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[Background]: In the search for novel antibody-drug conjugates (ADCs) with therapeutic potential, it is imperative to identify novel targets to direct the antibody moiety. CD13 seems an attractive ADC target as it shows a differential pattern of expression in a variety of tumors and cell lines and it is internalized upon engagement with a suitable monoclonal antibody. PM050489 is a marine cytotoxic compound tightly binding tubulin and impairing microtubule dynamics which is currently undergoing clinical trials for solid tumors., [Methods]: Anti-CD13 monoclonal antibody (mAb) TEA1/8 has been used to prepare a novel ADC, MI130110, by conjugation to the marine compound PM050489. In vitro and in vivo experiments have been carried out to demonstrate the activity and specificity of MI130110., [Results]: CD13 is readily internalized upon TEA1/8 mAb binding, and the conjugation with PM050489 did not have any effect on the binding or the internalization of the antibody. MI130110 showed remarkable activity and selectivity in vitro on CD13-expressing tumor cells causing the same effects than those described for PM050489, including cell cycle arrest at G2, mitosis with disarrayed and often multipolar spindles consistent with an arrest at metaphase, and induction of cell death. In contrast, none of these toxic effects were observed in CD13-null cell lines incubated with MI130110. Furthermore, in vivo studies showed that MI130110 exhibited excellent antitumor activity in a CD13-positive fibrosarcoma xenograft murine model, with total remissions in a significant number of the treated animals. Mitotic catastrophes, typical of the payload mechanism of action, were also observed in the tumor cells isolated from mice treated with MI130110. In contrast, MI130110 failed to show any activity in a xenograft mouse model of myeloma cells not expressing CD13, thereby corroborating the selectivity of the ADC to its target and its stability in circulation., [Conclusion]: Our results show that MI130110 ADC combines the antitumor potential of the PM050489 payload with the selectivity of the TEA1/8 monoclonal anti-CD13 antibody and confirm the correct intracellular processing of the ADC. These results demonstrate the suitability of CD13 as a novel ADC target and the effectiveness of MI130110 as a promising antitumor therapeutic agent., This work was partially supported by grant IPT-2012-0198-090000 (“MARINMAB” project) from Ministerio de Economía y Competitividad (MINECO) and European Regional Development’s funds (ERDF) and by CSIC grant 2019AEP146.

INTRODUCTION COBE SPECTRA [COBE] (Terumo, BCT Lakewood CO) apheresis system has been the most used device for hematopoietic progenitor cells (HPC) collection. Recently, it has been replaced by the SPECTRA OPTIA [OPTIA] (Terumo, BCT Lakewood CO) apheresis system. The aim of our study is to compare both methods for HPC collection. MATERIAL AND METHODS We retrospectively compared 302 HPC collection apheresis procedures (115 allogeneic donors and 187 autologous). The study cohort was divided according to the apheresis system used to analyze the differences between COBE and OPTIA, specifically efficacy of apheresis procedure and product characteristics. RESULTS OPTIA collections result in a higher CD34+ collection efficiency in both groups (autologous 45.3% vs 41%, P

Supplemental digital content is available in the text., Background/Objective There is evidence supporting that there are no relevant clinical differences between dosing rituximab 1000 mg or 2000 mg per cycle in rheumatoid arthritis (RA) patients in clinical trials, and low-dose cycles seem to have a better safety profile. Our objective was to describe the pattern of use of rituximab in real-life practice conditions. Methods Rituximab for RA in clinical practice (RITAR) study is a retrospective cohort study from 2005 to 2015. Eligibility criteria were RA adults treated with rituximab for active articular disease. Response duration was the main outcome defined as months elapsed from the date of rituximab first infusion to the date of flare. A multivariable analysis was performed to determine the variables associated with response duration. Results A total of 114 patients and 409 cycles were described, 93.0% seropositive and 80.7% women. Rituximab was mainly used as second-line biological therapy. On demand retreatment was used in 94.6% of cases versus fixed 6 months retreatment in 5.4%. Median response duration to on demand rituximab cycles was 10 months (interquartile range, 7–13). Multivariable analysis showed that age older than 65 years, number of rituximab cycles, seropositivity, and first- or second-line therapy were associated with longer response duration. The dose administered at each cycle was not significantly associated with response duration. Conclusions Our experience suggests that 1000 mg rituximab single infusion on demand is a reasonable schedule for long-term treatment of those patients with good response after the first cycles, especially in seropositive patients and when it is applied as a first- or second-line biological therapy.

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection. Neutrophils display circadian oscillations in numbers and phenotype in the circulation. Adrover and colleagues now identify the molecular regulators of neutrophil aging and show that genetic disruption of this process has major consequences in immune cell trafficking, anti-microbial defense, and vascular health., This study was supported by Intramural grants from A∗STAR to L.G.N., BES-2013-065550 to J.M.A., BES-2010-032828 to M.C.-A, and JCI-2012-14147 to L.A.W (all from Ministerio de Economía, Industria y Competitividad; MEIC). Additional MEIC grants were SAF2014-61993-EXP to C.L.-R.; SAF2015-68632-R to M.A.M. and SAF-2013-42920R and SAF2016-79040Rto D.S. D.S. also received 635122-PROCROP H2020 from the European Commission and ERC CoG 725091 from the European Research Council (ERC). ERC AdG 692511 PROVASC from the ERC and SFB1123-A1 from the Deutsche Forschungsgemeinschaft were given to C.W.; MHA VD1.2/81Z1600212 from the German Center for Cardiovascular Research (DZHK) was given to C.W. and O.S.; SFB1123-A6 was given to O.S.; SFB914-B08 was given to O.S. and C.W.; and INST 211/604-2, ZA 428/12-1, and ZA 428/13-1 were given to A.Z. This study was also supported by PI12/00494 from Fondo de Investigaciones Sanitarias (FIS) to C.M.; PI13/01979, Cardiovascular Network grant RD 12/0042/0054, and CIBERCV to B.I.; SAF2015-65607-R, SAF2013-49662-EXP, and PCIN-2014-103 from MEIC; and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) to A.H. The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).

Graft-versus-host-disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation. GvHD patients have aberrant T cell expansions, which are thought to drive pathological immune activation. Here we report mechanistic insights that somatic mutations may account for persistent clonal T cell expansions in chronic GvHD (cGvHD). In an index patient suffering from cGVHD, we discovered persisting somatic MTOR, NFKB2, and TLR2 mutations in an expanded CD4+ T clone. In the screening cohort (n=135), the MTOR P2229R kinase domain mutation was detected in two additional cGvHD patients, but not in controls. Functional analysis of the discovered MTOR mutation indicated a gain-of-function alteration in translational regulation yielding in up-regulation of phosphorylated S6K1, S6, and AKT. Paired single-cell RNA and T cell receptor alpha and beta sequencing strongly supported cytotoxicity and abnormal proliferation of the clonally expanded CD4+ T cells. Real-time impedance measurements indicated increased cytotoxicity of mutated CD4 + T cells against the patient’s fibroblasts. High throughput drug-sensitivity testing suggested that mutations induce resistance to mTOR inhibitors but increase sensitivity for HSP90 inhibitors. Our findings suggest a novel explanation for the aberrant, persistent T cell activation in cGvHD, and pave the way for novel targeted therapies.

Graft-versus-host disease (GVHD) is the main complication after allogeneic hematopoietic stem cell transplantation. We previously unveiled a correlation between proportions of C-C motif chemokine receptor 7 (CCR7)+ T cells in the apheresis and the risk of developing GVHD. We wanted to evaluate in vivo whether apheresis with low proportion of CCR7+ cells or treatment with an anti-human CCR7 monoclonal antibody (mAb) were suitable strategies to prevent or treat acute GVHD in preclinical xenogeneic models. Therapeutic anti-CCR7 mAb was the most effective strategy in both prophylactic and therapeutic settings where antibody drastically reduced in vivo lymphoid organ infiltration of donor CCR7+ T cells, extended lifespan and solved clinical signs. The antibody neutralized in vitro migration of naive and central memory T cells toward CCR7 ligands and depleted target CCR7+ subsets through complement activation. Both mechanisms of action spared CCR7− subsets, including effector memory and effector memory CD45RA+ T cells which may mediate graft versus leukemia effect and immunity against infections. Accordingly, the numbers of donor CCR7+ T cells in the apheresis were not associated to cytomegalovirus reactivation or the recurrence of the underlying disease. These findings provide a promising new strategy to prevent and treat acute GVHD, a condition where new specific, safety and effective treatment is needed.

Background and objectives: Percentages of blood CD19 + CD5 + B cells and CD8 + perforin + T lymphocytes can predict response to Interferon (IFN)-beta treatment in relapsing-remitting multiple sclerosis (RRMS) patients. We aimed to standardize their detection in a multicenter study, prior to their implementation in clinical practice. Methods: Fourteen hospitals participated in the study. A reference centre was established for comparison studies. Peripheral blood cells of 105 untreated RRMS patients were studied. Every sample was analyzed in duplicate in the participating centre and in the reference one by flow cytometry. When needed, participating centres corrected fluorescence compensations and negative cut-off position following reference centre suggestions. Concordance between results obtained by participating centres and by reference one was evaluated by intraclass correlation coefficients (ICC) and Spearman correlation test. Centre performance was measured by using z-scores values. Results: After results review and corrective actions implementation, overall ICC was 0.86 (CI: 0.81-0.91) for CD19 + CD5 + B cell and 0.89 (CI: 0.85-0.93) for CD8 + perforin + T cell quantification; Spearman r was 0.92 (0.89-0.95; p < 0.0001) and 0.92 (0.88-0.95; p < 0.0001) respectively. All centres obtained z-scores 5 0.5 for both biomarkers. Conclusion: Homogenous percentages of CD19 + CD5 + B cells and CD8 perforin + T lymphocytes can be obtained if suitable compensation values and negative cut-off are pre-established.

Reconstitution of B-cells after their therapeutic depletion with rituximab mimics the ontogeny of the B-cell linage in patients with rheumatoid arthritis. However, little is known about the effects of multiple cycles of treatment on the repletion kinetics and their long-lasting effects on the B-cell compartment. We therefore compared the recovery capacity of the B cell subpopulations between patients who experienced their first cycle of rituximab and those who experienced successive cycles.The distribution of the different B-cell subsets was characterised by multiparametric flow cytometry in the peripheral blood of 29 patients in the first rituximab course (naïve cycles) and 40 patients in successive cycles. Samples were obtained at baseline and at 3, 6, and 8 months of each cycle.The baseline percentage of B-cell subsets was similar among successive cycles. Therefore, successive cycles were grouped for comparison with naïve cycles. Patients in naïve cycles had higher percentages at baseline of both total and memory B-cells. However, the recovery of the different B-cell subsets was similar between naïve and successive cycles. In naïve patients the percentage of transitional B-cells significantly correlated with disease activity at baseline.Rituximab induces a long-term reshape of the B-cell compartment while multiple cycles of rituximab do not induce cumulative effects on B-cell subpopulations. Transitional B-cells seem to be associated with higher disease activity, although further studies are needed to determine if they can be used as a biomarker to predict the need for rituximab retreatment.

NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T-ALL), but crucial NOTCH1 downstream signals and target genes contributing to T-ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia-initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T-ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T-ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T-ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T-ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T-ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T-ALL pathogenesis. The observed therapeutic benefit of anti-CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T-ALL relapse., Ministerio de Economia y Competitividad (MINECO) PLE-2009-0110, SAF2010-15106, SAF2013-44857-R, and SAF2016-75442-R (Agencia Estatal de Investigacion/European Regional Development Fund, European Union), the Fundacion Sandra Ibarra, the Fundacion Asociacion Espanola Contra el Cancer (AECC CI13131229), the Instituto de Salud Carlos III (RTICC RD06/0014/1012), and the European Union Seventh Framework Programme (FP7/2007-20013) ThymiStem project (602587). Institutional grants from the Fundacion Ramon Areces and Banco de Santander

Purpose: Interleukin-8 (IL8) is a chemokine produced by malignant cells of multiple cancer types. It exerts various functions in shaping protumoral vascularization and inflammation/immunity. We evaluated sequential levels of serum IL8 in preclinical tumor models and in patients to assess its ability to estimate tumor burden. Experimental Design: IL8 levelsweremonitored by sandwich ELISAs incultured tumor cells supernatants, tumor-xenograftedmice serum, and in samples from126 patients with cancer. Wecorrelated IL8 serumlevels with baseline tumor burden and with treatment-induced changes in tumor burden, as well as with prognosis. Results: IL8 concentrations correlated with the number of IL8-producing tumor cells in culture. In xenografted neoplasms, IL8 serum levels rapidly dropped after surgical excision, indicating an accurate correlation with tumor burden. In patients with melanoma (n = 16), renal cell carcinoma (RCC; n = 23), non-small cell lung cancer (NSCLC; n = 21), or hepatocellular carcinoma (HCC; n = 30), serum IL8 concentrations correlated with tumor burden and stage, survival (melanoma, n = 16; RCC, n = 23; HCC, n = 33), and objective responses to therapy, including those to BRAF inhibitors (melanoma, n = 16) and immunomodulatory monoclonal antibodies (melanoma, n = 8). IL8 concentrations in urine (n = 18) were mainly elevated in tumors with direct contact with the urinary tract. Conclusions: IL8 levels correlate with tumor burden in preclinical models and in patients with cancer. IL8 is a potentially useful biomarker to monitor changes in tumor burden following anticancer therapy, and has prognostic significance. Fil: Sanmamed, Miguel F.. Universidad de Navarra; España Fil: Carranza Rua, Omar. Universidad de Navarra; España Fil: Alfaro, Carlos. Universidad de Navarra; España Fil: Oñate, Carmen. Universidad de Navarra; España Fil: Martín Algarra, Salvador. Universidad de Navarra; España Fil: Perez, Guiomar. Universidad de Navarra; España Fil: Landazuri, Sara F.. Universidad de Navarra; España Fil: Gonzalez, Alvaro. Universidad de Navarra; España Fil: Gross, Stefanie. University Hospital Erlangen; Alemania Fil: Rodriguez, Inmaculada. Universidad de Navarra; España Fil: Muñoz Calleja, Cecilia. Universidad Autonoma de Madrid. Hospital Universitario de la Princesa; España Fil: Rodríguez Ruiz, María. Universidad de Navarra; España Fil: Sangro, Bruno. Universidad de Navarra; España Fil: López Picazo, José M.. Universidad de Navarra; España Fil: Rizzo, Manglio Miguel. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mazzolini Rizzo, Guillermo Daniel. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pascual, Juan I.. Universidad de Navarra; España Fil: Andueza, Maria Pilar. Universidad de Navarra; España Fil: Perez Gracia, Jose Luis. Universidad de Navarra; España Fil: Melero, Ignacio. Universidad de Navarra; España

Background Imatinib, dasatinib, and nilotinib are tyrosine kinase inhibitors (TKIs) used as first-line treatment of chronic myeloid leukemia. Therapeutic drug monitoring is important to achieve treatment efficacy in the case of imatinib and nilotinib, and to control toxicity in the case of dasatinib. New high-sensitivity methods to monitor those drugs are needed, especially for dasatinib. Thus, a simple method to determine plasma levels of imatinib, dasatinib, and nilotinib for application in clinical practice was developed. Methods TKIs were eluted with a Poroshell 120 EC-C18 column (2.1 × 75 mm, 2.7 μm) at 0.5 mL/min and 60°C, under gradient conditions through a mobile phase consisting of 4 mmol/L ammonium formate, pH 3.2 (65%), and acetonitrile (35%). TKIs were detected and quantified by liquid chromatography in tandem with mass spectrometry (LC/MS-MS) with positive electrospray ionization and analytes were extracted using solid phase extraction (Versaplate-SCX). Internal standards were isotope-labeled for each analyte. Results The method was linear in the range of 2.5-5000 ng/mL for imatinib, 0.75-400 ng/mL for dasatinib, and 2-4000 ng/mL for nilotinib. The validation assays for accuracy and precision, matrix effect, extraction recovery, carryover, and stability of the samples for all the TKIs were appropriate according to regulatory agencies. Furthermore, imatinib plasma samples, stored for 4 years at -80°C were quite stable in approximately half of the samples. Conclusions The method enables rapid quantification of TKI concentrations and is being applied to therapeutic drug monitoring to adjust dose and to manage adverse reactions in clinical practice.

Mice deficient in P-Selectin presented altered immunity/tolerance balance. We have observed that the absence of P-Selectin promotes splenomegaly with reduced naïve T cell population, elevated activated/effector T cell subset, increased germinal center B and Tfh populations and high production of autoreactive antibodies. Moreover, 1.5-3-month-old P-selectin KO mice showed reduced IL-10-producing leukocytes in blood and a slightly reduced Treg population in the skin. With aging and, coinciding with disease severity, there is an increase in the IL17+ circulating and dermal T cell subpopulations and reduction of dermal Treg. As a consequence, P-Selectin deficient mice developed a progressive autoimmune syndrome showing skin alterations characteristic of lupus prone mice and elevated circulating autoantibodies, including anti-dsDNA. Similar to human SLE, disease pathogenesis was characterized by deposition of immune complexes in the dermoepidermal junction and renal glomeruli, and a complex pattern of autoantibodies. More important, skin biopsies of cutaneous lupus erythematosus patients did not show increased expression of P-Selectin, as described for other inflammatory diseases, and the number of vessels expressing P-Selectin was reduced.

The homeostatic CC-chemokine receptor 7 (CCR7) is highly expressed in many hematological malignancies including chronic lymphocytic leukemia (CLL). CCR7 surface expression levels are much higher than those of CD20, and are correlated with poor prognosis. Upon engagement by its ligands (CCL19 and CCL21), CCR7 controls trafficking of CLL cells to locations where these chemokines are expressed, such as the lymph node (LN). In this protective microenvironment CCR7 ligands contribute to CLL cell survival and proliferation. Indeed, high CCR7-mediated in vitro migration of patient CLL cells correlates with LN involvement and adverse prognostic factors. Thus, strategies targeting CCR7 could provide an additional therapeutic approach for CLL. Therefore, we have generated CAP-100, the first humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) that specifically binds to human CCR7 and neutralizes ligand-mediated signaling from both CCL19 and CCL21. The antibody effectively inhibited CLL cells migration and survival. Furthermore, in in vivo pre-clinical studies, CAP-100 was shown to inhibit entry of CCR7-expressing cells to LNs. The antibody showed potent cell killing activity against CLL cells while preserving normal blood cells. This Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) clearly outperformed standard-of-care in CLL, such as rituximab. ADCC and migration inhibition were both independent of prognostic markers for high risk CLL, such as TP53 mutation including 17p deletion and relapse/refractoriness to various standard therapies, including BTK-inhibitors. Finally, when given as monotherapy in disseminated NHL xenograft tumors in SCID mice, CAP-100 exhibited tumor growth inhibition in established tumors and provided significant survival. Collectively, our results demonstrate that CAP-100, the first-in-class anti-CCR7 mAb, is a potent antagonist with biological activity in CLL primary cells, including those from relapsed/refractory patients. Moreover, these results highlight the relevance of the CCR7-CCL19/CCL21 pathway as a therapeutic target in CLL. CAP-100’s unique propensity to block migration of CLL cells to the LN, in combination with its potent cell killing activity provides the biological rationale for use of CAP-100 in CLL, either as monotherapy or in combination with novel agents such as BTK-inhibitors. Clinical trials in CLL and other CCR7-expressing NHL will be initiated soon. Citation Format: Carlos Cuesta-Mateos, Cecilia Muñoz-Calleja, Javier Loscertales, Fernando Terron, Wim Mol. CAP-100: first-in-class anti-CCR7 antibody for CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4849.

The proportion of multiple myeloma patients in long-term complete response (LTCR-MM) for more than 6 years after autologous stem cell transplantation (ASCT) is small. To evaluate whether this LTCR is associated with a particular immune signature, peripheral blood samples from 13 LTCR-MM after ASCT and healthy blood donors (HBD) were analysed. Subpopulations of T-cells (naive, effector, central memory and regulatory), B-cells (naive, marginal zone-like, class-switched memory, transitional and plasmablasts) and NK-cells expressing inhibitory and activating receptors were quantified by multiparametric flow cytometry (MFC). Heavy/light chains (HLC) were quantified by nephelometry. The percentage of CD4+ T-cells was lower in patients, whereas an increment in the percentage of CD4+ and CD8+ effector memory T-cells was associated with the LTCR. Regulatory T-cells and NK-cells were similar in both groups but a particular redistribution of inhibitory and activating receptors in NK-cells were found in patients. Regarding B-cells, an increase in naive cells and a corresponding reduction in marginal zone-like and class-switched memory B-cells was observed. The HLC values were normal. Our results suggest that LTCR-MM patients express a particular immune signature, which probably reflects a 'high quality' immune reconstitution that could exert a competent anti-tumor immunological surveillance along with a recovery of the humoral immunity.

The pathogenesis of GvHD involves migration of donor T-cells into the secondary lymphoid organs in the recipient, which is steered by two homing molecules, CD62L and CCR7. Therefore, we investigated whether the migratory capacity of donor T-cells is associated with GvHD. This single center prospective study included 85 donor-recipient pairs. In vitro chemotaxis assays of the lymphocytes of the apheresis product were performed in parallel to the analysis of CD62L and CCR7 by flow cytometry. The migratory index to the CCR7 ligands, CCL19 and CCL21, was higher in T-cells from donors whose recipients will develop GvHD. Similarly, the acute GvHD (aGvHD) group received higher percentage of CD4+CCR7+ T-cells, whereas chronic GvHD (cGvHD) patients were transplanted with higher percentages of CD8+CCR7+ T-cells compared with the non-GvHD group. These results were confirmed when patients were subdivided according to degrees of severity. Further, multivariate analysis confirmed that the proportions of CCR7+ CD4+ and CCR7+ CD8+ T-cells are risk factors for the development and severity of aGvHD and cGvHD, respectively. Functional experiments demonstrated that CCR7+ T-cells exhibited higher potential for activation than CCR7- T-cells did. We therefore propose that the selective depletion of CCR7-expressing T-cells may be an effective preventive therapy for GvHD.

[Purpose]: Dasatinib is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which is frequently used to treat chronic myeloid leukemia. Although very effective, patients taking dasatinib often display severe adverse effects, including pleural effusions and increased risk of bleeding primarily in the gastrointestinal tract. The actual causes of these side effects are currently undetermined. We hypothesize that endothelial cells (ECs) that line the inner walls of blood vessels and control the traffic to the underlying tissues might be involved. [Experimental Design]: The effects of TKIs on ECs were studied by various assays, such as real-time cell impedance measurements, live-cell microscopy, wound healing, Western blot, and an in vivo model. [Results]: Dasatinib uniquely causes a profound, dose-dependent disorganization of the EC monolayers. Dasatinib promoted the disassembly of cell–cell contacts, altered cell–matrix contacts, and further altered the wound healing. A key observation is that this effect is fully reversible after drug washout. In line with these in vitro observations, intraperitoneal administration of dasatinib to mice caused significant vascular leakage in the intestine. The underlying molecular mechanism of dasatinib-induced reorganization of the actin involves ROCK activation, which increases the amount of the phosphorylation of myosin light chain and consequently activates the non-muscle myosin II. [Conclusions]: Our data are consistent with a scenario in which dasatinib triggers a transient increase in vascular leakage that probably contributes to adverse effects such as bleeding diathesis and pleural effusions., A. Kreutzman is a Sigrid Jusélius postdoctoral fellow. Grants PI12/00494P and PI15/02085 from the Fondo de Investigaciones Sanitarias to C. Muñoz-Calleja supported this work. C. Muñoz-Calleja was co-financed by FEDER funds. M. Vicente-Manzanares is an investigator from the Ramon y Cajal Program (RYC-2010-06094) and is supported by grants SAF2014-54705-R from MINECO, Marie Curie CIG-293719 from the EU, CIVP16A1831 from Ramon Areces Foundation and the BBVA Foundation. S. Mustjoki is supported by the Finnish Cancer Institute, Academy of Finland, Sigrid Juselius Foundation and Finnish Cancer Associations.

[Purpose]: Chronic lymphocytic leukemia (CLL) still is lacking a cure. Relapse and development of refractoriness to current treatments are common. New therapies are needed to improve patient prognosis and survival. [Experimental design]: Indole-3-carbinol (I3C) is a natural product with antitumor properties already clinically tested. The effect of I3C, F-ara-A, and combinations of both drugs on CLL cells from patients representing different Rai stages, IGHV mutation status, cytogenetic alterations, p53 functionality, and treatment resistances was tested, as well as the toxicity of these treatments in mice. [Results]: I3C induces cytotoxicity in CLL cells but not in normal lymphocytes. I3C strongly synergized with F-ara-A in all CLL cells tested, including those with p53 deficiency and/or F-ara-A resistance. The mechanism of cell death involved p53-dependent and -independent apoptosis. The combination of I3C + F-ara-A was equally effective in CLL cells irrespective of IGHV mutation stage and patient refractoriness. Moreover, CLL survival and treatment resistance induced by co-culturing CLL cells on stroma cells were overcome by the combinatory I3C + F-ara-A treatment. No toxicity was associated with the combined I3C + fludarabine treatment in mice. [Conclusions]: I3C in combination with F-ara-A is highly cytotoxic in CLL cells from refractory patients and those with p53 deficiency. The striking dose reduction index for F-ara-A in combination with I3C would reduce fludarabine toxicity while having a similar or better anti-CLL effectiveness. Moreover, the low toxicity of I3C, already clinically tested, supports its use as adjuvant and combinatory therapy in CLL, particularly for patients with relapsed or refractory disease., This work was supported by grants from the Ministerio de Economia y Competitividad (PI12/01135 to J.M. Zapata, PI12/00494 to C. Muñoz-Calleja, and PI11/00708 to I. Buño) and from the Asociacion Española Contra el Cancer and Fundacion LAIR (to I. Buño). G. Perez-Chacon was the recipient of a JAE Doc contract from CSIC. The cost of this publication was paid in part by FEDER funds.

Involved/uninvolved heavy/light chain index can predict progression in transplanted multiple myeloma patients

Chronic lymphocytic leukemia (CLL) with deletions of the p53 locus on chromosome 17 and/or refractory to fludarabine chemoimmunotherapy remains a major clinical problem with few therapeutic options. Currently, these types of CLL are treated with approaches that do not target the p53 pathway, such as small molecules and monoclonal antibodies (mAb). We have previously postulated anti-CCR7 mAb therapy as a novel CLL treatment. In the present study, we evaluated the in vitro efficacy of anti-CCR7 mAb as a single agent in CLL patients with high-risk cytogenetics and/or refractory to fludarabine, by measuring CCR7 surface expression and complement-dependent cytotoxicity. Our results demonstrate that CCR7 is highly expressed in challenging and heavily treated CLL patients. In addition, the complement-mediated mechanism of action of this mAb effectively eradicates CLL cells while sparing subsets of T cells in these patients. Moreover, this mAb outperformed the activity of alemtuzumab, the mAb with the highest efficacy in these groups. Finally, in vitro activity was also demonstrated in patients with a disease refractory to both fludarabine and alemtuzumab, and patients harboring 11q22 deletion. Our results propose that anti-CCR7 mAb is an effective and promising future treatment in high-risk CLL.

Background Patients with clinically amyopathic dermatomyositis (CADM) appear to be at risk for developing cancer and interstitial lung diseases, but population data to confirm this hypothesis are limited. Moreover, CADM presents cutaneous and histological findings that may overlap with subacute cutaneous lupus erythematosus (SCLE). Objectives To determine the association between myositis-specific autoantibodies, myositis-associated autoantibodies and CADM in Spanish patients. In addition, to study the usefulness of these autoantibodies in the differential diagnosis between CADM and SCLE. Methods Serum samples were tested for myositis-specific autoantibodies and myositis-associated autoantibodies through immunoprecipitation and other standardized methods. Results Anti-CADM-p140 and anti-p155 antibodies were the only myositis-specific autoantibodies found and were associated with interstitial lung diseases and cancer respectively. No myositis-associated autoantibodies were found in CADM. Moreover, clinical subsets and proportions seemed to differ from Asian cohorts, where anti-CADM-p140 is considered a CADM hallmark antibody and a risk factor for the development of interstitial lung disease. Interestingly, anti-SSA was highly associated with SCLE, whereas no myositis-specific autoantibodies were found in this entity. Limitations of the study Association between CADM and myositis-specific autoantibodies and differences between CADM and SCLE were tested on a relatively small cohort of patients. Conclusion There is an association between cancer-associated myositis and interstitial lung diseases and their hallmark autoantibodies in our cohort. In addition, the combined determination of myositis-specific autoantibodies and SSA autoantibodies may help to accurately discriminate SCLE from CADM.

[Background]: The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model. [Methods]: NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 μg) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively. [Results]: The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated. [Conclusions]: The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies., BSC is supported by the Fundación Leucemia Linfoma and Fundación Vistare. Grants from the Fondo de Investigaciones Sanitarias to CMC (PI09/01336 and PI12/00494), JMZ (PI12/01135) and EFR (PI11/00128) and from IMMED to CMC supported this work.

INTRODUCTION: In patients with so called "late suboptimal responses" (patient with complete cytogenetic response (CCyR) without major molecular response (MMR) after 18 months of imatinib) , the role of dasatinib has not been evaluated. Dasatinib has unique immunomodulatory effects especially on the proliferation and activation of T- and NK-cells. Yet, how dasatinib affects the migration of lymphocytes is unknown. DASAPOST is the first clinical trial evaluating efficacy and safety of dasatinib in patients with late suboptimal response (now considered as ELN2013 as warning). Another aim is to correlate new immunological aspects related to dasatinib and its possible correlation with responses. METHODS: We are presenting results of first 18 patients enrolled in the phase II DASAPOST study (NCT01802450). Main inclusion criteria were patients treated with late suboptimal response by ELN09 (CCyR without MMR after 18 months of treatment. Sokal risk groups were (L/I/H) 22.5%, 50% and 22.5%. All BCR-ABL/ABL (IS) measurements were centralized in an EUTOS laboratory. An exhaustive lymphocyte migration study was done, including immunophenotipying pre and post samples (CD 45, CD3,CD8, CD16, CXCR3, CXCR4, CD56 and CCR7), migration assay (chemokines CXCL10, CCL19+CCL21 and CXCL12) and CXCL10 plasma concentration measured by ELISA. RESULTS: - Clinical: Median follow up was 288 days (100-380). Three out of 18 (16%) patients had discontinued dasatinib due to side effects (pancreatitis, pleural effusion and low grade, persistant side effects (fever, arthralgias, anemia and astenia). All patients have been evaluated at 3 months, 17 at 6 months and 11 at 12 months. Cumulative incidences by ITT of MMR by 3 and 6 months were 50% and 81%. Cumulative incidences by ITT of MR4.5 by 3 and 6 months were 18% and 25%, respectively. - Inmunological: Dasatinib intake induced a significant increase of NK-cells and decrease of percentage of T-cells. Further, it increased CD8+ T cells, while reducing the proportion of CD4+ T-cells among the total T-cells. With the first dose of dasatinib (to),the percentage of CCR7 was lower in CD4+ and CD8+ T-cells in the post-samples. Lymphocyte migration was studied with transwell assays. At t0, post-samples showed a reduced migratory capacity towards the chemokines CCL19 and CCL21 in both CD4+ and CD8+ T-cell subsets. Patients were classified as mobilizers (n=14) or non-mobilizers (n=3) depending on whether they experienced an increase in the absolute lymphocyte counts after the first intake of dasatinib or not, showing different lymphocyte distribution and migratory capacity. In order to study the long term effects of dasatinib, we calculated the fold change (FC) of absolute lymphocyte counts pre- and post-dasatinib intake. Patients were divided into two groups based on whether in the 3 months samples (t3) had a higher ("increase group") or a lower ("decrease group") FC compared with t0. The migratory capacity of these two groups was studied in basal conditions and towards CCL19+CCL21 or CXCL10. We found no differences in basal migration in the "increase "group, while, the basal migration in the "decrease" group was quite promoted at t0 and t3. Further, migration towards CCL19+21 in post-samples is even more inhibited in "increase" patients at t3, whereas in the "decrease" patients the inhibition is diminished. The patients were divided into two groups based on the achievement of MMR at t3. At t0 both patient groups had similar migratory capacity, however, at t3, responders maintained significantly impaired migratory capacity to CCL19+21, compared with non-responders. CONCLUSIONS: Our study shows, for the first time to our knowledge, that in patients treated with Imatinib and with late warning responses, switch to Dasatinib induced MMR in 83% of the patients, although 16% discontinued treatment because of toxicity. We reported for the first time that dasatinib has significant effects on lymphocyte migration, and these are associated with early response. Disclosures García-Gutierrez: Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Casado:Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Sánchez-Guijo:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau. Boque:Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria. Muñoz-Calleja:BMS: Research Funding. Steegmann:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Ariad: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Background. Cytomegalovirus (CMV) is a widespread persistent β–herpesvirus, which can cause severe complications during primary infection or reactivation in immunocompromised patients, such as after allogeneic stem cell transplantation (alloSCT). Another major complication associated with alloSCT is graft-versus-host disease (GVHD). The pathogenesis of GVHD involves migration of the transplanted donor naïve T-cells into the secondary lymphoid organs in the recipient, which is mainly steered by CD62L and CCR7. As these homing molecules have been associated with both acute GVHD (aGVHD) and chronic GVHD (cGVHD), we studied whether the CMV serostatus of the donor affects the lymphoid composition of the graft product and whether this phenotype can predict CMV reactivation and GVHD. Methods. This single-center study included 77 donor-recipient pairs who underwent alloSCT. 64 pairs were HLA identical, 12 had 1 mismatch and 3 had 2 mismatch. 36 donors were related to their recipients. All recipients were followed at least for 100 (aGVHD) or 360 days (cGVHD) after transplantation. 43 donors were CMV-seropositive (CMVpos) and 34 were CMV-seronegative (CMVneg). 62 recipients were CMVpos, and 32 of them developed CMV reactivation, 25 aGVHD and 30 cGVHD. Samples from the graft product (donor) were phenotyped by flow cytometry (CD45, CD3, CD8, CD4, CD62L, CCR7) and both frequency (freq) and absolute number (abs) of each T-cell subpopulation were analyzed. Results. When the donors were divided based on their CMV serostatus, we observed that the grafts from CMVpos donors had a lower freq of naïve (CCR7+CD62L+) CD4+ T-cells (of lymphocytes p=0.06, of CD3 p=0.06, of CD4 p=0.07) and naïve CD8+ T-cells (of leukocytes p=0.03, of lymphocytes p=0.041, of CD3 p=0.011, of CD8 p=0.012) compared to CMVneg donors. Further, the abs of transplanted naïve CD8+ T-cells was significantly lower in the grafts from CMVpos donors (p=0.048). No differences were observed in T-cells (CD3+, CD4+, CD8+). We next studied if the CMV-serostatus and T-cell phenotype of the graft associates with GVHD. CMVpos donors whose recipients developed aGVHD had higher abs (p=0.05) and freq of naïve CD8+ T-cells (of lymphocytes p=0.08, of CD3 p=0.08, of CD8 p=0.11) compared to those without aGVHD. The same trend was observed with abs (p=0.11) and freq of CCR7+CD4+ T-cells (of leukocytes p=0.15). Similarly, those CMVpos donors whose recipients developed cGVHD had higher abs (p=0.05) and freq of CCR7+CD8+ T-cells (of leukocytes p=0.03, of lymphocytes p=0.06). Further, cGVHD patients who received the transplant from CMVpos donors were infused with a higher freq of CD3+ (of leukocytes p=0.03) and CD4+ T cells (of leukocytes p=0.04) than patients who received a graft from CMVpos donors but did not develop cGVHD. In contrast, CMVneg donors who developed aGVHD had a higher freq of CD3+ (p=0.018) and CD4+ T-cells (p=0.09), whereas no differences were seen in the T-cell subpopulations. Conversely, the abs (p=0.08) and freq of CCR7+CD4+ T-cells (of leukocytes p=0.11) were higher in those who later developed cGVHD. To study whether the graft lymphoid composition can be used to predict CMV reactivation, we analyzed the lymphoid composition in the graft product of those donors (both CMVpos and CMVneg) whose recipients were CMV seropositive but did not develop any form of GVHD (to avoid the influence of GVHD in the reactivation of CMV). Despite the low number of patients (CMV reactivation n=9, and no CMV reactivation n=13), we observed trends of higher portion of CD4+ T-cells (p=0.09 of lymphocytes, of CD3 p=0.20) and CCR7+CD4+ T-cells (of lymphocytes p=0.18, of CD4 p=0.16) in those grafts that were transplanted into CMV seropositive recipients who did not reactivate CMV. Conclusions. CMVpos donors whose recipient developed either aGVHD or cGVHD had a higher abs and freq of naïve CD8+ T-cells, which was not seen with CMVneg donors. This suggests that seropositivity sets the abs and freq of CD8 subpopulations near to a decisive cutoff for the development of GVHD. Conversely, other factors influences the development of GVHD in those patients whose donors were seronegative. In other words, seropositivity of the donor affects the graft composition and thus the risk of GVHD. Finally, our data indicate that a higher proportion of naïve or central memory CCR7+ CD4+ T cells in the donor graft could prevent CMV reactivation suggesting that graft composition affects also CMV reactivation. Disclosures No relevant conflicts of interest to declare.

e22135 Background: IL-8 is a chemokine that is produced by malignant cells of multiple cancer types and that exerts various functions in shaping pro-tumoral vascularization and inflammation/immunit...

Background Dasatinib is a second-generation tyrosine kinase inhibitor (TKI), which is used successfully in the treatment of chronic myeloid leukemia (CML). Dasatinib has two unique features when compared to other TKIs (imatinib, nilotinib); first, dasatinib has a significantly shorter half-life in the plasma and second, dasatinib inhibits a wider spectrum of kinases, including several kinases known to be important in the function of the immune system (src, tec, and syk families), which are not affected by the other TKIs. Interestingly, it has been recently shown that both short-term exposure to dasatinib in vivo and long-term treatment with dasatinib improves NK-cell cytotoxicity, however, the mechanisms are not known. To study the improved cytotoxicity observed in dasatinib-treated patients, we aimed to perform a complete NK-cell phenotyping in these patients. Finally, our goal is to correlate NK-cell phenotype with NK-cell cytotoxicity, and to study the possible correlation between phenotypical changes, NK-cell function and clinical outcome. Methods This study included 19 dasatinib-treated (DA) CML patients, both first-line (n=7) and second-line (n=12). To investigate the specificity of the immunomodulatory effects of dasatinib, a control group of 9 CML patients treated with imatinib (IM) and another group of 12 healthy donors (HD) were included. Peripheral blood samples obtained before the patients took their daily drug dose were phenotyped with a comprehensive 8-color flow cytometry panel (total 32 antibodies, table 1). To study the correlation between phenotypical changes and NK-cell cytotoxicity, we performed a standard CD107 degranulation assay. Mononuclear cells were incubated for 6 hours in the presence of the target cell line K562 and a CD107 antibody. CD107 positive NK-cells were then phenotyped with the same panel of 32 antibodies. Results All results are summarized in table 1. In brief, DA- and IM-treated CML patients and HD had equal proportions of NK-cells (CD3negCD56+) of total lymphocytes. Regarding trafficking molecules, NK-cells in both DA- and IM-patients had a lower frequency of the chemokine receptor CCR7 when compared to HD. This suggests a reduction in the NK-cell population that is able to migrate to lymph nodes, and is likely caused by the disease or TKIs in general. Moreover, DA-treatment specifically decreases the expression of the homing molecule CD62L in NK-cells. In addition, NK-cells in DA-patients, when compared to IM and HD, expressed less CD11b and significantly more often CD11c and HLA-DR, which reproduce the immunophenotypic changes that typically occurs in recently activated NK-cells and has been shown to associate with improved clinical benefits. Conversely, increased expression of CD57 together with a lower frequency of CD27 and CD28 were observed in both groups of patients and were similar to those typically observed in conditions of chronic NK-cell stimulation. In contrast, DA-patients had a lower frequency of most of the studied NK-receptors (Nkp30, Nkp46, NKG2D, CD94, CD161, KIR2DL1/S1) when compared to IM and HD. This suggests that NK-cells in DA-treated patients have a more mature phenotype, which is caused by the treatment. Conclusions NK-cells in TKI-treated CML patients display a mature phenotype, which is often observed after chronic stimulation suggesting that TKIs have immunomodulatory effects on NK-cells or the disease itself causes the changes. Interestingly, NK-cells in DA-treated patients express a highly differentiated phenotype characterized by high expression of CD57, and decreased expression of Nkp30, Nkp46 and CD161. Similar changes were not seen in IM-patients or HD. It is possible that NK-cells expressing this phenotype might also represent those NK-cells that have previously been driven into clonal expansion by encounters with pathogens because of the specific immunomodulatory effects of dasatinib. This phenotype of highly mature NK-cells, which is associated with high cytolytic potential, could be responsible for the previously described enhanced NK-cytotoxicity caused by dasatinib. In accordance, our preliminary results suggest that these unique phenotypic changes observed in DA-treated patients correlates with the cytotoxic potential. Studies to correlate these results with therapy outcome are ongoing. Disclosures: Garcia: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Research Funding. Steegmann:Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau.

El pdf del artículo es la versión pre-print., [Objective]: The CCR7 chemokine receptor has been reported to promote homing of B-cell chronic lymphocytic leukemia (CLL) cells into lymph nodes and support their survival, but the mechanisms mediating these effects are largely unknown. We investigated the role of different signaling pathways triggered by CCR7 engagement by its ligands, the chemokines CCL19 and CCL21, in the control of CLL migration and survival. [Materials and Methods]: Chemotaxis and apoptosis assays were performed in the presence of pharmacologic inhibitors and genetic mutants of the phosphatidylinositol-3-OH kinase (PI3K), Rho guanosine triphosphatase, and mitogen-activated protein kinase (MAPK) signaling cascades to assess the role of these pathways on primary CLL migration and survival in response to CCR7 activation. Kinase activation was determined by immunoblotting and pull-down experiments. [Results]: CLL chemotactic activity induced by CCL19 or CCL21 was markedly reduced by inhibitors of PI3K and the Rho effector molecule Rho-associated coiled-coil forming protein kinases (ROCK), and also by the expression of dominant negative forms of PI3K and RhoA, whereas constitutively activated PI3K and RhoA mutants strongly promoted CLL migration. In contrast, MAPKs were not significantly involved in CLL migration to CCL19/CCL21. Conversely, extracellular signal-regulated kinase and c-Jun-N-terminal kinase, along with PI3K, had a role in CCR7-mediated CLL cell survival. Biochemical experiments confirmed that CCL19/21 induced PI3K-dependent phosphorylation of Akt/protein kinase B, activation of the Rho/Rho-associated coiled-coil forming protein kinases/myosin light chain pathway and MAPKs phosphorylation. [Conclusions]: The role of PI3K, Rho guanosine triphosphatases, and MAPKs in CCR7-mediated CLL cells migration and survival suggests that these signal transduction pathways could represent promising targets for CLL therapy. © 2010 ISEH - Society for Hematology and Stem Cells., Funding was from Ministerio de Educación y Ciencia (PETRI2005_0908), IMMUNONET (SUDOE1/P1/E014) and Immunological and Medicinal Products Inc. (Madrid, Spain) (064700) to C.M-C, and FIS (PI080170) to JMZ. S.L-G. was supported by the Fundación de Investigación Biomédica, Hospital de La Princesa (Madrid, Spain). M.A-P. was supported by the Fundación LAIR (Madrid, Spain).