Circulating CLL Cells Research Articles

Low Dose Rituximab Efficiently Clears Circulating CLL Cells and Maintains Sensitivity to Antibody Dependent Cellular Phagocytosis (ADCP)

Introduction: We have recently shown that high frequency (2 x week) low dose (50 mg)(HFLD) rituximab (RTX) and acalabrutinib is highly effective initial therapy for patients (n=38) with progressive CLL (ClinicalTrials.gov NCT03788291, PMID:36689726). The first dose of 50 mg of IV rituximab administered at 25 mg/h decreased the median circulating CLL cell count by 84% from pre-treatment baseline at 1h with no further decrease in the count during the remainder of the infusion (PMID:37003030). During the first hour of infusion, median CLL cell membrane CD20 levels decreased to 65% of baseline and were 41% of baseline at the end of the infusion. Median serum rituximab concentration was 2 mcg/ml at 1h and 10 mcg/ml at the end of the infusion. Median complement (CH50) concentration decreased to 91% of baseline at 1 h and to 81% of baseline at the end of the infusion. The cessation of clearance of CLL cells from the circulation within 1h of starting rituximab therapy is thus unlikely to be caused by low rituximab levels, decreased CLL cell CD20 expression or low serum complement levels. We and others have previously shown that ADCP is the primary mechanism of clearance of anti-CD20 mAb opsonized B cells in both humans and mice. We hypothesized that clearance of circulating CLL cells decreases because residual circulating CLL cells are resistant to mAb induced ADCP. To test this hypothesis, we measured in vitro ADCP sensitivity of CLL cells sampled from patients before and after rituximab treatment. Methods: Blood specimens were collected from 13 patients prior to starting therapy (C1D1_pre), 1h after start of IV rituximab infusion (C1D1_1h), at end of rituximab infusion (C1D1_post), and 48 h after initiation of therapy (C1D3_pre). Sensitivity of circulating CLL cells to in vitro ADCP was measured using live cell time-lapse high-content microscopy imaging of phagocytosis of CLL cells by human monocyte derived macrophages (hMDM) as previously described (PMID:32005699). Phagocytosis was quantified using a void index and sensitivity of samples was compared using the area under the curve of the 2h void index plot (AUC). Experiments were done in triplicate with co-cultures of hMDM and CLL cells without mAb as controls. Rituximab (10 mcg/ml) was used to test CD20 mAb mediated ADCP and alemtuzumab (10 mcg/ml) to test non-CD20 mediated ADCP. Results: Figure (median and 95% confidence intervals) shows that neither rituximab nor alemtuzumab induced ex-vivo ADCP as measured by median void index AUC was significantly altered by prior in vivo exposure of CLL cells to rituximab (Wilcoxon Match-Paired Sign test; all p>0.05). Discussion: The rapid decrease in circulating CLL cells induced by IV rituximab occurred only within the first hour after initiation of mAb infusion. Our measurements show that serum rituximab and complement concentrations and CLL cell membrane CD20 levels should be adequate to support additional CLL cell clearance after 1h. We now demonstrate that residual circulating CLL cells remain sensitive to in vitro ADCP by both rituximab and alemtuzumab. This suggests that despite reduced levels of surface antigen, circulating cells remain sensitive to mAb mediated clearance. Alternative explanations for the limited clearance of circulating CLL cells include: 1. Saturation of the limited cytotoxic capacity of the innate immune effector cells (Kupffer cells and splenic macrophages) compatible with our previously reported data on finite macrophage ADCP capacity (PMID:32556153); 2. Mobilization of CLL cells from lymphoid tissue; and 3. Temporary sequestration of rituximab opsonized CLL cells in tissue (e.g. spleen or lungs), which is unlikely given the observed decrease in lymph node size following initiation of therapy. Ongoing research is testing these hypotheses.

Ibrutinib sensitizes CLL cells to venetoclax by interrupting TLR9-induced CD40 upregulation and protein translation

Chronic lymphocytic leukemia (CLL) cells upregulate Bcl-2 proteins within the lymph node (LN) microenvironment. Signaling via B-cell receptor, Toll-like receptors and CD40 collectively reduce sensitivity to the BCL-2 inhibitor venetoclax. Time-limited treatment with venetoclax plus the BTK-inhibitor ibrutinib results in deep remissions, but how this combination affects LN-related signaling is not yet completely clear. Therefore, samples obtained from the HOVON141/VISION phase 2 clinical trial were used to analyze this. Two cycles of lead-in ibrutinib monotherapy resulted in decreased protein expression of Bcl-2 proteins in circulating CLL cells. Strikingly, at this timepoint CD40-induced venetoclax resistance was strongly attenuated, as was expression of CD40. Since CD40 signaling occurs within the CLL LN, we tested various LN-related signals that could affect CD40 signaling. While BCR stimulation had only a minor effect, TLR9 stimulation via CpG led to significantly increased CD40 expression and importantly, reverted the effects of ibrutinib treatment on venetoclax sensitivity by inducing overall protein translation. Together, these findings identify a novel effect of ibrutinib: interruption of TLR9-induced CD40 upregulation and translation of pro-survival proteins. This mechanism may potentially further inhibit priming of CLL cells in the LN microenvironment for venetoclax resistance.

Paradoxical activation of chronic lymphocytic leukemia cells by ruxolitinib in vitro and in vivo.

Chronic lymphocytic leukemia (CLL) is characterized by an aberrant cytokine network that can support tumor growth by triggering janus kinase (JAK)/STAT pathways. Targeting cytokine-signaling should then be a rational therapeutic strategy but the JAK inhibitor ruxolitinib failed to control and seemingly accelerated the disease in clinical trials. The effect of ruxolitinib on primary human CLL cells was studied in vitro and in vivo. Ruxolitinib increased phosphorylation of IRAK4, an important toll-like receptor (TLR)- signaling intermediate, in circulating CLL cells in vitro. It also enhanced p38 and NFKB1 phosphorylation while lowering STAT3 phosphorylation in CLL cells activated with TLR-7/8 agonists and IL-2. Among the cytokines made by activated CLL cells, high levels of IL-10 contributed strongly to STAT3 phosphorylation and inhibited TLR7 activity. Ruxolitinib limited TLR-mediated IL10 transcription and markedly reduced IL-10 production in vitro. It also decreased blood levels of IL-10 while increasing TNFα along with phospho-p38 expression and gene sets associated with TLR-activation in CLL cells in vivo. The bruton's tyrosine kinase inhibitor ibrutinib decreased IL-10 production in vitro but, in contrast to ruxolitinib, blocked initial IL10 transcription induced by TLR-signaling in vitro, decreased TNFα production, and deactivates CLL cells in vivo. These findings suggest the possible benefits of inhibiting growth factors with JAK inhibitors in CLL are outweighed by negative effects on potential tumor suppressors such as IL-10 that allow unrestrained activation of NFκB by drivers such as TLRs. Specific inhibition of growth-promoting cytokines with blocking antibodies or infusing suppressive cytokines like IL-10 might be better strategies to manipulate cytokines in CLL.

Rituximab Induces an Inflammatory Cytokine Response with Increased IP-10 (CXCR10) in Patients with Infusion Reactions

Most patients initiating anti-CD20 monoclonal antibody (mAb) therapy for CLL have a disruptive infusion reaction that can cause serious complications and be fatal. A better understanding of the risk factors and etiology of infusion reactions is required to improve preventative measures and management. We report a study to test the hypothesis that IV rituximab induces an inflammatory cytokines response with differences in individual cytokine concentrations correlating with infusion reactions. We designed a clinical trial (NCT03788291) for initial treatment of patients with progressive CLL that provided clinical data and blood specimens from 37 patients treated with 50 mg of IV rituximab infused at 25 mg/h. Blood specimens were collected immediately prior to initiation of rituximab (baseline), one hour later (1h), at the end of the infusion (post-infusion) and at 48 h after initiation of therapy (48h). Infusion reactions (CTCAE grade ≥2 events) were managed by interrupting the infusion and initiating symptomatic treatment. We measured circulating CLL cell counts and CD20 levels, and serum rituximab concentration, complement levels, and cytokine concentrations of IFN-γ, IL-10, IL-18, IL-2, IL-4, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, and TNF-α using standard methods. Twenty-four (65%) patients had 25 infusion reactions (all CTCAE grade 2) at a median of 78 minutes (range 35-128) after initiation of rituximab therapy (median dose 32 mg, range 15-50). All patients completed the 50 mg rituximab infusion (median time 150 minutes, range 119-195). Patient and CLL characteristics including baseline CLL cell counts did not predict the risk of infusion reactions. Median CLL counts at 1h decreased to 14.7% of baseline with no further changes post-infusion and no significant difference between patients with or without infusion reactions (60x109/L vs. 45x109/L, p=0.61). Median CLL cell membrane CD20 levels (x103 molecules/cell) decreased from 4.77 at baseline to 3.10 at 1h (p=0.002) and 1.59 post-infusion (p<0.0001 vs. baseline and 1h) with no significant differences between patients with or without an infusion reaction. The median estimated number of circulating CLL cell membrane CD20 molecules (x1012 molecules/L) decreased from 325.8 at baseline to 34.1 at 1h (p<0.0001) and 14.5 post-infusion (p<0.0001 vs. baseline, p<0.03 vs. 1h) with no significant differences between patients with or without an infusion reaction. There was no correlation between risk of infusion reaction and serum levels of rituximab or serum complement. Thirty-five (95%) patients had a ≥4 fold increase in serum concentration of ≥1 cytokine with resolution of the cytokine response at 48h. All patients with an infusion reaction had significantly higher median post-infusion serum concentrations of IP-10, IL-6 and IL-8. All 17 patients with IP-10 serum concentrations above the limit of detection (40,000 pg/ml) post-infusion had an infusion reaction, and all 24 patients with an infusion reaction had a ≥4 fold increase in serum IP-10 concentrations. Of 13 patients without an infusion reaction, the highest measured serum IP-10 concentration was 22,013 pg/ml and only 3 (23%) had ≥4 fold increases in serum IP-10 concentrations. Slow infusion of a reduced dose of rituximab induced a cytokine response in 95% of patients and therapy disrupting infusion reactions occurred at a comparable rate (65%) to standard rituximab therapy. Infusion reactions were associated with increased levels of IP-10, IL-6 and IL-8. IP-10 levels were increased by ≥4 fold in all patients with an infusion reaction compared to 23% without an infusion reaction. These data suggest that rituximab infusion reactions are caused by inflammatory cytokines produced by cytotoxic effector cells activated by rituximab independent of the pre-treatment amount of circulating CLL cells or CD20 levels, or efficacy of rituximab in reducing the circulating CLL cell count. Infusion reactions occurred after at least 35 minutes of therapy concurrent with a rapid decrease in circulating CLL cells. This observation suggests that cytokine release could be a result of activation of effector cells responsible for CLL cell elimination. Our data provides novel insights into the potential mechanism of infusion reactions and could form the basis of further research to determine methods to control this clinically important and therapy limiting complication.

S144: IMMUNE RESTORATION AND SYNERGISTIC ACTIVITY WITH FIRST-LINE (1L) IBRUTINIB (IBR) PLUS VENETOCLAX (VEN): TRANSLATIONAL ANALYSES OF CAPTIVATE PATIENTS WITH CLL

Background: Ibr and Ven work synergistically through their distinct and complementary modes of action: BTK inhibition by Ibr decreases levels of anti-apoptotic MCL-1 and BCL-XL proteins, but not BCL-2, and therefore increases sensitization to BCL-2 inhibition by Ven in vivo. Single agent Ibr has been shown to normalize CLL-associated immune cell alterations in number and function but the impact of the Ibr plus Ven combination (I+V) on immune cells has not been evaluated. Aims: To 1) confirm BCL-2 sensitization by single-agent Ibr and 2) monitor changes in the cellular immune profile of patients (pts) treated with I+V in CAPTIVATE (NCT02910583), a multicenter phase 2 study evaluating 1L I+V treatment (tx) in CLL and SLL. Methods: Ibr (420mg po daily) effects on anti-apoptotic proteins were evaluated by flow cytometry with samples from 4 previously untreated pts with CLL treated for 1 cycle (28 days). Immune restoration was evaluated in 79 previously untreated pts with CLL enrolled in the CAPTIVATE Minimal Residual Disease (MRD) cohort. Pts received 3 cycles of Ibr, followed by 12 cycles of I+V (Ibr 420 mg/d orally; Ven ramp-up to 400 mg/d orally). Pts who met criteria for confirmed undetectable MRD (Confirmed uMRD) were then randomized 1:1 to placebo fixed-duration (FD) tx or Ibr at cycle 16; pts who did not meet Confirmed uMRD criteria (uMRD Not Confirmed) were randomized 1:1 to Ibr or I+V. Cryopreserved peripheral blood mononuclear cells at baseline (pre-dose cycle 1) and day 1 of cycles 4, 7, 16, 20, 23, and 29 in each of the 4 arms were analyzed by high-dimensional flow cytometry (34-color panel). Immune cell subset counts of B cells, T cells, monocytes, dendritic cells (DCs), myeloid-derived suppressor cells, natural killer cells, and innate lymphoid cells were calculated and compared to those of 20 age-matched healthy donors. Median changes from baseline are reported. Results: In pts treated with single-agent Ibr for 1 cycle, MCL-1, BCL-XL and BCL-2 expression decreased by 74%, 95% and 10%, respectively, in lymph node emigrant (CD5+ CXCR4dim) CLL cells, confirming increased BCL-2-dependence and corresponding inhibitor sensitization. In CAPTIVATE pts treated with I+V, a rapid and significant decrease in circulating CLL cells was observed within the first 3 cycles of Ven tx initiation (Fig 1A, cycle 7). From cycle 16 and onwards, Confirmed uMRD pts had CLL cell counts similar to healthy donors (≤0.8 CLL cell/mL), while in uMRD Not Confirmed pts counts were 1.5 to 22.9 CLL cell/mL. Subsequently, normal B-cell count restoration occurred in Confirmed uMRD pts randomized to placebo (FD tx) with a recovery to levels similar to healthy donors (+332% at cycle 29; Fig 1B). Across all arms, abnormal counts of T cell subsets, classical monocytes and conventional DCs characteristic of CLL were normalized to healthy donor levels (‒49%, +101% and +91% respectively) within the first 6 months of tx and were maintained thereafter regardless of randomized tx. Plasmacytoid DCs counts recovered by cycle 20 (+598%) in all 4 arms. Image:Summary/Conclusion: Sensitization to BCL-2 inhibitor effects by Ibr supports the synergistic activity of I+V. The FD regimen (Confirmed uMRD, placebo arm) of I+V effectively eradicated CLL cells to healthy donor levels and enabled sustained regeneration of normal B cell counts in contrast to ongoing therapy in the remaining tx arms. Combination I+V also allowed for normalization of other critical immune cells, including T cell subsets, classical monocytes, and conventional and plasmacytoid DCs. These data demonstrate promising evidence of immune restoration with FD I+V tx.

BCR Unresponsiveness and 13q14 Duplication Underpin Spontaneous Disease Regression in Chronic Lymphocytic Leukemia

Spontaneous regression of chronic lymphocytic leukemia occurs in less than 2% of cases and the mechanisms that underlie this rare event remain largely unknown. We reviewed 1425 patients with CLL across 4 centers and identified 20 who underwent spontaneous disease regression without treatment over a median period of 8.6 years. This included 12 patients with complete and 8 with partial (> 50%) resolution of lymphocytosis and clinical features. Residual circulating CLL cells remained detectable in all cases and phenotypic, functional and genomic analyses were performed on the residual CLL clone, as well as stored cells where available, to investigate potential mechanisms underlying regression.IGH sequence analysis of sequential samples confirmed that the CLL clone was identical between the time of diagnosis and regression (n=6). Moreover, all cases showed somatic mutation of the IGHV gene and VH3 or VH4 usage was seen in 17 cases. Flow cytometric analysis of spontaneous regression cases (n=17) showed that Ki-67 and CD49d expression was markedly decreased compared to stable indolent disease (n=54) and progressive disease (n=40) whereas CXCR4 and FasR expression was increased. No difference was observed with respect to the expression of CD38, ZAP-70, Bcl-2 and Mcl-1 between spontaneous regression and indolent cases.Sequential analysis revealed that these changes in Ki-67 and CXCR4 expression developed during the period of regression. Furthermore, telomere length analysis demonstrated that tumor cells generally had short telomeres (< 5 kb) after regression and this profile, together with the phenotypic progression, indicates that tumors have undergone an initial period of intense proliferation prior to the development of regression. Importantly, telomerase expression remained high in regression tumors and no evidence of cellular senescence was observed. Analysis of the immune compartment revealed a reduction in T cell and NK cell numbers during spontaneous CLL regression and a modest improvement in T cell function as revealed by increased T cell proliferation and decreased expression of PD-1.The response of tumor cells to stimulation through the B cell receptor (BCR) was also investigated. Surface expression of IgM and IgD was low in regression cases and basal Erk phosphorylation was increased. In addition, these tumors were uniformly unresponsive to stimulation separately with anti-IgM F(ab‘)2 antibodies and anti-IgD F(ab‘)2 antibodies, and combined stimulation with both antibodies. These findings indicate that spontaneously regressed CLL tumors are in a state of profound unresponsiveness to BCR stimulation. Consistent with reduced BCR responsiveness, RNA-seq analysis of sorted CLL cells revealed reduced expression of both MYC and Myc target genes in regression cases as well as downregulation of pathways involved in RNA processing, cellular metabolism and proliferation.Finally, the genomic landscape of tumor cells after regression was analyzed by whole exome sequencing and SNP array analysis on paired germline and CLL samples. CLL genomic complexity was generally low although mutations in TP53, MYD88 or HIST1H1B were observed in 5 tumors. Strikingly, 13q14 duplication was present in 13 of 19 tumors and qPCR analysis showed that this was associated with increased expression of miR-15a/16-1.This is the largest and most comprehensive analysis of spontaneous CLL regression to date and reveals a range of novel features. Tumors that have undergone spontaneous regression express somatically mutated immunoglobulin genes, demonstrate short telomeres with a phenotype of BCR unresponsiveness and have a very high prevalence of 13q14 duplication. This profile suggests a model in which CLL clones undergo substantial proliferation initially which subsequently subside into a state of BCR unresponsiveness and low proliferation. The identification of 13q14 duplication as a recurrent genetic abnormality indicates a potential molecular mechanism for this pattern but it is currently unclear if this lesion is established during tumorigenesis or is acquired during the initial proliferative phase. These findings identify CLL regression as a unique subset of B cell lymphoproliferative disease and suggest that therapies which upregulate the expression of miR-15a/16-1 may represent a mechanism to suppress B-CLL proliferation. [Display omitted] DisclosuresRawstron:Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; BD biosciences: Patents & Royalties; Roche: Consultancy, Honoraria; Gilead: Research Funding. Munir:Alexion Pharmaceuticals, Inc.: Honoraria; Roche: Honoraria; Gilled: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Hillmen:AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Honoraria, Research Funding; Celgene: Research Funding; GSK: Consultancy, Honoraria, Research Funding.

Anti-CD20 Therapy Reliance on Antibody-Dependent Cellular Phagocytosis Affects Combination Drug Choice

The efficacy of many therapeutic unconjugated monoclonal antibodies (mAbs), including those targeting CD20 in CLL, requires immune cell-mediated cytotoxicity. mAbs have often been optimized for natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC) activity. However, in vivo mouse studies have shown that antibody-dependent cellular phagocytosis (ADCP) by macrophages is the major mechanism of clearance of circulating B cells by anti-CD20 mAbs. To directly compare ADCC versus ADCP, we previously used a panel of anti-CD20 mAbs (rituximab, ofatumumab, obinutuzimab, ocaratuzumab) to test cytotoxicity of paired human NK cells and monocyte-derived macrophages (hMDM) against CLL cells in vitro. All mAbs demonstrated ADCP activity at least 10-fold greater than ADCC as measured by CLL cell depletion per effector cell. Moreover, ADCC and ADCP activity levels did not correlate, meaning that ADCC cannot be used as a surrogate measure of ADCP for these mAb. This could explain why mAb optimization for ADCC activity has often failed to translate into more efficacious treatment. Thus, ADCP may be an effective translational measurement of anti-CD20 mAb performance. Because of the clinical interest in combining anti-CD20 mAb with targeted small molecule inhibitors, we began studying the effects of the Bruton tyrosine kinase (BTK) inhibitors on anti-CD20 mAb-mediated ADCP. Our initial studies showed that ibrutinib (IBR), but not acalabrutinib (Acala), significantly decreased anti-CD20 ADCP as measured by a flow cytometry-based assay that measures single timepoint cell collections. These types of assays cannot easily determine the kinetics and individual effector cell activity of ADCP. Thus, to more fully study the BTK inhibitor effects on ADCP, we developed a live cell time-lapse imaging method for measuring ADCP, utilizing recent advances in microscopy, cellular dye labeling, digital imaging, imaging software and computing. Whole-cell labeling of macrophages enabled visualization of internalized CLL cells as regions of dye exclusion or "voids". Because of the vast number of images acquired during live cell time-lapse imaging, we utilized computer software-aided image recognition and enumeration to measure the number of macrophages and voids inside each macrophage. As a measure of phagocytic engulfment, we developed a void index, which provides a relative measure of phagocytic engulfment per macrophage. Measuring ADCP in this manner replicates clinical observation of mAb therapeutic activity. Clinically, intravenous anti-CD20 mAb therapy typically induces a rapid decrease in circulating CLL cells (within hours), followed by a long period (days) of stable to increased levels of circulating cells. Similarly, our live cell time-lapse video assay shows initial rapid ADCP over the first 2 hours followed by a prolonged period of "hypophagia" with little ADCP for the remainder of the assay (imaged every 2 minutes for 8 hours). This "hypophagia" phenomenon may explain the resistance to therapeutic mAb observed clinically. With these new tools for quantitation of ADCP, we compared the effects of serial dilutions of IBR or Acala on ADCP. Overall, as measured by Area Under the Curve (AUC) analysis, IBR decreased phagocytic capacity of anti-CD20 mediated CLL cell ADCP at concentrations of 0.41 μM and above. By contrast, Acala did not begin to decrease AUC measurements until 3.7 μM, and subsequent AUC values were higher in Acala versus IBR-treated ADCP assays up to the highest tested drug concentration (100 μM). Similarly, the initial ADCP kinetics (void index / min over the first hour) reflected a decrease with IBR treatment at 0.41 μM that continued until a nadir was reached at 33 μM. In contrast, Acala did not induce a decrease in this kinetic measurement until 3.7 μM and a nadir was not reached (up to 100 μM). Thus, IBR significantly decreases ADCP by hMDM at concentrations much lower than a more specific BTK inhibitor, Acala. This result suggests that BTK inhibition has little to no effect on ADCP and furthermore suggests that IBR off-target effects decrease ADCP. IBR off-target candidates include other tyrosine kinases in the TEC (tyrosine kinase expressed in hepatocellular carcinoma) family. These data suggest that a a highly selective BTK inhibitor with little effect on ADCP could be a more suitable drug to combine with therapeutic mAb(s). Disclosures Chu: Pfizer: Equity Ownership; Acerta Pharma: Research Funding. VanDerMeid:AstraZeneca: Research Funding. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents &amp; Royalties: Acalabrutinib patents; AstraZeneca: Equity Ownership. Munugalavadla:Acerta Pharma: Employment; AstraZeneca, Gilead Sciences: Equity Ownership. Barr:TG Therapeutics: Consultancy, Research Funding; Celgene: Consultancy; Pharmacyclics LLC, an AbbVie company: Consultancy, Research Funding; Seattle Genetics: Consultancy; Merck: Consultancy; Genentech: Consultancy; Verastem: Consultancy; Gilead: Consultancy; Astra Zeneca: Consultancy, Research Funding; Janssen: Consultancy; AbbVie: Consultancy. Elliott:Astra Zeneca: Research Funding. Zent:Mentrik Biotech: Research Funding; Astra Zeneca: Research Funding.

Constitutively Activated STAT3 Induces the Production of PTX3 That Contributes to the Induction of Bone Marrow Reticulin Fibrosis in Patient with CLL

Constitutively Activated STAT3 Induces the Production of PTX3 That Contributes to the Induction of Bone Marrow Reticulin Fibrosis in Patient with CLL

CLL Cells from Ibrutinib-Induced Lymphocytosis of Relapsed/Refractory Chronic Lymphocytic Leukemia Patients Are Responsive to Obinutuzumab, but Not Rituximab, Ex Vivo

Abstract Introduction: Ibrutinib is an irreversible first-in-class inhibitor of BTK (Bruton tyrosine kinase) approved for the therapy of relapsed/refractory chronic lymphocytic leukemia (R/R CLL). The drug mediates a transient increase in circulating CLL cells together with reduction in spleen and lymph node size, by both cellular mobilization and apoptosis of resident CLL cells (Herman SE, et al. Blood 2014;123:3286-95). These events occur with important patients' inter-variability (Herman SE, et al. Leukemia 2014;28:2188-96), one cluster of patients presents with greater peak lymphocytosis (resolving between 1 to more than 6 months), while another cluster presents with rapid resolution of lymphocytosis and lymph node/spleen size within 2 months. Upon such dramatic shifts in disease distribution the first 2 months of therapy (and sometimes lasting &gt;6-12 months), the question of phenotypic changes, sensitivity to monoclonal antibodies (MoAbs), and subclonal diversity of circulating cells remains central for further combination studies. In this study, we evaluated changes in CD5, CD19, and CD20 expression in vitro/in vivo, and peripheral blood side population (SP) cells (a fraction highly enriched in chemorefractory cells, Gross E, et al. Leukemia 2010;24:1885-92) upon ibrutinib therapy. We also investigated whether patterns of lymphocytosis may predict for response to rituximab (RTX) or obinutuzumab (GA101). Methods: R/R CLL patients (n=25) median prior lines=4, range=2-8), PBMCs were collected before ibrutinib initiation and after 1 and 2 months of therapy. PBMC were seeded at 10 x 106 cells/mL in culture medium and treated for 7 days with 10µg/mL control IgG1 (trastuzumab), RTX or obinutuzumab. The specific percentage of remaining B cells in MoAbs-treated samples was calculated as (absolute number in treated samples/absolute number in control samples) x 100. For each condition, absolute number of remaining B cells =total viable cell number (trypan blue exclusion determination) x % of viable CD19+/CD5+ lymphocytes (flow cytometry determination). For statistical analyses, Student's test (paired, two-sided) was used (*p&lt;0.05;**p&lt;0.01;***p&lt;0.001). Results: We firstanalyzed patterns ofabsolute lymphocytes count ( ALC) across 23 patients receiving ibrutinib (Fig 1a) to classify them into two clusters as previously published (Fig 1b): Cluster 1 and cluster 2 did not differ significantly in terms of initial lymphocytosis, line of therapy, gender, karyotype, IgHV. Interestingly, the SP fraction in peripheral blood was significantly increased (median: 5/microL before ibrutinib, 10/microL at peak lymphocytosis), suggesting mobilization of resident SP cells, although no apoptosis was detected (in vitro or in vivo) with ibrutinib. We next assessed CD5, CD19 and CD20 levels in vitro (n=22) and in vivo (n=15) upon ibrutinib therapy. In vitro, ibrutinib significantly reduced CD20 (Fig 2a) and CD19 surface expression, but not CD5; nevertheless anti-CD20 MoAbs still had activity in vitro (Fig 2b). Expression levels were not linked to clusters 1 or 2. Finally we compared RTX- and obinutuzumab-induced B-cell depletion before administration of ibrutinib, and at various sampling time points (1 to 6 months). Obinutuzumab induced significantly superior depletion at various timepoints than RTX. More interestingly, when analysis was performed from paired samples before/during ibrutinib therapy from the same ibrutinib-exposed patients, only obinutuzumab-induced depletion was increased in cluster 2 (Fig 3). Conclusions: Ongoing and planned clinical studies evaluate the combination of ibrutinib and obinutuzumab in CLL (first-line and relapsed). Some concerns have emerged due to published preclinical data showing that ibrutinib can interfere with efficacy of therapeutic antibodies. Here, we suggest that ibrutinib-exposed CLL cells, despite wide inter-patient heterogeneity, are targetable with obinutuzumab. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Klein: Roche: Employment.

Microenvironment Regulation of IL23R/IL-23 Axis Drives Chronic Lymphocytic Leukemia (CLL) Progression

Background: CLL displays a considerable degree of clinical heterogeneity, which is in part ascribable to clone-intrinsic biological features and that are also influenced by clone-extrinsic events related to the microenvironment. Among the dynamics-taking place within the CLL microenvironment, those finalized to the induction of an overly inflammatory milieu may significantly impact on the CLL natural history by hijacking the immunological microenvironment at the same time fostering clone fitness. IL-23 acts as a prototypical pro-inflammatory mediator representing a promising therapeutic target. We analyzed the ability of CLL cells to sense IL-23 through the IL-23R complex (consisting of IL12Rß1 and IL23R subunits) expression and correlated this feature with clinical outcome. Moreover, we investigated the synthesis of IL-23 within the CLL microenvironment, and tested the biological effects of the IL-23/IL-23R axis engagement and of its interference in vitro and in vivo.Methods: IL23R complex was detected by quadruple flow cytometry staining with CD19, CD5, IL23R, and IL12Rβ1 in prospectively enrolled CLL cases (O-CLL1 protocol, clinicaltrial.gov identifier NCT00917540). On human tissue specimens, lymph node and bone marrow samples from 16 CLL patients were selected for in situ immunolocalization analyses. NOD/Shi-scid/γcnull (NSG) mice were used for in vivo xenografts, in which activated autologous T cells (AAT), obtained by adding anti-CD3 and CD28 Dynabeads and rIL2 were co-injected with CLL cells. MiRNA analysis was performed by Agilent's Human V2 platform and by quantitative PCR. MirVANA microRNA mimics and inhibitors were purchased from Ambion, Inc. For 3'UTR luciferase reporter experiments, miRNA target reporter vectors were purchased from Origene.Results: By flow cytometry, circulating CLL cells of 281 cases variably expressed IL23R side chain while consistently lacking IL12Rß1 chain expression. The engagement of the uncoupled IL23R complex expression (i.e. IL23R but not IL12Rb1 expression) by IL23 did not activate downstream signaling pathways, such as the up-regulation of pSTAT3. The 3-year TTFT probability of patients with low IL23R expression (IL23R-low) was 91% as compared to 75% of IL23R-high cases [χ2 9.1, P=.003; HR=3.2, 95%CI (1.4-7.1)]; in a multivariate model, IL23R expression still remained independently associated with TTFT. We explored the potential control of IL23R expression in CLL cells by miRNA and found 15 miRNAs inversely associated with IL23R expression, five of which predicted as regulators (miRNA-146b-5p, miRNA-155, miRNA-324-5p, miRNA-532-3p and miRNA-630). Among these, miR-324-3p and miR-146b-5p were demonstrated to functionally regulate the expression of IL23R and IL12Rβ1 proteins in CLL cells, respectively. Within lymphoid tissues, in situ, CLL clones expressing IL23R side chain also showed expression of IL12Rß1, which varied according to the density of CD40L-expressing bystander elements suggesting a microenvironment-driven regulation of the IL-23R complex. To functionally test this hypothesis, CLL cells were co-cultured in the presence of NIH-3T3 transduced with CD40L or with AAT cells. A significant up-regulation was observed for both the IL12Rß1 and IL23R side chains, suggesting the environment co-stimulation as a mechanism of IL-23R complex regulation. Consistently, the IL-23R complex was upmodulated in CLL cells expressing IL-23R but not IL12Rß1, upon xenograft with autologous T cells into NOD-Scid mice. We then investigated the effect of IL-23R engagement by IL-23 in CLL cells and found that IL-23R activity correlated with CLL cell proliferation and survival in vitro via STAT3 phosphorylation. The trophic nature of IL-23-mediated stimuli over CLL cells was further demonstrated in vivo through the adoption of an anti-IL23p19 monoclonal antibody for clinical use, which proved to be effective in eradicating the xenografted CLL clone in the infiltrated tissues (spleen, liver and BM) by inhibiting proliferation and inducing apoptosis. Noteworthy, the therapeutic effect of IL-23 antagonism was demonstrated by histopathology, flow cytometry and BCR clonality.Conclusions: Overall, we demonstrated that IL-23/IL-23R axis is a novel microenvironment-regulated determinant in CLL pathobiology representing a strong prospect in disease prognostication and treatment. DisclosuresNo relevant conflicts of interest to declare.

Sphingosine Kinases (SK): Key Molecules Associated with the Activation, Proliferation and Ibrutinib-Induced Cell Death of Chronic Lympocytic Leukemia Cells

Leukemic cells from CLL patients can survive and proliferate within lymphoid tissues where the supportive microenvironment favors their accumulation. We have previously reported that the activation of CLL cells reduces the expression of the main receptor for sphingosine 1-phosphate (S1P) (Borge M, J Immunol, 2014), a bioactive phospholipid that participates in lymphocyte egress from lymphoid tissues. S1P also mediates several biological functions, including cell growth stimulation and protection of apoptosis, through receptor independent intracellular mechanisms. S1P is generated by two isoforms of sphingosine kinases (SK1/2) and its levels are tightly controlled via degradation by intracellular S1P lyases (S1PL). Several studies have implicated the SK/S1P/S1PL pathway as an essential regulator of cell proliferation and survival in different cancer cells. The aims of our study were: a) to evaluate the expression of SK and S1PL in CLL cells, b) to assess whether key microenvironment signals are able to modulate this expression and c) to evaluate the effect of SK inhibitors on the activation and survival of the leukemic clone.We measured the basal expression of SK and S1PL by qRT-PCR on purified B cells from CLL patients (n=22) and aged-matched healthy donors (n=9), and found that CLL cells express high levels of SK1, favoring an increased SK1/S1PL ratio in the malignant clone compared to healthy B lymphocytes ( p<0.05). Similar results were obtained when SK2 was evaluated. The in vitro activation of CLL cells with anti-IgM+CD40L increased SK1/S1PL ratio (n=10, p<0.01) and the expression of the activation marker CD69. To evaluate the expression of SK1/S1PL within in vivo activated CLL cell subpopulations, we segregated the proliferative fraction (PF) of circulating CLL cells from the quiescent fraction (QF) in the same sample. As it was previously described, this PF is characterized by the presence of an active class-switch recombination process and a high expression of proliferation-related genes, such as Ki-67, c-myc, CD49d, and p27-Kip1 (Palacios F, Blood, 2010). Interestingly, SK1/S1PL ratio was increased in the PF compared with the QF (n=3). Additionally, bone marrow leukemic cells expressing high levels of CD38, which defines a subpopulation of activated lymphocytes, showed a higher S1P/S1PL ratio compared to CD38 low or negative counterparts (n=3),showing that the in vivo activated CLL cells expressed higher ratios of SK1/S1PL compared with the rest of the leukemic clone. Finally, we wondered whether the inhibition of SK impairs the survival, activation and proliferation of the leukemic clone. To this aim we employed a commercial selective SK1 and SK2 inhibitor (SKI-II, 5 and 15μM), which did not affect cell viability (n=10, evaluated at 24, 48, 72 and 96hs). However, it was able to impair the expected upregulation of CD69 induced by IgM+CD40L at 24hs (n=10, p<0.001) and the leukemic cell proliferation evaluated by CFSE dilution assay at 96hs (n=10 , p<0.001). Moreover, while SKI-II did not increase the sensitivity of CLL cells to Fludarabine or Bendamustine, it was able to enhance the cell death induced by Ibrutinib (0.3 and 3μM) (n=5, p<0.05 and p<0.01 respectively).Taking together, our results suggest that SK/S1P/S1PL axis might participate in the accumulation of the malignant clone in CLL patients and the disruption of this pathway might be a potentially effective treatment option in the future. DisclosuresBezares:Janssen: Honoraria. Gamberale:Janssen: Honoraria.

CXCL12 Enhances CLL Cell and T-Cell Migration in a Dynamic Circulating Model of CLL That Can be Abrogated By the CXCR4 Antagonist ONO-7161

We have recently developed a circulating model of CLL that more accurately mimics the transient interactions that take place between lymphocytes and the vascular endothelium. Normal and malignant lymphocytes actively undergo transendothelial migration in our system in the absence of a chemoattractant. We have now refined our model to include a CXCL12 gradient and evaluated the ability of CXCR4 antagonists to modulate lymphocyte migration. Initially we demonstrated that a single deposition of CXCL12 (100ng/ml) into the extravascular space (EVS) of our model significantly increased CLL cell migration (P = 0.049, mean circulating CXCL12 level = 254pg/ml). Subsequently, we created a stable chemokine gradient by seeding CXCL12-secreting MRC5 cells into the EVS. Although this resulted in lower peak circulating levels of CXCL12 (as measured by ELISA, mean 23pg/ml), it significantly increased migration (P = 0.024) presumably because of the constant nature of the chemokine gradient. As previously shown CXCR4 expression is up-regulated on circulating CLL cells when compared to static cultures (P=0.023). Basal CXCR4 expression and expression after circulation for 48h, correlated with the degree of CLL cell migration into the EVS (r2=0.40, P=0.003; r2=0.31, P=0.009 respectively). It is worthy of note that as a proportion of all circulating peripheral blood mononuclear cells, CD3+ T-cells migrated significantly more than CD19+ CLL cells (T-cells: 12.72% ±13.47, CLL cells: 1.16% ±1.23 P =0.02, n = 10). This may be partly the result of the known defects in CLL cell migration but is also likely to be promoted by CLL cell secretion of T-cell attracting chemokines CCL3 (298pg/ml ±197.5, n=13) and CCL4 (56pg/ml ±115.6, n=13) in our model. Furthermore, migrated T-cells and CLL cells recovered from the EVS showed a marked increase in Ki-67 expression (P<0.0001 and P<0.0001 respectively) confirming that both subsets of cells are activated. We next explored whether we could block the CXCR4/CXCL12 axis using the CXCR4 antagonist ONO-7161. In transwell chambers (3μm pores) 30nM ONO-7161 resulted in significantly decreased CLL cell migration (54.1% ±25.53 of control, P=0.0003) and was more potent than Plerixafor (1μM) (P=0.06). Additionally, in the circulating system, ONO-7161 resulted in significantly reduced CLL cell migration into the EVS at 48h (P=0.0006). T-cell migration was also reduced by ONO-7161 but not significantly. In summary, we have enhanced our circulating model system of CLL by adding a physiologically relevant CXCL12 chemokine gradient. This then allowed us to dissect the role of the CXCR4/CXCL12 axis in modulating CLL cell migration. Finally we demonstrated that the CXCR4 antagonist ONO-7161 was a potent inhibitor of CLL cell migration in our model. Given that ONO-7161 was non-toxic to CLL cells at concentrations up to 10μM, it seems likely that it might be usefully combined with chemotherapy and biologically targeted agents in the treatment of this and other diseases. DisclosuresYoshizawa:Ono Pharmaceutical Co., Ltd.: Employment. Fegan:ONO PHARMA: Honoraria.

MicroRNA-21 Is Differentially Expressed in the Lymphoid Tissue and Modulated By Stromal Signaling in Chronic Lymphocytic Leukemia

MicroRNAs contribute to the initiation and dissemination of malignant clone and serve as important prognostic indicators. miR-21 overexpression was linked to fludarabine resistance and unfavorable prognosis in CLL. We and others have shown that B-cell receptor (BCR) signaling upregulates miR-21 in CLL. While recent literature addresses the impact of miR-21 in the circulating malignant B cells, few studies have focused on its expression in CLL lymph nodes, where cells receive stromal pro-survival signals including via BCR. In those tissues, some microRNAs demonstrate distinct expression patterns, e.g. miR-155 is highly expressed in the proliferation centers. Here we aimed to investigate miR-21 expression in the lymphoid tissue and assessed the effect of the modeled lymphoid niche on this MIR in CLL cells in vitro.Expression of miR-21, RNU6B and CD20 was assessed in 30 formalin fixed paraffin-embedded lymph nodes from patients with CLL and 4 control (tonsil) tissues using a modified combined FISH/IHC assay. miR-21 was differentially expressed in the lymphoid tissues of patients with CLL: miR-21 was detected in 13/30 lymph nodes (43%), with three demonstrating strong and ten - weak staining. The remaining 17 tissue specimens (56%) stained negative for miR-21. Where present, strong miR-21 expression followed focal rather than diffuse staining pattern. Foci of miR-21 did not localize to the proliferation centers as per morphologic assessment. Since microRNAs are also expressed in other cell types including stromal cells, we used CD20 to confirm that miR-21 expression was detected in the neoplastic CLL cells. miR-21 positivity in the lymphoid tissue did not predict time to first treatment or correlate with either CD38 or ZAP-70 expression. Interestingly, 2/4 control tissues demonstrated strong staining for MIR21, indicating that its expression in the lymphoid organs is not restricted to clonal neoplastic B-cells.We further studied whether miR-21 expression was modulated by stromal signaling. Peripheral blood CLL cells (n=33) and normal B-cells (n=7) were isolated using standard Ficoll-Hypaque techniques, B-cell isolation kit and CD19 MACS microbeads, followed by cDNA synthesis and RT-PCR with specific TaqMan probes. We modeled lymph node microenvironment in vitro using CD40L-expressing or control mouse L cells, thus promoting drug resistance and survival of the peripheral blood CLL; CD40L-expressing cells induced NFκB. Consistent with previous reports, we found increased levels of miR-21 in peripheral blood CLL cells compared with normal B-cells. Co-culture of CLL cells with stroma resulted in induction of miR-21. IGHV mutational status is closely associated with BCR signaling capacity in CLL and predicts cellular reliance on microenvironmental support. We did not find a correlation between baseline miR-21 expression and IGHV mutational status in circulating CLL cells. However, CLL cells with unmutated IGHV were stronger inducers of miR-21 in response to stromal signaling. Meanwhile, CD40L-expressing stroma led to further induction of miR-155, a recognized NFκB target, but not miR-21, indicating that alternative mechanisms are responsible for stromal modulation of this microRNA.In summary, here for the first time we successfully visualized miR-21 expression within the neoplastic B-cells in the lymphoid tissues from patients with CLL. miR-21 was induced in stromal CLL cell co-cultures, primarily in cells with unmutated IGHV. Our observations are particularly relevant in the current era where BCR signaling pathways have become key pharmacologic targets. These therapies disrupt CLL–stroma interactions leading to an egress of CLL cells to the periphery, where they are unable to proliferate. Analysis of expression of miR-21 in lymph nodes and peripheral blood of patients treated with the BCR-targeting agents may clarify its predictive role in this setting as well as shed additional light on the mechanisms involved in its regulation. DisclosuresNo relevant conflicts of interest to declare.

Infusion Of Donor-Derived CD19-Redirected-Virus-Specific T Cells For B-Cell Malignancies Relapsed After Allogeneic Stem Cell Transplant: A Phase I Study

▪Donor lymphocyte infusions (DLI) following hematopoietic stem cell transplantation may reduce or control opportunistic infections and leukemia/lymphoma relapse, but the associated graft versus host disease (GvHD) limits the clinical success of this procedure. Since T cell immunotherapy may be a safer alternative to DLI we have now used a single T cell platform that mediates both antileukemic and antiviral activity. Autologous T cells modified to express CD19-specific chimeric antigen receptors (CD19.CAR) have had clinical activity against CD19-expressing malignancies, but it is unknown if similarly modified allogeneic T cells will be equally effective. Allogeneic virus specific T cells (VSTs) directed to cytomegalovirus (CMV), adenovirus (Adv), and Epstein Barr virus (EBV) have been shown to be safe and effective in preventing and treating life-threatening viral infections post HSCT. Therefore, we sought to determine whether allogeneic VSTs could be engineered to express CD19.CAR and would retain the safety and effectiveness of unmodified VSTs whilst gaining anti-tumor activity.VSTs were expanded ex vivo using antigen presenting cells engineered to express adenovirus and cytomegalovirus (using an Ad5f35 adenoviral vector expressing the CMV pp65 gene), and Epstein Barr virus (using EBV-infected lymphoblastoid cell lines) antigens. After 3 stimulations, the VST's were modified to express CD19.CAR.28ζ using a retroviral vector encoding the CAR-CD19 receptor coupled to the CD28 co-stimulatory molecule and the T cell receptor zeta (ζ) chain. Nine CD19.CAR-modified virus specific T cell (CD19.CAR-VSTs) products were generated for infusion. All VST lines recognized at least one viral antigen as determined by Elispot or chromium release assays and 20% to 48% of cells expressed the CD19.CAR. All lines killed CD19-expressing cells in vitro.We treated nine patients with these CD19.CAR-VSTs, 3 months to 13 years after HSCT. Six patients received CD19.CAR-VSTs for relapsed disease and 3 patients received the T cells as adjuvant therapy to prevent viral infection and relapse after HSCT. Safety. There were no infusion-related toxicities. One patient presented with gastrointestinal symptoms following infusion subsequently determined to be unrelated to the T cells. Persistence. VSTs persisted a median of 8 weeks in the peripheral blood and up to 9 weeks at disease sites. In three patients (#1, #3 and #5), CD19.CAR signals were detectable in the bone marrow or the lymph nodes (44.8, 25.85, and 32 copies/1000 ng DNA) even when no signal was measurable in peripheral blood, indicating preferential accumulation of the infused T cells at the disease site. Anti-Tumor Activity. During the period of CD19.CAR-VST persistence, objective anti-tumor activity was evident in 2/6 patients with relapsed disease (patient # 1 had detectable blasts in the peripheral blood which disappeared within 1-2 weeks following infusion, patient # 2 had 16% circulating CLL cells which decreased within 2 weeks of T cell infusion) but disease recurred after 3 and 2 months, respectively. The two patients who received cells while in remission remain disease-free >3 and >9 months later. Anti-Viral Activity. In two patients with EBV reactivation, donor CD19.CAR-VSTs expanded concomitant with an increase in virus-specific T cell responses, and decreased viral load. A third patient had a rise in adenovirus specific VSTs during an episode of adenovirus associated diarrhea. Although the infection was controlled, there was no concomitant rise in CD19-CAR expressing T cells in this patient. No other patient had viral disease.In conclusion, allogeneic CD19.CAR-VSTs administered after allogeneic HSCT are safe and can exert both anti-tumor and anti-viral activity in the absence of GvHD. Earlier administration of CD19.CAR-VSTs after HSCT, when the host is lymphodepleted and the incidence of viral infection is higher, may allow these cells to better capture the potential advantages of native TCR stimulation (and associated co-stimulation) for expansion and persistence, and thereby produce a higher frequency of sustained tumor responses. Alternatively, intentional stimulation of the native TCRs by viral vaccines may produce equal benefit, with greater predictability. Disclosures:Savoldo:Celgene: Patents & Royalties, Research Funding. Heslop:Celgene: Patents & Royalties, Research Funding; Cell Medica: Patents & Royalties. Rooney:Cell Medica: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Patents & Royalties, Research Funding. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

An Effective and Tolerable Chemoimmunotherapy Regimen For Relapsed/Refractory and Very-High Risk Chronic Lymphocytic Leukemia Combining Alemtuzumab With Pentostatin and Low Dose Rituximab

Patients with very-high risk (purine analogue refractory and TP53 defective) CLL have limited treatment options. In these patients alemtuzumab can be effective against CLL cells in the circulation and bone marrow, and in combination therapy with fludarabine can be active in patients with bulky disease, but these regimens have a high risk of serious infections. Addition of rituximab to alemtuzumab can also improve efficacy but has limited activity against bulky disease. We conducted a phase II clinical trial to determine the efficacy and toxicity of therapy with pentostatin, alemtuzumab, and low dose higher frequency rituximab (PAR) in patients with relapsed/refractory or progressive CLL with 17p13 deletion. The rituximab schedule was designed to decrease the loss of CD20 expression by circulating CLL cells. MethodsThis two-stage phase II trial study (NCT00669318) conducted at the Mayo Clinic Rochester and University of Iowa with IRB approval had an accrual goal of 38 evaluable patients. Eligibility required a diagnosis of progressive CLL by standard criteria and either previous treatment for CLL (<4 purine analogue regimens) or 17p13 deletion (17p13-). Exclusion criteria were organ failure, poor performance status (ECOG >3), infection with HIV, hepatitis B, hepatitis C, active autoimmune cytopenia, or alemtuzumab therapy within the past 2 months. Rituximab 20 mg/m2 IV M-W-F started on day 1, alemtuzumab started on day 3 with an escalation of 3-10-30 mg/d SQ and then 30 mg M-W-F from day 8, and pentostatin 2 mg/m2 IV every 2 weeks started on day 8. Peg-G-CSF or GM-CSF was used after each dose of pentostatin and patients received Pneumocystis and Varicella prophylaxis. CMV PCR assays were done weekly during treatment and viremia was treated with either valganciclovir or ganciclovir. Cycle 1 was 5 weeks and subsequent cycles were 4 weeks. At the end of cycle 2 patients with a clinical CR had a CT scan and a bone marrow study with immunohistochemical (IHC) staining for residual CLL cells, and therapy was stopped if there was no radiological or IHC evidence of residual CLL (stringent CR). Patients with residual disease received a 3rd cycle of therapy. ResultsForty-one patients were enrolled (July 2008 - February 2013) and all 39 who started therapy were evaluable for response: Median age 61 years (range 47-78), 30 (77%) males, 36 (92%) relapsed/refractory CLL (median prior regimens = 2, range 1-10), 3 (8%) previously untreated, 23 (59%) advanced stage (Rai III-IV), 16 (41%) intermediate stage (Rai I-II). Prognostic factors: FISH (hierarchical classification) 15 (38%) 17p13-, 6 (15%) 11q22-, 5 (13%) 12+, 3 (8%) no defects, 8 (21%) 13q14-, and 2 (5%) other abnormalities, IGHV analysis (n=38) 27 (71%) unmutated (<2%), ZAP-70 (n=37) 28 (76%) positive (>20%).Thirty (77%) patients completed planned therapy (28 had 3 cycles, 2 had 2 cycles with stringent CR). Nine patients received one (n=4) or two (n=5) cycles of therapy because of disease progression or complications. Grade 3-4 hematological adverse events (n=37) at least possibly related to treatment included neutropenia (n=22), thrombocytopenia (n=11), anemia (n=2) and hemolysis (n=2). Non-hematological adverse events (n=17) included infections/neutropenic fever (n=8), fatigue (n=3), and hemorrhage (n=2). CMV reactivation was detected and treated in 14 patients (grade 1-2). No patients died during treatment or from treatment related complications.The overall response rate was 56% (95% CI 40-72) with 4 (10%) CR, 7 (18%) CRi, 11 (28%) PR, 7 (18%) SD, and 10 (26%) PD. Four patients (3 CR and 1 CRi) had IHC negative bone marrow studies. Thirteen (33%) patients have died due to progressive CLL (n = 11), sepsis (n=1), and pneumonia (n=1). Median follow up for surviving patients is 23 months (range 3-55). Seven (18%) patients proceeding to RIC allogeneic transplant were censored for time to next treatment. Twenty-one (54%) patients required therapy for progressive CLL and 7 (18%) have required no further therapy. Median progression free survival was 7 months (95% CI: 5-16), time to next treatment 9 months (95% CI: 6-27) and median overall survival has not been reached. DiscussionPAR was effective and tolerable therapy for high-risk CLL. This study suggests that alemtuzumab can be used safely in combination with a purine analogue in a short-duration regimen. Disclosures:Zent:Genentech : Research Funding; Genzyme: Research Funding; Biothera: Research Funding; GlaxSmithKline: Research Funding; Novartis: Research Funding. Off Label Use: Pentostatin therapy for CLL, use of lower doses of rituximab.

Dasatinib in combination with fludarabine in patients with refractory chronic lymphocytic leukemia: A multicenter phase 2 study

Resistance to chemotherapy-induced apoptosis in CLL is associated with overexpression of antiapoptotic proteins induced by signals from the microenvironment. In vitro, dasatinib effectively inhibits expression of anti-apoptotic regulators and restores fludarabine sensitivity in activated CLL. The aim of this study was to evaluate efficacy of one cycle of dasatinib monotherapy (100mg/day, days 1-28) followed by combination of dasatinib with fludarabine (40mg/m²/day, days 1-3 every 28 day) for a total of 6 cycles in fludarabine-refractory CLL. The primary endpoint was overall response rate according to the IWCLL'08 criteria. 20 patients were enrolled: 18 completed at least one cycle of treatment of which 67% finished at least 2 cycles of combination treatment. 3 of these 18 patients reached a formal PR (16.7%). Majority of patients obtained some reduction in lymph node (LN) size. Most frequent toxicity was related to myelosuppression. NF-κB RNA expression levels of circulating CLL cells decreased whereas the levels of pro-apoptotic NOXA increased during treatment. In conclusion, dasatinib/fludarabine combination has modest clinical efficacy in fludarabine-refractory patients.

Circulating CLL Cells Expressing CD49d Display a Phospho-Proteomic Profile Consistent with a Constitutive Receptor Engagement by Blood-Borne Ligands

AbstractAbstract 930Chronic lymphocytic leukemia (CLL) cell growth and survival occur in defined microenvironment niches controlled by several receptor/ligand interactions including those mediated by the α4β1 integrin. This integrin, in particular, is known to interact with both the extracellular matrix component fibronectin, via the non-RGD CS-1 fragments, and the bone marrow/stromal components, via the vascular-cell adhesion molecule-1 (VCAM-1). The α4 integrin chain (a.k.a. CD49d) has been previously shown to be associated with poor prognosis in CLL by marking a subset of CLL patients characterized by a more aggressive clinical course both in term of disease progression and overall survival. Despite the great deal of studies investigating CLL cell microenvironmental interactions in tissue sites, little is known regarding the constitutive engagement of adhesion receptors in circulating CLL cells, and the role in this context of plasma/plasma components. To address this issue, the proteomic profiles of circulating leukemic cells from 80 CLL cases expressing or not CD49d were explored using a reverse phase protein microarray (RPMA) approach quantitatively analysing 77 proteins (61 phosphoproteins) known to be involved in translational control, cell growth, apoptosis, B cell receptor and cytoskeletal signaling. Comparison of the signaling activation portrait between 40 CD49d pos and 40 CD49d neg CLL cases highlighted the over-expression in CD49d pos CLL of proteins involved in the regulation of integrin-mediated cytoskeletal dynamics, such as phospho-p21-activated kinase (PAK1 Ser199-204/PAK2 Ser192-197; p=0.0005), phospho-LIM kinase (LIMK1 Thr508/LIMK2 Thr505; p=0.00001) and the adaptor protein CrkII Tyr221 (p=0.039). Moreover, CD49d pos CLL cells were characterized by a high correlation between proteins involved in integrin-mediated signal trasduction, including the focal-adhesion kinase (FAK Tyr576-Ser577), the tyrosine kinase Src Tyr527 and the adaptor protein CrkL Tyr207. Since PAK and LIMK represent key players in the modulation of the structure and the activity of actin cytoskeleton, we focused on these proteins for validation experiments. The constitutive pPAK and pLIMK overexpression in the CD49d pos CLL group was confirmed by western blot analysis comparing purified CLL cells from 3 CD49d pos versus 3 CD49d neg cases (mean fold increase >100 for both proteins). The results obtained suggest that integrin signalling is constitutively active in CD49d-expressing circulating CLL cells, pointing to a constitutive receptor engagement by CD49d ligands allegedly present in plasma. To test whether plasma constituents could modulate integrin-signaling proteins, CLL cells from 5 cases, expressing or not CD49d, were challenged with autologous plasma (1:3 dilution) for 30 seconds, 1 and 3 minutes. The presence of plasma induced a strong increase of PAK and LIMK phosphorylation intensities in CD49d pos CLL cells, starting at 30 seconds upon stimulation (mean fold increase >10 as compared to control for both proteins), and increasing after 1 and 3 minutes (mean fold increase >40 and >50 for both the proteins, respectively). Conversely, plasma stimulation did not induce pPAK and pLIMK expression modulation in CD49d neg CLL cells. Of note, pretreatment of CD49d-expressing CLL cells with the anti-CD49d HP1/2 blocking antibody, resulted in lower up-regulation of pPAK and pLIMK with an overall 60% inhibition for both the proteins (p=0.01), confirming the involvement of CD49d triggering in the observed activated signaling. Given the above results, we investigated plasma from 24 CLL patients, expressing CD49d at different levels, for the presence of the CD49d ligand fibronectin using an ELISA detection kit. All samples tested showed more than 1 mg/ml fibronectin concentration, without differences between CD49d-expressing and CD49d neg CLL cases. Altogether these results sustain the hypothesis of an active role of plasma/plasma components in the activation of CD49d-mediated integrin pathway, thus favoring the delivery of chemoresistance/pro-survival signals even in the context of circulating CLL cells. Our results may be of interest in the perspective of novel therapies (e.g. Bruton tyrosine kinase inhibitors) known to provoke a massive egress of neoplastic cells from tissue sites into the blood stream. Disclosures:No relevant conflicts of interest to declare.

Fludarabine, Bendamustine, and Rituximab (FBR) Chemoimmunotherapy Is a Safe and Active Regimen for Relapsed/Refractory CLL with in Vivo Mechanism of Action for Combination Chemotherapy

Abstract 437Chemoimmunotherapy (such as fludarabine, cyclophosphamide, and rituximab) is highly effective CLL therapy; adding novel agents or replacing standard with more effective agents could improve outcomes. Bendamustine (B) is a potent alkylating agent that induces DNA damage and repair response. Marked DNA damage response (H2AX phosphorylation) was seen with activation of p53 protein and PUMA, and cell death when fludarabine was combined with bendamustine in vitro. Base on this, we are conducting a phase I/II trial of escalating doses of bendamustine at 20 (n=6), 30 (n=17), 40 (n=6), or 50 (n=6) mg/m2 on D1, 2, 3 with fludarabine 20 mg/m2 administered prior to bendamustine on D2 & 3. Rituximab 375–500 mg/m2 was given D3. Courses were repeated each 28 days for 6 planned courses to assess the safety and tolerability, clinical efficacy, and pharmacodynamics (PD) in previously treated CLL. Response assessment (IWCLL 2008 criteria) was after 3 courses and end of treatment; bone marrow residual disease was assessed by 4-color flow. We previously reported (ASH 2011) that no MTD was identified in phase I and identified bendamustine 30 mg/m2 as safe for phase II expansion. We now report on efficacy of this FBR regimen in 35 pts treated in phase I & II who have response data available. The median age was 62 yrs; number of prior treatments was 3 (1-6); and number of FBR courses was 3 (1-6). Dose-reduction after course 1 occurred in 10/35 pts. Responses are shown (Table) and were seen across all dose levels. Time-to-event endpoints will be presented. Pts had high-risk features: median b-2 microglobulin was 4.1 (1.8–10.4); 18/35 were Rai stage III-IV; 23/29 were ZAP70+; 23/29 had unmutated IGHV; and FISH identified 3 pts with del17p and 13 with del11q. Myelosuppression was the most common treatment-related toxicity considering all courses given (n=106). Grade(G) 3&G4 neutropenia occurred in 26&29% of courses, respectively; thrombocytopenia G3&G4 occurred in 14&9% of courses, respectively; and anemia G3&G4 occurred in 15&2% of courses, respectively. There were no treatment-related deaths.To test fludarabine triphosphate-mediated mitigation of DNA repair response induced by bendamustine, on D1 bendamustine was infused alone and on D2, fludarabine was administered 2 hours prior to second bendamustine infusion; circulating CLL cells from 11 pts at different bendamustine doses (3 at 20, 3 at 30, 3 at 40, and 2 at 50 mg/m2) were evaluated. Phosphorylation of histone 2A variant × (H2AX) was used as damage response marker. There was heterogeneity in extent of DNA damage response elicited after first bendamustine infusion. Considering basal phosphorylation level in the pretreatment sample on D1 as 1.0, the H2AX phosphorylation at D1-6hr (bendamustine alone) ranged between 0.2–8 (n=11). Median intracellular fludarabine triphosphate level at the start of bendamustine infusion was 12 μM (range 5–21 μM). This was sufficient to increase H2AX phosphorylation in all 11 pts tested. At the end of D2-4hr (bendamustine combined with fludarabine) the range was between 0.9–22 and remained the same on D2-6hr. In 1 pt sample, the phosphorylation persisted at 26 until D3-pretreatement, while in others it ranged between 3–12. Consistent with H2AX data, molecular markers of DNA damage response showed activation of ATM measured in 5 pt samples as ser1981 phosphorylation and phosphorylation of p53 at ser15. In parallel, there was a decrease in anti-apoptotic proteins Mcl-1 and Bcl-2 at the end of D2-6hr; however protein levels were retained on D3.In conclusion, this FBR regimen was tolerated up to the highest evaluated bendamustine dose; efficacy was demonstrated in previously treated pts with CLL. DNA damage and repair response biomarkers validated the hypothesis that fludarabine triphosphate inhibited bendamustine-induced DNA repair resulting in increased or sustained DNA damage. We continue to extend the clinical and PD investigations in phase II.TableCharacteristicn% CR/CRi% OR%MRD NegAll Pts35267111Rai StageIII-IV181767110-II17357612No. Prior Rx>219165851-216388819B2M34 mg/l19115311<4 mg/l16449413FISH17p del333100011q del1323690+127578643None30333313q del301000IGHVUnmutated2326709Mutated63310033ZAP70Positive23227013Negative6178317CD38 (>7%)Positive2623738Negative8387525CR, complete remission; CRi, CR with incomplete recovery of cytopenias; OR, overall response; MRD, minimal residual disease. Disclosures:No relevant conflicts of interest to declare.

Resistance to Complement Dependent Cytotoxicity in CLL Cells From Patients Treated with Ofatumumab

Abstract 2836Monoclonal antibodies (mAb) are an effective but non-curative treatment of CLL. Although the mechanisms of action of mAb in vivo are not fully defined, complement dependent cytotoxicity (CDC) appears to play an important role. We have previously shown that addition of ofatumumab (OFA) significantly increases alemtuzumab (ALM) CDC in vitro and identified a subpopulation of CLL cells that are intrinsically resistant to activated complement. We propose that resistance to CDC could be an important cause of mAb treatment failure in CLL patients. To test the hypothesis that CLL cells treated in vivo with OFA would be resistant to subsequent OFA retreatment in vitro, we studied CLL cells and serum from 10 previously untreated patients with progressive CLL treated with pentostatin, cyclophosphamide, and ofatumumab (PCO). Methods.Patients received 300 mg of OFA followed by 2 mg/m2 of pentostatin and 600 mg/m2 of cyclophosphamide on day 1 of therapy. Samples were taken prior to treatment (S1), immediately after the OFA infusion and prior to chemotherapy (S2), and post-chemotherapy before the second dose of OFA on day 2 (S3). Mononuclear blood cells isolated from EDTA anticoagulated blood by Ficoll-Paque centrifugation were purified to achieve a concentration of > 90% CLL. CDC was assayed at a concentration of 2 × 106/ml CLL cells with 10 mg/ml of ALM (Genzyme), OFA (G.S.K.) or rituximab (RTX)(Genentech) in AIM V medium (Invitrogen, CA) and 10% normal human serum (10%NHS)(Sigma, MO) as a source of complement for 1 hour at 37°C. Absolute viable cell counts were measured by flow cytometry using counting beads (Trucount, BD, CA) and propidium iodide staining (Sigma, MO) with a FACSCalibur (BD, CA) and CellQuest Pro software (BD, CA). Percent CDC was calculated relative to counts for CLL cells treated with only 10%NHS. Binding of mAb and C3b to CLL cells was measured by flow cytometry with mouse anti-human Fc antibody FITC-HB43 and anti-C3b antibody FITC-7C12. C5 deficient serum (C5-serum)(Sigma, MO) was used to examine mAb and C3b binding without CDC. Results were expressed as delta mean fluorescent intensity (dMFI) relative to cells treated without mAb in 10%NHS or C5-serum as appropriate. Complement (CH50) was measured based on lysis of opsonized sheep red cells using standard methods. OFA concentrations in serum samples were determined by measuring the level of binding to Daudi cells compared to standards as previously described. Results:S1 CLL cells were significantly more susceptible to in vitro OFA CDC (median 30%) than S2 and S3 cells (both 0%, p=0.002)(Fig. 1). In contrast, high levels of CDC (median 86 – 88%) were induced in vitro by ALM in all these samples and prior in vivo exposure to OFA did not affect susceptibility to ALM CDC in vitro (p>0.1). CLL cells from S2&S3 had low levels of OFA binding both prior to and post in vitro exposure to OFA (median dMFI 2 and 3 respectively) which was significantly less than OFA binding for S1 (median dMFI 38 p=0.002) likely reflecting in vivo trogocytosis of bound OFA and CD20, as previously described for RTX. In vitro ALM binding was higher than OFA (median dMFI>150) in all specimens. C3b binding was low in S2&S3 both before and after in vitro OFA exposure (median dMFI 3 and 4 respectively) with significantly higher levels in S1 (median dMFI 94, p=0.002). C3b binding was higher in all CLL cells treated with ALM (median dMFI>358). OFA treatment resulted in marked decreases in serum complement levels in S2 (median 86%, range 65–98%) and S3 (median 78% range 61–88%) compared to S1. The median serum OFA concentration was 18.5 μg/ml (range 7.3–50.4) in S2 and 19.8 μg/ml (range 0–32.3) in S3. Conclusions:Circulating CLL cells from patients treated with OFA are resistant to in vitro OFA CDC primarily because of low levels of expression of CD20. The reduction in complement titers could limit in vivo CDC but we found no evidence that low levels of OFA were important. Cells surviving OFA in vivo do retain sensitivity to ALM CDC. Our data support the previous description of trogocytosis in CLL patients treated with RTX and suggest that lower doses of these mAb, that promote far lower levels of trogocytosis, could be more effective in sustaining CDC. In addition, our study provides pre-clinical data to support a clinical trial of combination therapy with OFA and ALM for CLL. Acknowledgment:This study was supported by funding from GlaxoSmithKline and the University of Iowa/Mayo Clinic Lymphoma SPORE (CA097274). [Display omitted] Disclosures:Taylor:Genmab: Consultancy; Glaxo Smith Kline: Research Funding. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

A Phase I/II Trial of Fludarabine, Bendamustine, and Rituximab (FBR) Chemoimmunotherapy for Previously Treated Patients with CLL,

Abstract 3901Chemoimmunotherapy (such as fludarabine, cyclophosphamide, and rituximab) has been the most significant advance in treatment for patients with CLL, achieving the highest complete remission rates, longest progression-free and overall survival compared to chemotherapy combinations or monotherapy. Bendamustine (B) is a well-tolerated, alkylating agent that induces a DNA damage and repair response. In vitro data in 30 CLL patient (pt) samples suggested an increased DNA damage response (measured as H2AX phosphorylation), activation of p53 protein and PUMA, and cell death when fludarabine was combined with bendamustine (El-Mabhouh, A, unpublished). To translate this observation to the clinic, we are conducting a phase I/II trial of escalating doses of bendamustine at 20, 30, 40, or 50 mg/m2 on D1,2,3 with fludarabine 20 mg/m2 administered prior to bendamustine on D2&3. Rituximab 375–500 mg/m2 was given on D3. Courses were repeated each 28 days to assess the safety and tolerability, clinical efficacy, and pharmacodynamics (PD) in previously treated pts with CLL. Responses were assessed after 3 courses and end of treatment. We report results of the phase I portion of this study. For phase I, dose-limiting toxicities (DLT) were assessed in course 1 and were Grade (G) ≥3 treatment-related, non-hematologic adverse event (AE), and hematologic toxicity G≥3 that lasted beyond D42 of course 1. MTD was defined as the cohort with ≤1 DLT in 6 treated pts. All pts (n=19) had active CLL and were previously treated; median number of prior treatments was 2 (1–6). Pts had high-risk features, median >102<−2 microglobulin was 4 (2.4–8.7); Rai stage III-IV was 10/19; 13/15 were ZAP70+; 12/15 had unmutated IGHV; and FISH identified 2 with del17p and 7 with del11q. 19 patients were evaluable for course 1 toxicities and DLT. Course 1 toxicities were predominantly G1-2 and most common were nausea, fatigue, and hyperglycemia. One of 6 pts experienced DLT (G3 nausea/vomiting/dehydration) in the B-20 cohort; 0 of 3 pts experienced DLT in the B-30 cohort; 1 of 6 pts experienced DLT (G4 sepsis) in the B-40 cohort; and 1 of 4 pts experienced DLT (G3 neutropenia) in the B-50 cohort. Pts continued on treatment, 5 with dose reduction, (Table) for up to 6 courses. The B-50 cohort continues enrollment and treatment, all other cohorts completed treatment. Among 14 pts evaluable for response, there were 5 complete responders (3 MRD negative by 4-color flow cytometry) and 8 partial responders (2 PRs were CRi by IWCLL 2008 criteria); only 1 pt was a non-responder (Table). Considering all courses given, the most common G3-4 AEs that occurred in more than 10% of courses (n=56) were: neutropenia (30%) and thrombocytopenia (13%). All other AEs were G1-2 and resolved. There were no treatment-related deaths. More frequent AEs with higher doses of bendamustine supports selection of the 30 mg/m2 dose level to move forward in phase II. To test fludarabine triphosphate-mediated mitigation of DNA repair response induced by bendamustine, on D1, bendamustine was infused alone and on D2, the fludarabine dose was given 2 hours prior to bendamustine infusion. Circulating CLL cells from 7 pts (3 B-20 and 3 B-40, and 1 B-50) were evaluated for PD endpoints. Median intracellular fludarabine triphosphate level at the start of bendamustine infusion was 12 μM (range 5–21 μM). This was sufficient to increase by 3–5-fold the H2AX phosphorylation response. Molecular markers of DNA damage response and cell death (ATM, p53, PUMA, Mcl-1) are being evaluated.In conclusion, the FBR regimen was tolerated up to the highest bendamustine dose evaluated, with significant efficacy in previously treated patients with CLL. We are extending the clinical and PD investigations in a phase II study with B-30 dose.TableCohort*nMedian coursesTotal coursesTotal AEs per Cohort (C1)Eval for ResponsePercent RespondersG1-2G3-4nCRORB-2063 (2–6)2222465083B-3034 (3–5)1218130100B-4062.5 (1–4)164514540100B-5041.5 (1–2)**6**318–––AEs, adverse events; G, grade; n, number; CR, complete remission; OR, overall response*Bendamustine dose mg/m2 daily × 3;**Treatment continues Disclosures:No relevant conflicts of interest to declare.