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B Gellen and L Messonnier, and L Feasson and P Bartolucci equally contributed to this work Introduction Sickle cell disease (SCD) is the most common inherited disease worldwide. Because favoring the risk of vaso-occlusive crises (VOC), SCD patients are banned from strenuous exercise. We hypothesized that a well-controlled moderate-intensity endurance exercise training (EET) program could improve SCD patients without provoking VOC. Patients and Methods: Weconducted aprospective, multicenter, controlled study with individually tailored Blood lactate-guided (BL) endurance training on a cycloergometer for 8 weeks in adult SCD patients. The primary end point was the submaximal incremental cardio-pulmonary exercise testing (CPET) at a BL concentration of 4 mmol.L-1. Secondary endpoints were: CPET at a BL 2.5 mmol/L and at the first lactic threshold (LT1), BL levels at given workload level (Female: 40W and Male: 60W), safety (pain, VOC and acute chest syndrome), biological and echocardiography parameters, pulmonary function test (PFT), 6-minute walk test (6MWT), quality of life (QoL) and muscle properties and vascularization (biopsy of vastus lateralis muscle). Results: Forty homozygous SCD patients (33±10 years, 23 male) were included. Four patients (3 EET and 1 control) were lost to follow-up. Data of 33 patients (15 EET and 18 controls) were analyzed. EET and control patients were comparable at baseline with regard to CPET, clinical, biological, and resting cardiorespiratory parameters. EET patients presented a significant increase of CPET workload at BL4 (p=0.031), BL2.5 (p=0.003) and LT1 (p Conclusion In SCD patients, EET based on Blood lactate guidance is safe and yields a significant functional benefit. This benefit is linked essentially to improved oxidative function of the peripheral skeletal musculature. Disclosures Galactéros: Addmedica: Membership on an entity's Board of Directors or advisory committees. Bartolucci: GBT: Membership on an entity's Board of Directors or advisory committees; Addmedica: Research Funding; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

Purpose: The total metabolic tumor volume at baseline (T-MTV0) computed on PET has been proposed as a prognostic factor at staging in Hodgkin lymphoma (HL) and diffused large B cell lymphoma (DLBCL). It has also been described to play a role in non-HL treatment by monoclonal antibodies since it could influence antibody exposure and efficacy in a murine model (Dayde D, et al. Blood 2009) that has been confirmed in DLBCL patients study (Casasnovas O, et al. Abstract Lugano 2015). We hypothesized the metabolic tumor burden could influence the efficacy of a monoclonal antibody-drug conjugated (ADC) such as monotherapy based anti-CD30 ADC brentuximab-vedotin (BV). In the present study, we assessed the pre-treatment baseline total metabolic tumor volume based on PET evaluation in the response to BV treatment as a single agent in patients with RR-HL, as well as its prognostic value. Methods: A retrospective multi-center study was built from January 2011 to June 2015. Forty-one consecutive heavily pre-treated patients with a diagnosis of relapsed refractory HL (RR-HL) were included. PET was performed at baseline (PET0) and after 4-6 cycles of BV defining two groups of responders, good responder group achieving a complete metabolic response (CMR group, 23 patients), and non-responder group (no-CMR group, 18 patients), using the revised Lugano classification with the 5-point scale visual analysis PET/CT. T-MTV0 was measured with a semiautomatic method using a 41%-SUVmax-threshold. SUVmax at PET0 (SUVmaxPET0) was measured in each patient and represented the hottest lesion independently from the site. To assess the influence of the T-MTV0 on BV efficacy, we compared the baseline metabolic tumor volume between the two groups. The ROC curve was established to determine the optimal cut-off of T-MTV0 to predict treatment failure. Results: T-MTV0 ranged from 14 cm3 to 213 cm3 in the 41 patients (median 62 cm3; 25th - 75th percentiles 25 - 94 cm3) and SUVmaxPET0 ranged from 4,1 to 19,3 (median 10,4; 25th-75th percentiles 7,8 - 13,7). T-MTV0 was significantly higher in the no-CMR group as compared with the CMR group (median 96 and 30 cm3; 25th-75th percentiles 90 - 139 cm3 and 18 - 38 cm3, respectively; p< 0.01). Considering metabolic sites, the nodal metabolic tumor volume (N-MTV0) represented the main component of T-MTV0 and was significantly higher than the extra nodal metabolic tumor volume (EN-MTV0) in the CMR group (p = 0.025). However, the EN-MTV0 dominated in the no-CMR group and was significantly higher than the N-MTV0 (p = 0.01). SUVmaxPET0 as well as clinical characteristics were not significantly different in the two groups. Furthermore the TMTV0 cut-off value was 62 ml and predictive of treatment failure. Conclusion: In the present study, we demonstrated that tumor metabolic burden and nodal or extra-nodal localizations influence response to BV as a single agent in RR-HL patients, offering some future research directions in the development of new schedules of antibodies administration in anticancer therapy such as the concept of the individual adjustment of treatment dose to tumor burden. This may guide clinicians in their choice of therapeutic strategy. Disclosures Thieblemont: St. Louis Hospital, Paris, France: Employment. Cartron:Sanofi: Honoraria; Gilead: Honoraria; GSK: Honoraria; Roche: Consultancy, Honoraria; Celgene: Honoraria.

Background: PET with fluorodeoxyglucose combined with computed tomography is a major tool for the diagnosis, staging and monitoring of treatment response in clinical oncology. 3′-18Fluoro-3′-deoxy-L-thymidine (18F-FLT) is a nucleoside analog that quickly accumulates in proliferating cells, evaluated in clinical studies in various cancers as a PET radiotracer offering non invasive assessment of cell proliferation in vivo. Preliminary results of a pilot study suggested that this technique could be useful to assess bone marrow (BM) activity and extramedullary hematopoiesis in patients with myelofibrosis (MF). We present the final analysis of the FLT-MF-2009 study (EudraCT Number: 2009-016804-21) confirming that FLT PET could be a new technique useful for MF management. Methods: Main inclusion criteria were: a diagnosis of MF according to WHO criteria; BM biopsy available for centralized review; written informed consent. 18F-FLT PET was performed 1 hour after injection of 18F-FLT (provided by AAA), and consisted in a whole-body acquisition. Two nuclear physicians interpreted images in a blinded fashion independently, qualitatively and according to a visual scale, and patients were classified according to 3 distinct patterns. 18F-FLT uptake was quantified using maximum standardized uptake value (SUVmax) in several sites of the skeleton (axial skeleton, proximal and distal territories of upper and lower limbs), in the spleen and the liver. Analysis of factors affecting the intensity of 18F-FLT uptake in these sites (using PET pattern and SUVmax as the endpoints) was performed by using non-parametric tests. Results: 15 patients (mean age: 62 years) were included between Apr 2011 and Jul 2012. 8 patients had primary (PMF), 3 post-polycythemia vera (PV), and 4 post-essential thrombocythemia (ET) MF. 13 patients had a palpable splenomegaly. 10 patients had the JAK2V617F mutation with a median mutant allele burden of 59% (interquartile range: 29-80%), 1 had a MPL515 mutation. Therapies at time of PET included hydroxyurea (n=1), androgens (n=1), interferon (n=4) and ruxolitinib (n=5); 4 patients had no specific therapy for MF. Three patterns of 18F-FLT PET images were observed. Pattern A (n=3) showed a normal BM activity in the central skeleton, a mild expansion to distal extremities and normal splenic uptake. Pattern B (n= 9) displayed a rather normal pattern of BM activity in the central skeleton associated with marked expansion of BM activity to distal extremities and intense uptake of the tracer in the spleen. Pattern C (n=3) showed a marked reduced hematopoietic activity in the central skeleton but a high uptake in spleen, suggesting the existence of myeloid metaplasia. Quantitative analyses of 18F-FLT uptake using SUVmax permitted the search for correlations with clinical and biological characteristics. Grade 3 fibrosis in BM biopsy was significantly associated with low SUVmax values measured in axial skeleton (p=0.019), proximal upper limbs (p=0.016) and proximal lower limbs (p=0.019). Significantly higher SUVmax values in proximal upper (p=0.014) and lower limbs (p=0.021) were associated with the diagnosis of post-PV- vs. post-ET- or PMF. SUVmax values in proximal upper limbs also correlated with the hemoglobin level (Spearman correlation coefficient: 0.53; p=0.038). No significant correlation was found between SUVmax values in any territory and platelet values, JAK2V617F positivity or allele burden, disease duration. Significantly lower SUVmax values were measured in proximal upper limbs of patients treated with ruxolitinib (3.9 ± 2) compared to patients treated with interferon (11.2 ± 5) or untreated patients (7.7 ± 3.1) (p=0.043). Conclusion: The results of this pilot study suggests that 18F-FLT PET could be a new, objective, non invasive technique useful for the evaluation of malignant hematopoiesis in MF, in terms of diagnosis, staging and for monitoring response to therapy. SUVmax values may discriminate post-PV MF from other forms of MF, and may distinguish patients with grade 3 fibrosis. If this latter finding is confirmed in a larger study, 18F-FLT PET could become a convenient substitute to sequential biopsies for the staging of BM fibrosis during the course of the disease. In addition, distinct SUVmax values were found in patients treated with ruxolitinib, suggesting that 18F-FLT PET could also be useful to monitor the efficacy of therapies in MF. Disclosures Off Label Use: 18F-FLT is an investigational isotope not approved for the imaging of myelofibrosis. Patients treated off-label with interferon were included in the study..

Although previous findings have suggested that some adult stem cells are pluripotent and could differentiate in an appropriate microenvironment, the fate conversion of adult stem cells is currently being debated. Here, we studied the ability of mobilized stem cells to repair cardiac tissue injury in a nonhuman primate model of acute myocardial infarction. Mobilization was carried out with stem cell factor, 25 mcg/Kg/d (D), and granulocyte-colony-stimulating factor, 100 mcg/Kg/D administered 5 days before (D - 5 group; n = 3) or 4 hours after (H + 4 group; n = 4) circumflex coronary artery ligation; no growth factor was administered to 3 baboons of the control group. No adverse effect relating to growth factor administration was observed. Flk-1 and transcription factors of cardiac lineages could be detected in peripheral blood only by reverse transcriptase-polymerase chain reaction. When comparing positron emission tomography (PET) with [11C]-acetate between examinations from D2 and D30, a relative increase (perfusion ratio between infarct and noninfarct regions) of 26% ( P = .01) in myocardial blood flow was found in the H + 4 group; the relative rate of oxidative metabolism remained unaltered in the 3 groups. No change was observed in the echographic indices of the left ventricular enlargement or systolic function in the 3 animal groups during the 2-month follow-up. The PET findings concurred with the immunohistochemistry analysis of left ventricular myocardial sections with evidence of endothelial cells but no myocyte differentiation; few cycling cells were observed at this time. Thus, the present data suggest that, in nonhuman primates submitted to coronary artery ligation, mobilization by hematopoietic growth factors could promote angiogenesis in the infarcted myocardium, without detectable myocardial repair. (Blood. 2003;102:4361-4368).

Abstract 1741 Background: Positron emission tomography (PET) generally employing fluorodeoxyglucose (FDG) combined with high-resolution structural imaging using computed tomography (CT) is regularly used in the diagnosis, staging and monitoring of treatment response in clinical oncology. 3′-18Fluoro-3′-deoxy-L-thymidine (18F-FLT) is a nucleoside analog that quickly accumulates in proliferating cells, more recently evaluated in various cancers including hematologic malignancies like acute leukemias or lymphomas as a PET radiotracer offering non invasive assessment of cell proliferation in vivo. Published results suggest that this technique could be useful to assess bone marrow (BM) activity and extramedullary hematopoiesis (EMH). However, to our knowledge, only 3 patients (pts) with myelofibrosis (MF) have been explored with 18F-FLT PET/CT (FLT PET). This pilot study aimed to establish proof-of-concept that FLT PET could be a new non invasive technique useful for MF management, in terms of diagnosis, staging and for monitoring response to therapy. Methods: Pts were evaluated using 2 different techniques. First, conventional BM scintigraphy (BMS) was performed: on day 1, pts were injected with 99mTechnetium-nanocolloids and a planar image of the reticuloendothelial system was performed 30 min after injection; pts were then injected with 111Indium-Cl3 and planar imaging of the erythroid BM was performed after 48h. Secondly, FLT PET was performed 1 hour after injection of 18F-FLT (provided by AAA), and consisted in a whole-body acquisition. Images were interpreted in a blinded fashion independently by two nuclear physicians, qualitatively and according to a visual scale for both examinations. In addition, 18F-FLT uptake was quantified using standardized uptake value (SUV) in several sites of the skeleton, spleen and liver. Results: 15 pts (9 men, 6 women, mean age: 62 years) were included between Apr 2011 and Jul 2012 (14 evaluable at time of abstract submission). 7 pts (47%) had primary (PMF), 4 post-polycythemia vera (PV), and 4 post-essential thrombocythemia (ET) MF, respectively (WHO criteria). All the pts had a BM biopsy with quantification of fibrosis. 11 pts (73%) had a JAK2V617F mutation, 1 a MPL515 mutation, and 3 had neither of these mutations. Therapies included hydroxyurea (n=1), androgens (n=1), interferon (n=4) and ruxolitinib (n=5); 4 pts had no specific therapy for MF. Three distinct patterns of FLT PET images were observed. 3 pts showed a marked reduced hematopoietic activity in the central compartment of the skeleton but a high uptake in spleen, suggesting the existence of myeloid metaplasia (Fig 1A). 8 pts had a rather normal pattern of BM activity in the central skeleton associated with marked expansion of BM activity to distal extremities and intense uptake of the tracer in the spleen (Fig 1B). 3 pts showed a relatively normal pattern of BM activity in the central skeleton, a mild expansion to distal extremities with no splenic abnormality (Fig 1C). FLT interpretation in myeloid malignancies is not standardized and we used comparisons with BMS to establish interpretation guidelines. Qualitative FLT PET results were equivalent to the 111In-Cl3 imaging in most cases, but in 2 pts FLT uptake was normal when BMS showed reduced 111In-Cl3 uptake. Compared to BMS, PET will also provide much more information including: (i) quantitative analyses of 18F-FLT uptake using SUV (preliminary results show that SUV ranges are [1.8 – 18.4] and [2.3 – 19.8] in BM and spleen, respectively); (ii) precise evaluation of malignant myelopoiesis in the different anatomical sites using coupled CT images. These analyses, and correlation with clinical and biological characteristics, BM histopathology and type of therapy received are ongoing. Conclusion: FLT PET is a new, convenient non invasive technique for evaluation of malignant hematopoiesis in MF, including BM activity and EMH. Distinct patterns of FLT uptake may help in the diagnosis and staging of MF. In addition, ongoing correlation studies with histological BM fibrosis could provide evidence for a role of this non invasive technique in the assessment of the evolution of fibrosis over time without the need for sequential biopsies. A subsequent clinical trial will determine in a larger cohort of MF pts the usefulness of PET for evaluation of tumor response to therapy and prediction of early response using sequential evaluation of FLT uptake in BM and spleen. Disclosures: Off Label Use: 3′-18Fluoro-3′-deoxy-L-thymidine (18F-FLT) is a nucleoside analog tested as a PET radiotracer in patients with myelofibrosis.

Abstract 3835 Background: Activation of the RAS pathway plays an important role in the pathogenesis of myeloid malignancies. RAS mutations occur in some 10% of MDS and acute myelogenous leukemia (AML) cases. We previously showed in mouse models of NRASD12/BCL-2 MDS/AML that RAS and BCL-2 co-operate in vivo. These genes co-localize at the plasma membrane in the MDS model (MRP8NRASD12/MMTVLTRTetoBCL-2) with increased apoptosis and in mitochondria in the AML post-MDS model (MRP8[NRASD12/BCL-2] with reduced apoptosis (Omidvar Cancer Res 2007). Here, we screened MDS patients for the cellular co-localizations of RAS:BCL-2 and the consequent apoptosis status. Methods: 39 MDS and AML post-MDS patients were studied, including (WHO classification): 3 RCMD-RS, 3 RCMD, 1 RARS, 8 RAEB I, 9 RAEB II, 4 CMML I, 1 CMML II, 10 AML post-MDS and 2 normal individuals. 17 patients had cytogenetic abnormalities. Bone marrow (BM) mononuclear cells (MNC) were assayed for RAS:BCL-2 localization by confocal and immunofluorescence microscopy. IL-3 dependent mouse hematopoietic FDCP-1 cells were infected with NRASD12 or BCL-2 alone or in combination. Co-localization was measured using anti-NRAS, anti-BCL-2, anti-mitochondrial (Tom 20) and anti-ezrin or anti-wheat germ agglutinin (WGA) plasma membrane antibodies. Apoptosis in patient samples was detected using the Mebstain apoptosis kit and visualized using FITC-dUTP on cytospun cells; apoptosis in the mice was detected by single photon emission computed tomography (SPECT) using technicium labeled annexin-V (99mTc-AnnexinV). NRAS mutations were analyzed by exon direct sequencing or by WAVE, a denaturing high-performance liquid chromatography (DHPLC) based assay. Lineage negative Lin-/Sca-1+/c-Kit+ (LSK) spleen cells were purified by automacs followed by flow sorting. Western blots were probed with anti-caspase 3 and 9 specific antibodies. Results: RAS and BCL-2 were co-expressed in the BM (MNC) of all 39 cases. The intensity (low or high) of RAS:BCL-2 co-localization measured by confocal microscopy significantly correlated with % BM blasts (p=0.0283) and WHO classification (p15% marrow blasts. The intensity of the complex was independent of karyotype and NRAS mutation (found in 3 of 30 (10%) patient samples). The 2 normal samples had low intensities of RAS:BCL-2 co-localizations and wild type NRAS. Further analysis of RAS and BCL-2 co-localizations was carried out in 13 of these patients. In 6 of the 7 patients with BM blasts Conclusion: These findings illustrate the prognostic impact of the intensity of the RAS:BCL-2 complex in MDS/AML post-MDS patients, and further suggest that localization of RAS:BCL-2 at the plasma membrane correlates with less advanced disease while its presence at mitochondria correlates with more advanced MDS. Therapeutic targeting of the RAS:BCL-2 complex could potentially prevent transformation in MDS patients. Disclosures: No relevant conflicts of interest to declare.

OBJECTIVES: Activating RAS mutations and over-expression of BCL-2 are prognostic features of myelodysplastic syndromes/acute myeloid leukemia (MDS/AML) transformation. Using NRASD12 and BCL-2, we created two distinct transplantable in vivo models of MDS and AML. Expression of hBCL-2 in a primitive compartment by MMTV-LTR results in a disease resembling human MDS with bone marrow blasts of 15% with increased apoptosis assayed by TUNEL on liver sections, whilst the myeloid MRP8 promoter induces a disease with characteristics of human AML with marrow blasts of up to 90% with liver apoptosis patterns similar to wild type. In MDS mice RAS and BCL-2 do not co-localize in the mitochondria, but localize to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localize in the mitochondria, where BCL-2 is normally found; consistent with its anti-apoptotic properties. We next examine the mouse hematopoietic FDCP-1 in vitro model with stable exogenous expression of NRASD12, hBCL-2 or NRASD12 and hBCL-2. Furthermore, we report the in vivo effects of the BH-3 mimetic inhibitor ABT-737. RESULTS: FDCP-1 lines demonstrate that whilst hBCL-2 alone prevents staurosporine-induced mitochondrial-dependent (caspase-9-mediated intrinsic) apoptosis, both NRAS-D12 and NRASD12/hBCL-2 cell lines were pro-apoptotic. No significant difference was observed in cell-death receptor (caspase-8-mediated extrinsic) apoptosis between the cell lines following pharmacological induction with Fas-ligand or TRAIL. Annexin-V/Propidium Iodide flow cytometry and caspase activity assays on hematopoietic primary cells from the mouse models recapitulated the findings; namely the MDS-model demonstrates increased caspase-9-mediated apoptosis (within the Lin−/Sca-1+/KIT+ (LSK) sub-population), whilst the AML-model illustrate anti-apoptotic activity. This is concordant with the signaling profile where phosphorylated AKT is reduced in the RAS-mediated MDS mice and active-AKT is increased in the BCL-2 mediated AML disease. Expanded leukemic stem cell LSK populations had increased hBCL-2 expression in the RAS-GTP complex in both MDS/AML diseases. Thus we suggest that this complex can be a specific target for therapy. When hBCL-2 is switched off with doxycycline in the conditional MDS mice, only partial reversal of the phenotype was observed as RAS recruits endogenous mouse BCL-2 to remain active; thus demonstrating the role of the complex in the disease. In order to target both human and mouse BCL-2 the efficacy of in vivo treatment with the specific BH-3 mimetic inhibitor ABT-737 was determined. We show that the treatment significantly reduced the progression of the disease, increased the peripheral blood platelet counts, decreased the bone marrow blast and cleared the tissue invasion in the MDS mice or reduced tissue infiltration of the AML. In vivo imaging by single-photon emission computed tomography (SPECT) using 99mTc-labelled Annexin-V shows that ABT-737 induces apoptosis of the blast cells that infiltrate the liver and the spleen of the treated mice, which was confirmed by TUNEL on liver sections. Additionally, ABT-737 can reduce the myeloid colony growth and the LSK expansion in these mice; whilst abrogating BCL-2:RAS-GTP complex formation illustrated via biochemical and confocal assays. CONCLUSION: This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex rescued by ABT-737 via intrinsic apoptosis and thus support the case for BH-3 mimetic therapy.