ALBERT

Description: Earlier evaluation of therapy effect in patients with CML would assist in optimal use of available tyrosine kinase inhibitors (TKI). Single cell analysis by mass cytometry has enabled the quantification of up to 46 antibody epitopes, making it ideally suited for exhaustive immunophenotyping of the haematological hierarchy, and evaluation of associated dynamic signal transduction events, in a clinical setting. By integrating time resolved single cell signalling data with clinical parameters, we searched for prognostic and efficacy-response mass cytometry biomarkers within a month of TKI therapy. We report data from experiments used to validate the custom panels of antibodies, highlighting the power of mass cytometry in the analysis of primary patient material obtained on clinical studies. Peripheral Blood (PB) samples were collected before, 3 hours, 7 days and 28 days, after start of nilotinib (300 mg BID) treatment in a subset of patients (n=55) enrolled in the ENEST1st trial. PB cells were stained with two panels of antibodies, allowing a comprehensive immunophenotyping of numerous cellular subsets, and also the evaluation of intracellular phosphorylation status of several epitopes. Moreover, using a straightforward barcoding scheme, the time-resolved samples from each individual patient were pooled after barcoding and stained with the antibody panels to minimize sample variation. In a pilot study, 7 and 10 cell subsets were identified in PB samples from 4 untreated healthy donors and 2 complete sets of 4 patients enrolled in this sub study, respectively. Furthermore, a robust signal was measured for pCrkL, pStat5, pStat3, pCreb, pAbl Y412 and pAbl Y245. The two sets of samples from study patients showed substantial changes in activation status over the course of therapy. Some changes, such as pStat3 alterations are only detectable in neutrophils and monocytes, while the activity of others i.e. pCreb was found to be ubiquitous. CD34+ cells indicated decreased phosphorylation of CrkL, Stat5, and Abl Y412/245. To increase the immunophenotyping resolution of the myeloid lineage, 3 additional cell surface markers were incorporated into the cell surface panel. In 1 healthy donor, and in diagnostic samples from three patients enrolled in this sub study, this allowed the identification of 13 cell subsets: CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-) (Figure 1 A,B). With respect to the relative number of cells identified for each cell type, the three diagnosis samples differed from the single healthy control. In the patients, we observed an expansion of the granulocytic compartment, as well as the emergence of CD34+ progenitor and stem cells in the peripheral blood. In conclusion, the here presented developed assay is able to resolve most of the cell subpopulations found in the hematopoietic tree, and also robustly measure the activity of central signalling substrates known to be involved in CML pathogenesis. With the addition of new phospho-specific antibodies, the methodology may facilitate the detailed characterization of CML in an immunological context, and may shed new light on both the disease and therapeutic mechanism. Analysis of variation in signal responses and immune profile are now in progress in the subset of patients (n=55) in the ENEST1st trial. Figure 1. Manually annotated SPADE tree from healthy donor and patient (3581_0002). With the incorporation of additional cell surface markers, the protocol was able to identify 13 cellular subsets in healthy donors (A) and a typical CML patient (B): CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-). Figure 1. Manually annotated SPADE tree from healthy donor and patient (3581_0002). With the incorporation of additional cell surface markers, the protocol was able to identify 13 cellular subsets in healthy donors (A) and a typical CML patient (B): CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-). Disclosures Thaler: AOP Orphan: Research Funding. Lang:Celgene: Consultancy. Hjorth-Hansen:Bristol-Myers Squibb: Research Funding; Ariad: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding. Hellmann:Novartis: Consultancy, Other: funding of travel, accomodations or expenses, Research Funding, Speakers Bureau; BMS: Consultancy, Other: funding of travel, accomodations or expenses, Speakers Bureau. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Janssen:ARIAD: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Ossenkoppele:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Signe and Ane Gyllenberg Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Pfizer: Honoraria, Research Funding; the Finnish Cancer Societies: Research Funding; Academy of Finland: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Gjertsen:Bergen University Hospital: Research Funding.

Description: Background and Aim: Tyrosine kinase inhibitors (TKI) imatinib and dasatinib modulate immune responses in vitro and in vivo. Immunological surveillance in the MRD-situation might be of particular relevance for long-term control or even elimination of CML-repopulating stem cells. Moreover, baseline immunological characteristics may be associated with response to TKI therapy. Little is known about potential immune-modulatory effects of nilotinib in vivo. The ENEST1st study (NCT01061177) evaluated the role of first-line nilotinib therapy in CML-CP. The primary endpoint was the MR4 rate at 18 months. A comprehensive immunological monitoring program within this ENEST1st substudy characterized baseline and therapy-induced immunological variables to correlate them with biological disease characteritics and clinical response parameters. Methods: Peripheral blood was taken prior to treatment initiation and after 6 and12 months (mo) from 52 patients. Samples were analyzed by nine colour flow cytometry employing six panels of optimized antibodies to determine various leukocyte populations (e.g. T cell subpopulations including Treg and NKT cells, NK cells, B cells, monocytes, MDSC, dendritic cell subsets). Plasma concentrations of soluble CD62L (sCD62L) and TACE (tumor necrosis factor-α-converting enzyme; ADAM17, CD156b), the metalloproteinase inducing proteolytic cleavage of CD62L from the cell surface, were either measured by ELISA or (in case of the enzymatic activity of TACE) using a fluorogenic assay. Changes in immune cell parameters were correlated to biological disease features and clinical endpoints. Results: The most striking finding of this study is the drastic loss of the lymph-node homing marker CD62L on immune cells (T cell subsets and granulocytes) at baseline (basCD62L), which increased back to normal levels during nilotinib therapy. The proportion of basCD62L+ cells among both CD4+ and CD8+ T cell subsets significantly correlated with Sokal score (both as continous and categorial variable, i.e. high vs. low/int). Low basCD62L expression levels on both T cell subsets correlate with increased spleen size, higher BM and PB blast and WBC counts as well as it correlates to higher BCR-ABL copy numbers at almost all time points during treatment. Similarly, lower basCD62L on either CD4+ or CD8+ T cells is linked to a longer duration to reach the respective molecular endpoint. Patients reaching MR4 at 18 months (primary study endpoint) had significantly higher levels of basCD62L on both CD4+ (p=0.02) and CD8+ (p=0.008) T cells. Consequently, MR4 at 18 months was attained in a significantly higher percentage of patients in the basCD62hi compared to the CD62lo patients (63% vs. 13.0%). Vice versa, patients who reached MR4 at 18 months had significantly higher proportions of basCD62L expressing cells among both CD4+ and CD8+ T cells. Moreover, as depicted by a cumulative response rate, patients with high proportions of basCD62Lhi T cells, achieved MMR and MR4 significantly earlier and in a higher proportion throughout the observation period. A detailed characterization of other T cell differentiation marker (CD45RA, CD45R0, CD28, CD27, and CD95) did not reveal significant baseline T cell subset alterations as explanation for altered CD62L expression. In contrast to low basCD62L surface expression levels, its shed form sCD62L is significantly increased at diagnosis but subsequently drops back during nilotinib therapy. Similar to surface CD62L expression, also sCD62L associates with biological disease features and molecular response to nilotinib. Finally, low CD62L surface expression was associated with elevated sCD62L levels and increased proteolytic activity but not total amount of TACE. Conclusion: At baseline, increased proteolytic activity of TACE sheds CD62L from the immune cell surface. During nilotinib therapy, TACE activity gets normalized leading to re-expression of CD62L on T cells and vice versa a drop of sCD62L. Low baseline T cell expression levels of CD62L and increased sCD62L levels correlate to a more aggressive CML phenotype and are linked to inferior molecular response to nilotinib in early CML-CP. Larger prospective studies including also other TKIs are needed to confirm the prognostic relevance of sCD62L/CD62L expression as response-prediction marker, as this marker is easy to measure by ELISA in plasma samples or flow-cytometry. Disclosures Mustjoki: Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding. Loskog:RePos Pharma AB: Membership on an entity's Board of Directors or advisory committees; Vivolux AB: Membership on an entity's Board of Directors or advisory committees; Lokon Pharma AB: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; NEXTTOBE AB: Membership on an entity's Board of Directors or advisory committees; Alligator Bioscience AB: Patents & Royalties. Gjertsen:Haukeland University Hospital: Research Funding. Giles:Novartis: Consultancy, Honoraria, Research Funding. Ossenkoppele:Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.

Description: Every second more than 2 million new erythrocytes are released from the bone marrow of human adults, highlighting the tremendous turn-over of these cells. In parallel to hematopoietic stem cell niches, the last stages of erythropoiesis take place in specialized bone marrow niches, termed 'erythroid niches'. Concretely, the erythroid niche is composed of a 'central macrophage' which is surrounded by erythroid progenitor cells. Regardless of steady-state or stress erythropoiesis, iron availability is, beside erythropoietin, a key factor determining erythroid output and red blood cell quality, as reflected by hemoglobin content of these cells. It is well established that systemic iron availability for erythropoiesis, in the form of iron saturated transferrin (Tf), is mainly maintained via a recycling process of senescent red blood cells, which takes place in macrophages of the reticuloendothelial system. Yet, it is still a matter of debate if also central macrophages are involved in iron supply for red blood cell development in a more direct way due to their close proximity to developing red blood cells. Using a myeloid-specific knockout mouse strain, lacking the solely known iron exporter ferroportin (Fpn; Fpnfl/flLysMCre+/+ mice) and specific reporter mice (ROSA26tdTomatofl/fl Cx3cr1CreERT2 mice), we examined the connection between iron metabolism, erythropoiesis and central macrophages. Analysis of Fpnfl/flLysMCre+/+ animals at steady state revealed microcytic anemia, higher tissue iron loading, reduced hepatic hepcidin expression and distorted erythroid precursor population distribution in the bone marrow with no significant chances in Tf saturation (Tf-Sat). The latter is giving a first hint, that local bone marrow Fpn expression on macrophages may be important for iron supply for erythropoiesis. Strikingly, further work up via flow cytometry demonstrated that disturbances seen in bone marrow erythropoiesis were accompanied by nearby loss of resident bone marrow macrophages (defined as CD11blo, F4/80pos, MerTKpos). In parallel, a CD11bhi, F4/80pos, MerTKpos population came into existence, suggesting that these cells may compensate for the loss of 'canonical' central macrophages. Attempting to explain these intriguing results, we sought to investigate differentiation pathways and turnover of bone marrow central macrophages. First, we used the ROSA26tdTomatofl/fl Cx3cr1CreERT2 monocyte-specific reporter mice and techniques of transient monocyte labelling in utero and in adult phlebotomized animals to determine the origin of central macrophages. We could show that those cells undergo constant replenishment by circulating monocytes. Notably, the rate of this process got markedly increased upon recovery from blood loss and concomitant expansion of the central macrophage population. Second, by administration of a CCR2/CCR5 inhibitor (cenicriviroc), diminishing monocyte egress from the bone marrow and tissue infiltration, we could demonstrate decreased reticulocyte count during stress erythropoiesis, thus strengthening the direct impact of macrophages to support effective erythroid output. Next, effects of stress-induced erythropoiesis were investigated in Fpnfl/flLysMCre+/+ compared to Fpnfl/flLysMCre-/- mice. Amelioration of anemia after phlebotomy was extended, microcytosis was more pronounced and reticulocyte egress was diminished but prolonged. Of interest, Fpnfl/flLysMCre+/+ mice on a diet containing an 8-times higher iron content during phlebotomy, thus transiently increasing Tf-Sat, recovered from anemia wildtype-like. These results indicate that stress erythropoiesis with a high iron demand depends, under normal iron availability, in part on central macrophages and their nursing function to overcome the increased demand of iron. Ongoing experiments aim to identify how recruited bone marrow macrophages, i.e. central macrophages, contribute to erythropoiesis during stress - if central macrophages directly supply developing erythroid cells with iron in a Tf-free fashion or, if they are suppliers of additional growth factors that work synergistically with the Tf-bound iron to drive hemoglobin production. In summary our data clearly show that macrophages need to be recruited to the bone marrow for effective erythroid output during stress erythropoiesis. Disclosures Weiss: Kymab Ltd.: Consultancy. Theurl:Kymab Ltd.: Consultancy, Research Funding.

Description: Recombinant erythropoietin (EPO) and iron substitution are a standard of care for treatment of anemias associated with chronic inflammation, including anemia of chronic kidney disease. A black box warning for EPO therapy and concerns about negative side effects related to high-dose iron supplementation as well as the significant proportion of patients becoming EPO resistant over time explains the medical need to define novel strategies to ameliorate anemia of chronic disease (ACD). As hepcidin is central to the iron-restrictive phenotype in ACD, therapeutic approaches targeting hepcidin were recently developed. We herein report the therapeutic effects of a fully human anti-BMP6 antibody (KY1070) either as monotherapy or in combination with Darbepoetin alfa on iron metabolism and anemia resolution in 2 different, well-established, and clinically relevant rodent models of ACD. In addition to counteracting hepcidin-driven iron limitation for erythropoiesis, we found that the combination of KY1070 and recombinant human EPO improved the erythroid response compared with either monotherapy in a qualitative and quantitative manner. Consequently, the combination of KY1070 and Darbepoetin alfa resulted in an EPO-sparing effect. Moreover, we found that suppression of hepcidin via KY1070 modulates ferroportin expression on erythroid precursor cells, thereby lowering potentially toxic-free intracellular iron levels and by accelerating erythroid output as reflected by increased maturation of erythrocyte progenitors. In summary, we conclude that treatment of ACD, as a highly complex disease, becomes more effective by a multifactorial therapeutic approach upon mobilization of endogenous iron deposits and stimulation of erythropoiesis.

Description: Introduction:Indoleamine 2,3 dioxygenase (IDO1) is the rate-limiting enzyme during metabolism of the essential amino acid tryptophan (TRP). IDO1 is up-regulated mainly by interferons during infection and inflammation and depletes tryptophan, which results in reduced T cell activation and proliferation as well as expansion of immunosuppressive regulatory T cells. Deregulation of IDO1 activity has been implicated in cancer immune evasion, but its role in chronic phase (CP) CML remains elusive so far. Methods:A large panel of circulating pro-inflammatory cytokines and components of the IDO-pathway (soluble IDO1=sIDO1 and kynurenine/tryptophan ratio=KYN/TRP as a product of IDO1 activity) as well as plasmacytoid dendritic cells (pDC) were analyzed alongside the prospective pan-european ENEST1st clinical study (NCT01061177). This substudy included 52 nilotinib-naïve chronic phase (CP)-CML patients that were subsequently treated with 300 mg BID nilotinib and analyzed at months 6 and 12. Molecular responses were quantified in central EUTOS reference laboratories. Results: Soluble IDO (sIDO1) levels and KYN/TRP ratio are significantly up-regulated in newly diagnosed CP-CML and significantly drop during nilotinib therapy. sIDO1 levels significantly correlate with increased KYN/TRP, suggesting increased IDO1 activity at diagnosis. Increased sIDO is linked to a pro-inflammatory status in CML patients, as it positively correlates to increased serum neopterin levels as well as to various other pro-inflammatory markers, such as IFN-g, IL-8, IL-10, IL-17A, sVEGF-A, sVCAM-1 and sTNFR-1. Interestingly, albeit being an IFN-regulated gene, IDO1 activity (KYN/TRP) negatively correlated with the proportion of pDC, the main producers of IFN-a. Interestingly, a higher KYN/TRP is linked to superior molecular response, as demonstrated by a significant correlation of the KYN/TRP ratio to BCR-ABL transcript levels. Patients having a high KYN/TRP ratio (〉 mean +2SD of post therapy levels) reach deep molecular response rates (i.e. MR4.5) significantly earlier and at higher rates. Conclusions: CML diagnosis in CP is linked to an inceased inflammatory status, as shown by increased levels of sIDO and its metabolites kynurenine leading to an increased KYN/TRP ratio. In solid cancer increased IDO expression/activity is linked to inferior outcome by favoring immune evasion. In contrast, in CML an increased KYN/TRP ratio is associated with improved molecular outcome during nilotinib 1st-line therapy. One reason could be that IDO activity may reflect endogenous IFN-α production, a known factor favoring immune-mediated CML-control. Disclosures Sopper: Novartis: Other: Travel costs reimbursement. Mustjoki:BMS, Novartis, Pfizer: Consultancy, Honoraria, Research Funding. Gastl:Novartis: Consultancy, Research Funding. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Janssen:BMS: Honoraria; Pfizer: Honoraria; ARIAD: Consultancy; Novartis: Research Funding.

Description: Background Leukemic stem cells (LSCs) are considered to be very important therapeutic targets in CML, as it has been shown both in vitro and in vivo that tyrosine kinase inhibitor (TKI) therapy is not able to eradicate these entirely. We previously reported that LSC burden at diagnosis has significant prognostic impact on therapy outcome in imatinib and dasatinib treated CML patients. The ENEST1st study (NCT01061177) is focused on examining the role of first-line nilotinib therapy in patients in CML-CP. This translational, multinational, multicenter ENEST1st substudy addresses the predictive value of the stem cell profile in these patients. Patients and methods Bone marrow (BM) and/or peripheral blood (PB) samples were collected from newly diagnosed CML-CP patients before and after 1 and 3 months of nilotinib treatment. The LSC burden was analyzed by FISH and flow cytometry (FC). With the FISH method, CD34+ cells were pre-selected with paramagnetic beads after which they were fractionated into CD38+ (upper 80%, “progenitor”) and CD38- (lowest 5%, “stem cell”) pools using flow sorting. The proportion of Ph+ cells was assayed by counting 1,000 cells with interphase FISH using specific BCR-ABL1 probes. In the FC method, the stem cell compartment was defined as the lowest 1% of CD34+CD38- cells. LSCs were distinguished from normal hematopoietic stem cells (nHSCs) by either higher light scatter properties, and/or aberrant expression of CD7, CD11b and CD56, and/or higher CD45 and CD90 expression. Patient samples were assigned to as “residual nHSCs present” or “no residual nHSC present” at diagnosis and as “residual LSCs present” or “no residual LSCs present” at 1 and 3 months. Altogether, 48 patients from 6 European countries were investigated. Results By FISH analysis at diagnosis, the proportion of BCR-ABL+ cells in the stem cell compartment varied markedly in individual patients (1%-100%) and was lower (85%) than in progenitor (96%) or whole BM fractions (96%, p80% of BCR-ABL+ cells in the stem cell fraction at diagnosis. When LSCs were analyzed by the FC method, patients with residual nHSCs at diagnosis had significantly lower BCR-ABL levels at 3 (0.27 vs 1.74, p=0.025) and 6 (0.13 vs 1.21, p=0.03) months, but this was not reflected in lower BCR-ABL levels at 12 or 18 months. During nilotinib therapy, the proportion of BCR-ABL+ cells by FISH decreased rapidly both the in progenitor and stem cell compartments. At 3 months the median LSC percentage was 0.28%, and it did not differ significantly from the values detected either in the progenitor fraction (0.34%) or whole BM (0.29%). Similarly, only a few patients had detectable LSCs left during follow-up time-points with the FC method. Conclusions LSC burden at diagnosis reflects the biology of the disease in newly diagnosed CML-CP patients. It also carries a prognostic value in first-line treated nilotinib patients, and a substantial number of patients not meeting the optimal response criteria at the 12-month time-point have high LSC burden at diagnosis. Furthermore, the presence of residual nHSCs at diagnosis is predictive for molecular response at 3 and 6 months. Nilotinib therapy markedly decreases LSC pool during the first 3 months of treatment. Noteworthy, the reduction is as potent in the LSC compartment as it is in more mature progenitor cell or whole BM fractions. Disclosures: Richter: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Fioretos:Novartis: unrestricted research grant Other. Giles:Novartis: Consultancy, Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Ossenkoppele:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Porkka:Novartis: Consultancy, Research Funding, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau. Wolf:Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Bristol-Meyers Squibb: Honoraria. Janssen:Novartis: Consultancy, Research Funding. Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

Description: Background: In CML, immunological surveillance in the MRD-situation might be of particular relevance for long-term control or even elimination of CML-repopulating stem cells. Little is known about potential immune-modulatory effects of nilotinib in vivo. A comprehensive monitoring program to characterize nilotinib-induced immunological changes was set up as a substudy to the ENEST1st study (NCT01061177), which is focused on examining the role of first-line nilotinib therapy in CML-CP. Aims: The identification of immunological changes induced by nilotinib and definition of immunological surrogates for response prediction in newly diagnosed CML-CP patients. Methods: Peripheral blood was taken prior to treatment initiation and after 6 and12 months (mo) from 50 patients treated on the ENEST1st study. Whole blood was phenotyped by 9-color flow cytometry employing 6 panels of antibodies to determine various leukocyte populations and expression of differentiation and activation markers. Soluble factors in plasma were measured by ELISA. Changes in immune parameters were correlated to clinical endpoints. Results: 55% of the patients included in this substudy achieved MMR at 6 mo, 75% at 12 mo and 79% at 24 mo of therapy. MR4.5 was achieved by 5%, 18%, 24% of patients at 6, 12 and 24 mo of therapy, respectively. Expression of L-selectin (CD62L), a known lymph node homing marker, was very low on T cells at baseline (median 5.7%) and the proportion of CD62L-expressing cells among CD4+ and CD8+ T cells negatively correlated with SOKAL score and spleen size. During treatment, CD62L expression on both T cell subsets significantly increased (median 72.3%) to normal levels at mo 6. Moreover, higher proportions of CD62L+ cells among both CD4+ and CD8+ T cells at baseline were negatively associated with lower BCR-ABL1 mRNA burden at 3, 6, 9, 15 and 18 mo. Cumulative response rates for MMR and MR4 were significantly higher in patients with high proportions (cutoff: 〉25%) of CD62L expressing T cells when compared to patients with low proportions of CD62L+ cells. A detailed characterization of other T cell differentiation marker, such as CD45RA, CD45R0, CD28, CD27 and CD95, did not reveal a reduction of cells usually expressing high CD62L levels, such as naïve T cells. CD62L expression levels on granulocytes were also low at baseline and increased to normal levels during nilotinib therapy. Low levels of CD62L expression were associated with elevated plasma levels of soluble CD62L before treatment initiation. Washing whole blood from treatment naïve patient several times before staining partially restored immunoreactivity for CD62L, and plasma taken at baseline but not after treatment initiation reduced CD62L signal on normal T cells, indicating that interference by increased levels of soluble CD62L with assessment of surface CD62L at least in part contributes to low CD62L expression. Importantly, ADAM17 (TACE, CD156b), the metalloproteinase responsible for CD62L shedding was increased on granulocytes and monocytes but not on T cells of CML patients when compared to normal controls suggesting that reduced levels on CD62L on T cells were mediated by ADAM17 on myeloid cells. Conclusion: We here show for the first time the prognostic impact of reduced CD62L expression levels at baseline for later molecular response to nilotinib in early CML-CP. Decreased expression of CD62L is at least in part due to increased CD62L-cleavage, probably by ADAM17, which is aberrantly expressed on the clonal myeloid cells. One may speculate that decreasing CD62L expression on T cells may interfere with immune surveillance of CML cells and that ADAM17 might be a target for adjunctive therapy. Larger prospective studies are needed to confirm the prognostic relevance of increased soluble CD62L and reduced T cell CD62L expression levels for molecular response in TKI-treated CML-CP. Disclosures Sopper: Novartis: Travel Grants Other. Mustjoki:Novartis: Research Funding. Gjertsen:Novartis: Research Funding. Mark:Novartis: Employment. Haenig:Novartis: Employment. Jurjonas:Novartis: Employment. Giles:Novartis: Honoraria, Research Funding. Hochhaus:Novartis: Research Funding; BMS: Research Funding; MSD: Research Funding; Ariad: Research Funding; Pfizer: Research Funding. Ossenkoppele:Novartis: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Wolf:Nocartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Introduction Tyrosine kinase inhibitor (TKI) trough plasma level testing and pharmacodynamic evaluation (i.e. quantification of phosphorylated BCR-ABL downstream targets) may help to optimize patient-tailored dosing in CML-CP. For 2nd generation TKIs the clinical value of TKI plasma level and protein phosphorylation quantification remains unclear. The ENEST1st study (NCT01061177) is focused on examining the role of first-line nilotinib therapy in CML-CP. This ENEST1st substudy addresses the value of plasma-level testing and protein single cell phosphorylation pharmacodynamics as predictors of response and long-term outcomes. Methods Nilotinib levels were centrally quantified by means of mass spectrometry (HPLC-MS/MS) in 54 patients at 3 hours after first drug intake at day (d) 1 (Cmax) and at d7, d28 as well as at months (mo) 3, 6, 12 prior to morning drug intake (Cmin=trough level). Single cell quantification of intracellular protein phosphorylation was performed by flow cytometry (phosphoflow) in the myelocyte cell population after full blood fixation/cell permeabilization/erythrocyte lysis and analyzed 3 hours after first drug intake at d1 and on d7 and d28. Primary endpoint of the study was MR4 (BCR-ABL IS 10% BCR-ABL. On d1 at 3 hours after intake of the first tablet the median peak plasma levels reached 408 ng/ml increasing to a median trough level of 834 ng/ml at d7, 888 ng/ml at d28, 919 ng/ml at 3mo, 1122 ng/ml at 6m and 1003 ng/ml at 12mo. Initial Cmax-levels at d1 significantly correlated with steady state levels thereafter (e.g. with trough levels at 12mo: r=0.56, p=0.004). Three mo drug levels were significantly negatively associated with lower BCR-ABL mRNA burden at 12m and 24m (r=-0.44, p=0.011 for m12 and r=-0.35, p=0.014 for m24). When median steady state drug levels were compared in different response categories (yes/no: MR4, MR5, MMR, 〉IS 10% at 3mo), significantly higher nilotinib trough levels at day 90 and mean trough level (day 28-180) were seen in patients achieving the primary endpoint of the study (MR4 at 18mo). Similarly, 3mo and mean through levels were also significantly higher in patients achieving MR5 at 12 or 18mo. As only one patient did not reach

Description: Background Imatinib and dasatinib modulate immune responses in vitro and in vivo. Immunological surveillance in the MRD-situation might be of particular relevance for long-term control or even elimination of CML-repopulating stem cells. Little is known about potential immune-modulatory effects of nilotinib in vivo. The ENEST1st study (NCT01061177) is focused on examining the role of firstline nilotinib therapy in CML-CP - this ENEST1st substudy involves a comprehensive immunological monitoring program.