the risk of CLL (OR 3.860, 95% CI 1.827-8.153, p < 0.001). In addition, combining C/G with G/G, C/C genotype also significantly increased the risk of CLL (OR 3.820, 95% CI 1.885-7.742, p < 0.001). The comparison between frequencies of C and G allele of GLI1 was also statistically significant (p = 0.004). In the entire cohort, SNP1100 genotypes were found in association with Binet stage (p = 0.045) and trisomy 12 (p = 0.036). By comparing C/C with G allele (C/G+G/G) genotype, there was a significant correlation with trisomy 12 (p = 0.013). This study demonstrated that GLI1 Q1100E polymorphism was closely associated with CLL. C/C genotype contributes to the risk of developing CLL and correlates with trisomy 12. Patients with trisomy 12 are susceptible group of CLL.

Decreased dickkopf-1 levels in chronic lymphocytic leukemia and increased osteopontin levels in non-Hodgkin's lymphoma at initial diagnosis: Could they be playing roles in pathogenesis?

AimsWe determined plasma levels of dickkopf-1 (DKK-1) and osteopontin (OPN) which have roles in the Wnt pathway in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) patients and in healthy controls. We also tested whether DKK-1 and OPN levels could be of clinical or prognostic significance in CLL and NHL.MethodsWe included 36 CLL, 24 NHL patients, and 21 healthy controls. Patients’ clinical and demographic features, treatment modalities, and response to treatment were recorded. DKK-1 and OPN levels in plasma obtained at initial diagnosis were determined with enzyme-linked immunosorbent assay.ResultsCLL patients had significantly lower DKK-1 levels than NHL and control groups (P levels, respectively, 0.048 and 0.017). OPN level was significantly higher in NHL group than in CLL and control groups (P values, 0.017 and <0.001). CLL patients with early and late Rai stages of disease had similar DKK-1 and OPN levels. After a median follow-up of 48 months, 13 CLL patients died. Univariate analysis showed that advanced Rai stages and older age were significantly poor prognostic factors. DKK-1 level in CLL patients who have died was significantly lower than those who were alive (P = 0.035). NHL patients with extranodal involvement had significantly higher OPN levels than those with no involvement (P = 0.04).ConclusionsOur results demonstrated that the Wnt pathway inhibitor DKK-1 was decreased in CLL. OPN was increased in NHL and associated with extranodal involvement. In order to reveal the pathogenic and clinical roles of DKK-1 and OPN in CLL and NHL, larger studies need to be conducted.

Entering the era of targeted therapy for chronic lymphocytic leukemia: impact on the practicing clinician.

Chemoimmunotherapy has been the standard of care for chronic lymphocytic leukemia (CLL). However, the introduction of B-cell receptor (BCR) kinase inhibitors such as ibrutinib has the potential to eliminate the role of chemotherapy in the treatment of CLL. How to best incorporate old and new therapies for CLL in this landscape is increasingly complex. This article reviews current data available to clinicians and integrates these data to provide a strategy that can be used to approach the treatment of CLL in the era of BCR signaling inhibitors. Current strategies separate patients based on age or functional status as well as genetics [presence or absence of del(17)(p13.1)]. In the era of targeted therapy, this will likely continue based on current available data. Phase III studies support chemoimmunotherapy as the initial standard therapy for patients without del(17)(p13.1). Choice of chemotherapy (fludarabine plus cyclophosphamide, bendamustine, or chlorambucil) and anti-CD20 antibody (rituximab, ofatumumab, or obinutuzumab) varies based on regimen and patient status. For patients with del(17)(p13.1), no standard initial therapy exists, although several options supported by phase II clinical trials (methylprednisolone plus alemtuzumab or ibrutinib) seem better than chemoimmunotherapy. Treatment of relapsed CLL seems to be best supported by ibrutinib-based therapy. Completion of trials with ibrutinib and other new agents in the near future will offer opportunity for chemotherapy-free treatment across all groups of CLL. Therapy for CLL has evolved significantly over the past decade with introduction of targeted therapy for CLL. This has the potential to completely transform how CLL is treated in the future.