ALBERT

Description: B-Cell Receptor (BCR) triggering and responsiveness play a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones. In the recent past, several groups including ours have investigated the activation status of the signaling pathways originating from the leukemic BCR. Specifically we found that around 50% of CLL patients display a biochemical signature characterized by constitutive phosphorylation of ERK1/2 (pERK(+)) and constitutive nuclear translocation of NF-ATc1. These cases are unable to respond in vitro to BcR stimulation and are resistant to spontaneous apoptosis, thus resembling B lymphocytes previously anergized in vivo. Similar biochemical and functional features have been recently demonstrated in B leukemic cells persisting in the blood in patients treated with the BTK inhibitor, Ibrutinib, thereby making anergy an attractive target on the way to obtain eradication of the disease. CLL-associated B cell anergy can be specifically targeted by using different MAPK-inhibitors that have been shown to induce apoptosis selectively in the group of pERK(+) CLL. These data suggested that MAPK signalling can be efficiently inhibited in CLL for therapeutic purpose and that the phosphorylation status of ERK1/2 may represent a reliable biomarker to predict and monitor treatment response. However, even if the tested compounds were shown to be extremely efficient in inhibiting ERK1/2 phosphorylation in vitro, a lack of clinical activity was reported for many of them when tested in patients, mostly with solid tumors. In the present work, we used Trametinib, a specific MEK1/2 inhibitor, recently approved as a single-agent for the treatment of V600E mutated metastatic melanoma, and we investigated, at preclinical level, its activity in both primary CLL samples and a xenograft leukemic mouse model. Trametinib treatment completely inhibited constitutive ERK1/2 phosphorylation in 10 pERK1/2(+) samples at 3uM after 30 minutes treatment. Additionally, in 23 patients Trametinib treatment for 48 hours reduced cell viability in the cells from all 12 pERK1/2(+) patients (28,2% ± 3,5 mean survival) tested as compared to those from the pERK(-) group (11 cases, 58,1% ± 3,8 mean survival, p〈 0,0001). To strengthen our in vitro data, we evaluated the effect of Trametinib administration in the xenograft Rag2-/-gc-/- mouse model subcutaneously transplanted with the CLL cell line MEC1, characterized by specific features of anergy. Mice were subcutaneously injected with 10x106 cells and then challenged with Trametinib (oral gavage with 1mg/kg or with vehicle alone) starting from day 21 after tumour injection for 14 days. The effect of the inhibitor was monitored by tumour volume growth. Trametinb administration delayed tumour growth (p

Description: Key Points HS1 protein activation is differentially regulated by LYN kinase in CLL subsets. Dasatinib targets cytoskeletal activity, BCR signaling and survival of a sizable portion of patients with activated LYN/HS1.

Description: Easily reproducible animal models that allow for study of the biology of chronic lymphocytic leukemia (CLL) and to test new therapeutic agents have been very difficult to establish. We have developed a novel transplantable xenograft murine model of CLL by engrafting the CLL cell line MEC1 into Rag2−/−γc−/− mice. These mice lack B, T, and natural killer (NK) cells, and, in contrast to nude mice that retain NK cells, appear to be optimal recipient for MEC1 cells, which were successfully transplanted through either subcutaneous or intravenous routes. The result is a novel in vivo model that has systemic involvement, develops very rapidly, allows the measurement of tumor burden, and has 100% engraftment efficiency. This model closely resembles aggressive human CLL and could be very useful for evaluating both the biologic basis of CLL growth and dissemination as well as the efficacy of new therapeutic agents.

Description: The function of the intracellular protein hematopoietic cell–specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1−/− mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2−/−γc−/− mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eμ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eμ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.

Description: Abstract 1775 The immunoglobulin gene repertoire in CLL is remarkably restricted with greater than 30% of cases carrying quasi-identical (stereotyped)heavy complementarity-determining region 3 (VH CDR3) sequences. Indeed, cases can be clustered into different subsets based on shared, subset-biased motifs within the clonotypic VH CDR3s, with, notably, only a handful of subsets accounting for almost 10% of all CLL. VH CDR3 stereotypes are more frequent in cases with unmutated IGHV genes (U-CLL) who are associated with adverse prognosis. In principle, VH CDR3 stereotypy might allow to exploit these IG motifs as candidate Tumor Associated Antigens (TAA) for targeted immunotherapy of CLL. The aim of our study was to validate as potential TAA subset-specific IG motifs from major CLL subsets, focusing especially on subsets #1 and #2 that are the largest overall and both associated with aggressive clinical course. We have so far identified, by in silico analysis, 1–3 long peptides (15-mer) encompassing the VH CDR3 protein regions of subsets #1, 2, 4, 6, 8, 10 with (i) high binding score to MHC class II molecules and (ii) also containing minimal HLA class I-specific epitopes (HLA-A2, -A3, -A24, DR1, DR7, DR13 that are most frequent in the Caucasian population). Blood lymphocytes from 18 CLL patients were collected and phenotyped by flow cytometry with appropriate antibodies to assess the expression of stimulatory, co-stimulatory and negative regulatory molecules on both T and B cells. In addition, HLA typing of CLL patients was performed to select patients expressing the aforementioned HLA molecules. Overall, 13/18 patients matched the defined HLA class I and/or class II molecules. Negatively purified T cells from 11 CLL patients expressing HLA-A2 and/or DR13 have been then stimulated in vitro with the synthesized peptides of the specific stereotype (subset #1 and 2) in the presence of culture medium containing 5% of human serum plus IL-2 (20 IU/ml) and IL-15 (10 ng/ml). These T lymphocytes were then weekly stimulated with autologous irradiated antigen presenting cells (APC; monocytes, B cells, etc.) pulsed with the peptides. Starting from the third week of culture, the specific recognition of CDR3-derived TAAs and of tumor cells (autologous CLL cells) by the T cell cultures has been assessed by in vitro functional assays (ELISPOT assay). We were able to isolate CDR3- (subsets #1 and #2) and tumor-specific T cells from 5/11 CLL patients. In addition, in 4 selected patients the Ag- and tumor specific T lymphocytes have been expanded in vitro by Rapid Expansion Protocol (REP), based on the stimulation of T cells with allogeneic irradiated PBMCs from healthy donors plus OKT3 and high doses of IL-2. Using this protocol we were able to obtain large numbers (2–10 ×109) of anti-CDR3 T cells in all 4 cases tested, thereby, in principle, achieving the potential to use this protocol for expanding sufficient cells for clinical applications. Interestingly, post-REP T cell cultures showed enrichment (85–90%) of CD3+CD8+ T cells and down-modulation of negative regulatory molecules, such as CTLA-4, as compared to pre-REP in vitro stimulated T cells. These cells could be expanded in vitro for up to 6 weeks without any decay in proliferation. Taken together, these results indicate that stereotyped VH CDR3 peptide sequences can represent candidate antigens to elicit T cell-mediated anti-CLL responses, especially in poor prognosis cases, where therapeutic innovation is more urgently needed. After validation of this protocol in a larger series, our results may provide the proof of principle for the design of new immunotherapy protocols for CLL, including both active vaccination and adoptive cell therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2440 Monoclonal B-cell Lymphocytosis (MBL) is a preclinical hematologic condition wherein small B-cell clones are detectable in the peripheral blood of otherwise healthy individuals. Monoclonal B-cell expansions are rather heterogeneous in terms of phenotype, but in two thirds of cases clonal B-cell populations share the same unique immunophenotypic profile of Chronic Lymphocytic Leukemia (“CLL-like MBL”). Based on the number of B cells per μl, MBL cases might be further split into those associated with lymphocytosis, usually diagnosed in a clinical setting (“Clinical MBL”) when clonal B cells reach a concentration 〉1500/μL, and those detected in the general population (“low-count MBL”), usually characterised by 20% of people older than 60 years using highly sensitive techniques). We took advantage of our cohort of 138 MBL cases previously described among 1779 healthy individuals, living in a rural valley in Northern Italy (Val Borbera Valley) that included 96 CLL-like (69.6%), 20 Non-CLL (14.5%), 22 atypical CLL (15.9%) MBL. Of the 138 originally diagnosed MBL subjects, 76 individuals participated to a second visit after a median follow up of 34 months (range 11–50 months). 93.1% (54/58) of CLL-like MBL clones were confirmed, while only 44.5% and 66.7% of Atypical CLL-like and Non-CLL MBL, respectively, persisted over time. The few CLL-like clones that were not confirmed had a very low concentration at the initial visit (median number of clonal B-cells: 0.46 per μl) being proximal to the detection limit of the flow cytometric technique. Among the confirmed CLL-like cases, 1/54 was a Clinical MBL (1764 cells/μL), 3 subjects had 97, 190 and 265 cells/μl, while the vast majority of participants (50/54, 92.6%), had a number of monoclonal B-cells

Description: B cell receptor (BCR) signaling is a driving event in Chronic Lymphocytic Leukemia (CLL) progression. Most CLL cells express both surface IgM (sIgM) and sIgD molecules, and previous reports showed heterogeneous responses to anti-IgM stimulation: CLL cells carrying IGHV unmutated-BCRs are highly responsive in terms of intracellular signaling activation, while cells with IGHV-mutated receptors are usually anergic to BCR cross-linking. In contrast, it has been described that almost all CLL cells retain responsiveness to sIgD, suggesting that sIgD signaling may not be the most relevant event in disease progression and heterogeneity. As the clinical significance of these mechanisms still needs to be unraveled, in the present study we analyzed the kinetics of BCR signaling activation following either sIgM or sIgD stimulation in CLL cells. The activation of HS1 protein, along with BCR proximal kinases (i.e. SRC/LYN and BTK) and the downstream effector ERK, was tested by Western Blot and flow-cytometry analysis up to 60 minutes following receptor engagement. HS1 protein was chosen as an early marker of BCR signaling and cytoskeletal activation (ten Hacken E et al., Blood 2013), also in the light of its prognostic relevance, as HS1 hyperphosphorylation correlates with a dismal clinical outcome of patients. Purified B cells from 16 primary CLL samples were stimulated with either soluble anti-IgM or anti-IgD and the level of HS1 activating tyrosine (Y)397 phosphorylation was analyzed at different time points (2, 5, 15, 30, 60 minutes) by densitometric analysis of Western Blot results or Mean Fluorescent Intensity analysis of flow cytometry data. HS1 activation peaked after 2-5 minutes following stimulation, and then returned to baseline at later time points with different patterns. In 13/16 samples, we observed a protracted HS1 phosphorylation after anti-IgM stimulation. In contrast, 6/16 samples showed protracted HS1 phosphorylation after anti-IgD stimulation, while 10/16 samples showed a very transient HS1 activation that rapidly returned to baseline. The activation kinetics of the other signaling molecules (i.e. SRC/LYN, BTK and ERK) was in line with that of HS1. As protracted BCR signaling activation is linked to cell cycle entry and proliferation, the protracted IgD signaling observed in some patients suggests that not only IgMs, but also IgDs may have pathogenetic relevance in CLL progression and heterogeneity. We then analyzed how the BTK inhibitor ibrutinib affects BCR signaling activation kinetics and analyzed phosphoprotein activation patterns in 10 CLL samples after 1 hour pretreatment with ibrutinib and up to 60 minutes following anti-IgM and anti-IgD stimulation. Both IgM and IgD-induced phosphorylation of HS1, SRC/LYN, BTK and ERK was reduced by ibrutinib treatment, demonstrating that ibrutinib is effective in blocking signaling pathways downstream not only of IgM receptors, as previously demonstrated, but also downstream of IgDs. Interestingly, in 7/10 samples, HS1 partially retained the ability to be phosphorylated after BCR triggering following 1-hour ibrutinib treatment and 5-minute anti-IgM or anti-IgD stimulation, suggesting that HS1 activation can bypass BTK kinase inhibition at least at early time points following receptor stimulation. A broad analysis on a large cohort of patients will further allow the correlation between BCR signaling molecule activation and cell viability with molecular/cytogenetic prognostic factors. Overall, these results show that sIgM stimulation of CLL cells promotes protracted activation of BCR signaling, which can only be observed in a restricted set of patients after sIgD stimulation. These observations support the notion that sIgM signaling may be more relevant for CLL progression, but also suggest that sIgD signaling may contribute to CLL pathogenesis in selected patients. Both signaling pathways can be inhibited by ibrutinib, further supporting the effectiveness of ibrutinib in inhibiting BCR signaling in CLL. Disclosures: O'Brien: Pharmacyclics: Research Funding. Burger:Pharmacyclics: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Description: Background Olaptesed pegol (NOX-A12) is a novel, potent, L-stereoisomer RNA aptamer that binds and neutralizes CXCL12/SDF-1, a chemokine which attracts and activates immune- and non-immune cells via interaction with the receptors CXCR4 and CXCR7. Signaling of CXCL12 is pivotal to the interactions of leukemic cells with bone marrow microenvironment. The therapeutic concept of olaptesed is to inhibit such tumor-supporting pathways and thereby mobilizing and sensitizing CLL cells to therapy. Here we aim to assess the activity and safety of olaptesed in combination with bendamustine and rituximab (BR) in patients with relapsed / refractory CLL. Methods Twenty-eight relapsed or refractory CLL patients were enrolled and treated in this open-label, single-arm Phase IIa study. To investigate PK/PD, a pilot dose of 1 to 4 mg/kg olaptesed alone was administered to 3 patients per dose group (plus one additional replacement pt) before start of the regular treatment regimen (pilot group). Patients were treated using a dose titration design with intravenous (IV) olaptesed at doses increasing from 1 mg/kg to 2 mg/kg and 4 mg/kg at cycles 1, 2 and 3, respectively, at 1 hour before rituximab treatment. During cycles 4 to 6, olaptesed was dosed at the highest individually titrated dose. Rituximab was administered IV at doses of 375 mg/m² on day 1 of the 1st28-day cycle and 500 mg/m² on day 1 of subsequent cycles. Bendamustine (70 - 100 mg/m²) was given IV on days 2-3 (cycle 1) or days 1-2 (cycles 2-6) of each 28-day cycle following administration of rituximab. Clinical response was assessed according to NCI-WG Guidelines (Hallek M et al. Blood 111; 2008: 5446-56). Results To date, 24 patients completed treatment (12 women, 12 men) with a median age of 68.5 years (range 52 to 79). At screening 5, 9 and 10 patients presented with Binet stage A, B and C, respectively. The median number of prior treatment lines was 1 (range 1-2). Seven high-risk patients presented an unfavorable disease state being relapsed within 24 months after fludarabine/bendamustine treatment (5 pts) and/or presenting a deletion/mutation of the TP53 gene (3 pts). Most patients (19 of 24) were previously treated with fludarabine or bendamustine. A flow cytometric analysis of CD19+/CD5+CLL cells showed a rapid mobilization into the peripheral blood by a single dose of olaptesed which lasted throughout the observational time of 72h. Interestingly, CXCR4 expression levels increased on CLL cell surface in the periphery after olaptesed treatment. This increase, which peaked at 24h, likely reflects the extended circulation of CLL cells in the periphery due to the sustained blockade of CXCL12 by olaptesed. Reduction of lymphadenopathy by ≥ 50% was achieved in 14 out of 21 evaluable patients with reported enlarged lymph nodes by the end of treatment. Concomitantly, rapid reduction of lymphocytosis in peripheral blood with normalization by treatment cycle 2 – 3 was observed and the CLL to leukocyte ratio significantly improved. Efficacy was assessed at the end of cycles 3 and 6. In the full-analysis-set, which excludes two non-evaluable patients (drop-out after the 1st cycle due to adverse events), the overall response rate was 96%: Three patients (14%) achieved a complete response at end of cycle 6 (2 confirmed, 1 investigator reported) and eighteen patients (82%) achieved a partial response (fifteen at end of cycle 6 and three at end of cycle 3). Notably, all seven high-risk patients (defined as relapsed within 24 months after fludarabine/bendamustine treatment and/or presenting a deletion/mutation of the TP53 gene) responded to treatment with olaptesed + BR with a partial response. One patient had a progressive disease. Olaptesed at 1, 2 and 4 mg/kg at a single dose and in combination with BR was safe and well tolerated. The observed adverse reactions were qualitatively and quantitatively as expected for patients treated with BR. Conclusion Olaptesed in combination with BR was safe and well tolerated. Compared with historical data, olaptesed showed superiority over baseline therapy with regards to overall response rate and increasing rates of complete remission, warranting further development of this Spiegelmer in CLL. Disclosures Montillo: Janssen: Honoraria. Kruschinski:NOXXON Pharma AG: Employment. Dümmler:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment.

Description: Chronic Lymphocytic Leukemia (CLL) is a chronic lymphoid malignancy characterized by immune suppression that is responsible for an increase in infection susceptibility but also concurs to a reduced ability of the immune system to promote an effective response against the leukemic cells. Tumor-immunosuppressive mechanisms are essentially due to the capacity of CLL cells of modifying the surrounding microenvironment including immune effectors likely contributing to disease progression but also to limited effectiveness of current immunotherapy approaches. Lenalidomide is an immunomodulatory agent (IMID) able to induce significant long-lasting responses in CLL patients. The exact mechanism of anti-tumor activity of lenalidomide remains undefined, but it also implies the modulation of tumor microenvironment through down-regulation of critical cytokines and activation of immune effector cells. In addition, lenalidomide was shown to reverse, in vitro, defects in immunological synapse formation between T cells and CLL cells, by interfering with several cytoskeletal molecules. Chimeric antigen receptors (CARs) molecules are emerging as a powerful tool to redirect T-cell specificity against leukemia. CARs are artificial molecules constituted by an extracellular-antigen-binding domain consisting of the variable chains of a monoclonal antibody, linked together as a single chain Fv (scFV), and an intracellular signaling region, usually the zeta chain of the TCR/CD3 complex, that is immediately triggered after antigen recognition. Therefore, CARs take advantage of both the antigen binding non MHC-restricted-properties of monoclonal antibodies and of the typical T-cell mediated effector functions. Given the characteristic T cell defects occurring in vivo in CLL patients, it becomes very intriguing to explore the possibility of a novel CLL therapy combining a CAR-based immunotherapy with low doses of lenalidomide, in order to maximize the effect of the immune attack by reverting in vivo the acquired T cell defects. We studied the in vivo cytotoxic effects on the tumor microenvironment upon lenalidomide treatment utilizing the Rag2-/-γc-/--xenograft model of human CLL based on transplantation of the CLL cell line MEC1 into Rag2-/-γc-/--mice. Utilizing the CAR.CD23 tool as previously published by our group, we also performed experiments where MEC-1-trasplanted-Rag2-/-γc-/- mice were injected with CAR.CD23 T cells from CLL patients together with lenalidomide at low concentrations, uneffective in monotherapy. In these animals, a decrease of the percentage of CD19+leukemic cells was observed in all lymphoid and non-lymphoid tissues after 20 days of treatment, as compared to controls treated with CAR.CD23 T cells or lenalidomide alone. This combination resulted also in improved survival of the treated cohort (NT+lenalidomide vs CAR+lenalidomide: p