ALBERT

Description: 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.

Description: Abstract 2782 Background: The inhibition of oncogenic BCR-ABL1 kinase with tyrosine kinase inhibitors (TKIs) has significantly improved the prognosis of CML, but the majority of patients need life-long therapy. Before the TKI era, CML patients were treated with interferon-α (IFN-α), and a minor proportion of patients (10–20%) achieved prolonged complete cytogenetic remissions (CCyR). Interestingly, many of the patients in prolonged CCyR were able to discontinue the treatment without imminent disease relapse. As the ultimate goal of current CML therapy is cure, the deeper understanding of the cellular mechanisms behind successful IFN-α discontinuation are of outmost importance. In this study, we analyzed the function of immune effector cells (NK- and T-cells) derived from CML patients in prolonged molecular remission after IFN-α monotherapy in order to understand the putative anti-leukemic effects. METHODS: The study cohort included 13 CML patients treated with IFN-α who were in complete molecular remission (CMR) at the time of blood sampling. Five patients were still on IFN-α therapy (IFN-ON, median duration of the treatment 13.7 years) and 8 had stopped treatment successfully (IFN-OFF) and were off from any treatment (median time without treatment 6.3 years). None of the patients had used any TKIs. Samples from 10 healthy volunteers were used as controls. The cytotoxicity of NK-cells in blood was studied by measuring the direct killing of K562 cells and by degranulation assay (CD107). The cytokine secretion (TNF-α and IFN-g) of NK-cells was measured with flow cytometry after stimulation with K562. The function of T-cells was studied by antibody (OKT3) stimulation and measuring TNF-α and IFN-g production by flow cytometry. In addition, NK-cells were phenotyped by multi-color flow cytometry. RESULTS: IFN-OFF patients had a larger proportion of NK-cells from lymphocytes than IFN-ON patients or healthy controls (IFN-OFF median 23.5 %, IFN-ON 7.0 %, healthy 13.2 %; p=0.0136). Based on the subset analysis, expanded NK-cells in IFN-OFF patients had mature phenotype CD56dimCD62LlowCD27lowCD57high. On the contrary, IFN-ON patients had a larger proportion of immunoregulatory CD56bright NK-cells (20.2% vs. 6.5% in healthy and 3.0% in IFN-OFF, p=0.0035). When the direct killing of K562 cells was studied, NK-cells from healthy controls killed better than NK-cells from either of the patients groups (in healthy 55 % of K562 cells were alive compared to 79% in IFN-ON and 91% in IFN-OFF, p=0.0042). Similar trend was also observed in degranulation assay (median degranulation in healthy controls 8.3 % compared to 4.3 % in IFN-OFF and 3.6 % in IFN-ON group, p= 0.39). The direct killing capability tended to correlate negatively r=-0.60, p=0.07) with the NK-cell proportion (ie. patients with high NK-cell counts had worse killing). Instead of cytotoxicity, NK-cells from IFN-OFF patients seemed to secrete more efficiently cytokines (IFN-γ/TNF-α) than NK-cells from healthy controls, but due to low number of patients no firm conclusions can be made (IFN-OFF median 6.9 %, healthy 0.7 %; p= 0.25). The proportion of potentially cytotoxic CD4+GrB+ T-cells did not significantly differ between the groups, but wide variation was observed between the individual patients (IFN-ON median 9.0 %, IFN-OFF 2.6 %, and healthy controls 3.5 %; p=0.4142). However, the secretion of TNF-α and IFN-γ by CD4+ cells seemed to be increased in IFN-OFF patients when compared to healthy controls (IFN-OFF mean 5.9 %, IFN-ON 3.7 %, healthy 2.5 %; p=0.1055). CONCLUSIONS: CML patients who have been able to discontinue IFN-α therapy (IFN-OFF group) had an expansion of mature CD56dimCD62LlowCD27lowCD57high NK-cells in peripheral blood. Although CD56dim NK-cells have typically been suggested to act as cytotoxic cells, the killing in response to K562 cells was lower in IFN-OFF patients than in controls. It is possible that the expanded NK-cells in IFN-OFF patients represent exhausted terminal stage cells (CD57+) or memory cells that do not react properly against third party target cells (K562). Moreover, they may have an immunoregulatory function instead of cytotoxicity. Further follow-up studies with other patient cohorts (TKI monotherapy and TKI+IFN-α combination therapy treated patients) are ongoing to clarify the issue further. Disclosures: Porkka: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.

Description: Abstract 1204 Background: Dasatinib is a potent tyrosine kinase inhibitor (TKI) used in the treatment of Philadelphia-chromosome positive (Ph+) leukemia. We and others have recently observed that in a proportion of patients, dasatinib induces an absolute, oscillating blood lymphocytosis characterized by an expansion of pre-existing clonal cytotoxic memory cells (Kreutzman et al. Blood 2010). Lymphocytosis has been correlated with autoimmune adverse effects (pleural effusions, colitis) and with exceptionally good therapy responses in patients with advanced leukemia. The factors causing lymphocytosis are unknown. Aim: To study the cellular and molecular mechanisms of lymphocytosis in dasatinib treated patients and to assess the relation of drug intake, plasma level and lymphocyte counts. Patients and methods: Blood samples from 23 consecutive Ph+ leukemia (CML, Ph+ALL; first and second line therapies) patients treated either with dasatinib (n=17, daily doses 50–100 mg), imatinib (n=2, 400 mg), nilotinib (n=2, 300 mg) or bosutinib (n=2, 500 mg) were collected before drug intake and after 1, 2, 4 and 6 hours after a single oral dose. Similar data was collected from 1 healthy control after 100 mg of dasatinib. Immunophenotype and T-cell receptor (TCR) V beta expression were determined with flow cytometry and a multiplex bead assay was used for the measurement of plasma cytokines. A differential gene expression analysis was done with an Agilent 44k oligonucleotide array. Plasma dasatinib concentrations were measured by HPLC/MS. Result: In all patients and in one healthy control dasatinib induced a rapid and marked mobilization of blood leukocytes with peak values after 1–2 hours (h) of oral drug intake. Most substantial mobilization was observed for lymphocytes (median fold change [mFC] 2.15, range 1.05–5.12, 0h vs. 1h, P

Description: In a proportion of patients with chronic myeloid leukemia (CML) being treated with dasatinib, we recently observed large granular lymphocyte (LGL) expansions carrying clonal T-cell receptor (TCR) γ/δ gene rearrangements. To assess the prevalence and role of clonal lymphocytes in CML, we collected samples from patients (n = 34) at the time of diagnosis and during imatinib and dasatinib therapies and analyzed lymphocyte clonality with a sensitive polymerase chain reaction–based method of TCR γ and δ genes. Surprisingly, at CML diagnosis, 15 of 18 patients (83%) had a sizeable clonal, BCR-ABL1 negative lymphocyte population, which was uncommon in healthy persons (1 of 12; 8%). The same clone persisted at low levels in most imatinib-treated patients. In contrast, in a distinct population of dasatinib-treated patients, the diagnostic phase clone markedly expanded, resulting in absolute lymphocytosis in blood. Most patients with LGL expansions (90%) had TCR δ rearrangements, which were uncommon in patients without an LGL expansion (10%). The TCR δ clones were confined to γδ+ T- or natural killer–cell compartments and the TCR γ clones to CD4+/CD8+ αβ+ fractions. The functional importance of clonal lymphocytes as a part of leukemia immune surveillance and the putative anergy-reversing role of dasatinib require further evaluation.

Description: Abstract 3760 Background. Tyrosine kinase inhibitors (TKIs; imatinib, dasatinib, nilotinib) have dramatically improved outcome of CML. Besides inhibiting target kinases in leukemic cells, off-target kinases in immune effector cells are also affected. We have previously described that dasatinib therapy induces an oligoclonal expansion and mobilization of large granular lymphocytes (LGLs; CD8+ T-cells or NK-cells) in Ph+ leukemia patients. Importantly, LGL expansion is associated with improved therapy responses, but the actual mechanisms are unknown. In this study, we explored the function and anti-leukemic properties of LGLs. Methods. Peripheral blood samples from CML patients treated with dasatinib (n=10), imatinib (n=4), or nilotinib (n=7), or healthy controls (n=6) were used to analyze the activation and cytotoxicity of T- and NK-cells. Samples were collected before and after drug intake. The number of LGLs was determined from MGG stained blood smears and compared with granzyme B (GrB) positivity analyzed by flow cytometry. Th1-type cytokine (TNF-a, IFN-g) production was measured by flow cytometry after stimulation of mononuclear cells (MNCs) with a-CD3/CD28-antibodies. Unpurified and purified NK cells were cultured with K562 cells, and degranulation (CD107 analysis) and cytotoxicity were measured. Results. As GrB positivity correlated well (r=0.95, p

Description: Abstract 3729 Comprising

Description: Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion protein are an effective therapy for Philadelphia chromosome positive (Ph+) leukemia. Dasatinib, a 2nd generation pan-TKI, also inhibits wild-type kinases, which may result in unexpected clinical responses. Recent data suggest that dasatinib has a potent immunosuppressive effect on T- and NK-cells in vitro. In contrast, we have noticed a marked expansion of lymphocytes in blood in a subset of dasatinib patients, but its clinical significance and molecular mechanisms are unclear. In this multicenter study, 19 Ph+ leukemia patients with marked lymphoproliferation in blood while on dasatinib (9 CML CP, 2 CML AP, 2 CML BC, 6 Ph+ALL) were identified. Clonality, immunophenotype, and intracellular signaling was analyzed and related clinical information was collected. In addition, prevalence and prognostic significance of the phenomenon was retrospectively assessed in a Phase II clinical study on 46 Ph+ ALL patients. An abrupt lymphocytosis (peak count range 4–20×109/L) with large granular lymphocyte (LGL) morphology was observed after a median of 3 months from the start of therapy (range 1–8 months). In most patients, LGL lymphocytosis was long-lasting and continued throughout dasatinib therapy, albeit with marked fluctuations in absolute lymphocyte count (Fig. 1a). In all evaluable patients (n=4), lymphocyte counts normalized after drug discontinuation. All patients were previously exposed to imatinib without similar changes in lymphocyte count or morphology. By immunophenotyping, 14 patients had a cytotoxic T-cell and 5 patients a NK-cell phenotype. After initiation of dasatinib therapy, the CD4/CD8 ratio shifted, expression of activation antigens HLA-DR and CD57 increased, expression of homing antigen CD62L decreased and relative numbers of regulatory T-cells were significantly decreased (p

Description: Background: Tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia (CML) are not entirely selective for the BCR-ABL1 kinase but also inhibit a variety of other kinases, sometimes triggering unpredicted biological effects. As an example, the TKIs dasatinib and bosutinib both inhibit Src-kinases, which are important mediators of T-cell function. Earlier in vitro data has shown that dasatinib can suppress activation and proliferation of T and NK cells, but it can also elicit signs of immunostimulation in patients, including rapid mobilization of lymphocytes and LGL lymphocytosis. No extensive analyses of the immunological in vivoeffects of bosutinib have been performed thus far. Therefore, we aimed at characterizing T and NK cell phenotypes and functional features in CML patients in a clinical setting in the context of first-line bosutinib and imatinib treatment. Methods:Peripheral blood samples were obtained from newly diagnosed CML CP patients enrolled in the BFORE clinical trial (NCT02130557), receiving bosutinib (n=13) or imatinib (n=20) as frontline TKI treatment. Samples were drawn at diagnosis and following 3 and 12 months of therapy. Detailed immunophenotyping of NK and T cells was performed with multicolor flow cytometry. In addition, mononuclear cells were used to study the function of NK and T cells (CD107ab degranulation upon stimulation with K562 cells and detection of IFN-γ/TNF-α secretion after stimulation with anti-CD3/anti-CD28 antibodies, respectively). Moreover, blood differential counts were taken before and 2 hours after drug intake at 3 and 12 months to examine the direct effects on lymphocyte counts (mobilization). Results: No significant changes were observed in absolute white blood cell or lymphocyte counts directly (2 hours) after bosutinib or imatinib intake, in contrast to what has been observed in dasatinib treated patients. Analysis of T cell subsets during bosutinib treatment revealed that the proportion of CD4+ cells increased after the start of treatment (median dg. 60.0% vs. 3 months 62.0% p=0.06; vs. 12 months 72.8% p=0.03), but no significant changes were observed in the phenotype. Correspondingly, the proportion of CD8+ T-cells decreased moderately (dg. 31.6% vs. 3 months 25.5% p=0.01) after the therapy start. Interestingly, the proportion of PD1+ (dg. 19.6% vs. 3 months 11.9%, p=0.06; vs. 12 months 14.3%, p=0.11) and DNAM+ CD8+ T-cells decreased (dg. 73.1% vs. 3 months 66.2% p=0.004; vs. 12 months 64.6% p=0.02). No changes in the cytokine production of any of the studied subgroups of T-cells was observed. Moreover, the proportion, phenotype and function of NK-cells were not affected by bosutinib treatment. In contrast, during imatinib treatment the proportion of CD56+CD16+ NK-cells significantly increased (dg 4.3% vs. 3 months 9.9% p=0.0005; vs 12 months 14.4% p=0.002; 8.1% in bosutinib treated patients). Moreover, in imatinib patients NK-cells downregulated CD27 (dg 9.0% vs. 3 months 5.2% p=0.004; vs. 12 months 4.9%; p=0.002). Further, NK-cells from imatinib-treated patients expressed more CD107ab upon stimulation with K562 at 3 and 12 months, when compared to samples from diagnosis (dg 13.0% vs. 3 months 16.1%, p=0.01; vs. 12 months 23.2%, p=0.008). The proportion of CD4+ T-cells increased 3 months after the start of imatinib treatment (dg 60.1% vs. 3 months 63.5% p=0.01), whereas the percentage of CD8+ T-cells decreased (dg. 38.6% vs. 3 months 31.5% p=0.02). Decreased expression of DNAM (dg 73.5% vs. 3 months 67.9% p=0.0008; vs. 12 months 62.4% p=0.002) was observed in the CD4+ T-cells. Similarly as in bosutinib treated patients, the proportion of PD1+ CD8+ cells decreased during imatinib treatment (dg 18.2% vs. 3 months 14.7%, p=0.02; vs. 12 months 14.8%, p=0.03). Both CD4+ and CD8+ T-cell subsets from imatinib-treated patients secreted less cytokines after the start of treatment when compared to the pre-treatment samples. Conclusions: Despite of the Src-kinase inhibitory profile of bosutinib, no major changes were observed in T- or NK-cell phenotype or function during first-line bosutinib treatment. In contrast, in imatinib treated patients the proportion of NK-cells increased and their degranulation responses were significantly higher than in untreated CML patients. Comparison of these data with the clinical variables and treatment outcome is warranted. Disclosures Stentoft: Novartis: Research Funding; Bristol-Myers-Squibb: Research Funding; Pfizer: Research Funding; Ariad: Research Funding. Gjertsen:BerGenBio AS: Consultancy, Research Funding. Janssen:Pfizer: Honoraria; Novartis: Research Funding; Ariad: Honoraria; BMS: Honoraria. Brümmendorf:Pfizer: Research Funding; Novartis: Research Funding. Richter:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Description: 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.