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Introduction: The Philadelphia-negative myeloproliferative neoplasms are associated with chronic inflammation and accumulation of reactive oxygen species (ROS). Whole blood transcriptional profiling studies have most recently identified deregulation of several oxidative and anti-oxidative stress genes. Amongst the genes significantly downregulated is TP53 and the NFE2L2 or Nrf2 gene, the latter having a key role in the regulation of the oxidative stress response and in modulating both migration and retention of hematopoietic stem cells (HSCs). During MPN-disease progression, the HSC pool is steadily expanding with the egress of CD34+cell from stem cell niches into the circulation. In addition to Nrf2 several other genes are involved in this process, including CXCR4, which is also significantly downregulated in MPNs. Interferon-alpha2 (IFN) is recognized as a highly efficacious and promising agent in the treatment of MPNs. Taken into account that chronic inflammation with ROS accumulation is likely involved in the pathogenesis of MPNs, ultimately implying the induction of an altered redox balance of pivotal significance for stem cell mobilization in MPNs, we herein report the first study on the impact of IFN upon oxidative stress and anti-oxidative defence genes. Methods: The HG-U133 Plus 2.0 microarray was used to profile expression of 38.500 genes in whole blood from ET (n=19), PV (n=41), PMF (n=9) and controls (n=21) (data set 1) and in ET (n=8), PV (n=21), and PMF (n=4) before and during treatment with IFN (data set 2). All patients received treatment with IFN, in the large majority in a dosage ranging from 45-90 ug x 1 sc/week. Total RNA was purified from whole blood amplified to biotin-labeled RNA, and hybridized to microarrays. The R statistical software was applied to perform data preprocessing and statistical analysis of microarray data. Results: Single gene analysis of 154 genes found to be included in previous studies focusing on deregulation of oxidative stress genes in various diseases were chosen for further analysis. In response to treatment with IFN, 19 genes were upregulated in ET including CXCR4 and TP53, and 11 genes were downregulated including FOXO3, PRDX2, and PRDX6. In patients with PV, ATOX1, CXCR4, SEPP1, and TP53 were among the 29 upregulated genes, and FOXO3 and PRDX2 were among the 14 downregulated genes. In response to treatment with IFN, 2 genes were upregulated in PMF which were CYBB and MSRA, and 9 genes were downregulated including PRDX2, and FOXO3 (all P Discussion and Conclusions: The present study has shown IFN to have a major impact upon deregulated oxidative stress genes and antioxidative defence genes, implying a gene signature that mostly corresponds to decreased oxidative stress and enhancement of antioxidative defence genes. In regard to TP53 - being significantly downregulated before IFN treatment - this gene was significantly upregulated in ET and PV and no longer deregulated in PMF during IFN treatment. Downregulation of TP53 implies genomic instability due to an increased burden of oxidative stress upon the genome, which accordingly is reduced by IFN treatment. In addition to being the master regulator of the antioxidant response, Nrf2 also has a major role for normal stem cell function. Thus, our findings of a change in the Nrf2 gene expression from being significantly downregulated before IFN exposure to not being deregulated during treatment with IFN may imply an improvement in the antioxidant defence mechanisms against increased oxidative stress and accumulation of ROS. In conclusion, our findings of a major impact of IFN upon several oxidative stress genes and antioxidative defence genes may imply an IFN mediated dampening of genotoxic damage to hematopoietic cells and stromal cells as well, thereby ultimately diminishing the risk of additional mutations that might drive the malignant clone towards myelofibrotic and leukemic transformation. Disclosures Hasselbalch: Novartis: Research Funding.

The literature contains little on the prevalence and causes of high predonation haemoglobin levels among blood donors. This study aimed to characterize and develop an algorithm to manage would-be donors with polycythaemia. Between November 2009 and November 2011, we offered haematology consultations to blood donors with repeated haemoglobin concentration (Hb) above the WHO limit for polycythaemia vera (PV) (10.2 and 11.5 mm ⁄ 16.5 and 18.5 g/dl for women and men, respectively). Investigation of such donors included Hb, haematocrit, mean cell volume, erythropoietin, ferritin, platelet count and leucocyte count, JAK2 V617F and JAK2 exon12 analysis, as well as other routine measurements. Among 46 such donors, 39 had a history of smoking, which contributes to erythrocytosis. Two had PV, five had severe hypertension, one of them because of renal artery stenosis, and two had diabetes mellitus. Thus, we found a high morbidity among such donors. Of the 36 others, 30 donated again before May 2012, at which time the Hb was significantly lower. We recommend JAK2 V617F and JAK2 exon12 screening and clinical investigation for donors with concurrently high Hb, high haematocrit and iron deficiency. We also recommend that they stop or cut down on smoking to reduce the risk of thrombosis in general. We disqualified 10 of the donors.

Chronic myeloid leukemia (CML) stem cells appear resistant to tyrosine kinase inhibitors (TKIs) in vitro, but their impact and drug sensitivity in vivo has not been systematically assessed. We prospectively analyzed the proportion of Philadelphia chromosome-positive leukemic stem cells (LSCs, Ph+CD34+CD38=) and progenitor cells (LPCs, Ph+CD34+CD38+) from 46 newly diagnosed CML patients both at the diagnosis and during imatinib or dasatinib therapy (ClinicalTrials.gov NCT00852566). At diagnosis, the proportion of LSCs varied markedly (1-100%) between individual patients with a significantly lower median value as compared with LPCs (79% vs 96%, respectively, P = 0.0001). The LSC burden correlated with leukocyte count, spleen size, hemoglobin and blast percentage. A low initial LSC percentage was associated with less therapy-related hematological toxicity and superior cytogenetic and molecular responses. After initiation of TKI therapy, the LPCs and LSCs rapidly decreased in both therapy groups, but at 3 months time point the median LPC level was significantly lower in dasatinib group compared with imatinib patients (0.05% vs 0.68%, P = 0.032). These data detail for the first time the prognostic significance of the LSC burden at diagnosis and show that in contrast to in vitro data, TKI therapy rapidly eradicates the majority of LSCs in patients.

Biologic and clinical observations suggest that combining imatinib with IFN-α may improve treatment outcome in chronic myeloid leukemia (CML). We randomized newly diagnosed chronic-phase CML patients with a low or intermediate Sokal risk score and in imatinib-induced complete hematologic remission either to receive a combination of pegylated IFN-α2b (Peg–IFN-α2b) 50 μg weekly and imatinib 400 mg daily (n = 56) or to receive imatinib 400 mg daily monotherapy (n = 56). The primary endpoint was the major molecular response (MMR) rate at 12 months after randomization. In both arms, 4 patients (7%) discontinued imatinib treatment (1 because of blastic transformation in imatinib arm). In addition, in the combination arm, 34 patients (61%) discontinued Peg–IFN-α2b, most because of toxicity. The MMR rate at 12 months was significantly higher in the imatinib plus Peg–IFN-α2b arm (82%) compared with the imatinib monotherapy arm (54%; intention-to-treat, P = .002). The MMR rate increased with the duration of Peg–IFN-α2b treatment (< 12-week MMR rate 67%, > 12-week MMR rate 91%). Thus, the addition of even relatively short periods of Peg–IFN-α2b to imatinib markedly increased the MMR rate at 12 months of therapy. Lower doses of Peg–IFN-α2b may enhance tolerability while retaining efficacy and could be considered in future protocols with curative intent.

Chronic cold agglutinin disease (CAD) is an acquired autoimmune hemolytic anemia. Previous therapeutic modalities, including alkylating cytostatics, interferon and prednisolone, have been disappointing. However, several case reports and small-scaled studies have demonstrated promising results after treatment with rituximab. We performed a phase II multicentre trial to investigate the effect of rituximab in CAD, including 20 patients studied from October 2002 until April 2003. Thirteen patients had idiopathic CAD and seven patients had CAD associated with a malignant B-cell lymphoproliferative disease. Rituximab was given in doses of 375 mg/m(2) at days 1, 8, 15 and 22. Sixteen patients were followed up for at least 48 weeks. Four patients were excluded after 8, 16, 23 and 28 weeks for reasons unrelated to CAD. Nine patients (45%) responded to the treatment, one with complete response (CR), and eight with partial response. Eight patients relapsed, one patient was still in remission at the end of follow-up. There were no serious rituximab-related side-effects. Our study confirms previous findings of a favourable effect of rituximab in patients with CAD. However, few patients will obtain CR and, in most patients, the effect will be transient.

Imatinib mesylate targets the adenosine triphosphate (ATP)-binding sites of the protein tyrosine kinase domains associated with Bcr-abl, the platelet-derived growth factor (PDGF) and c-kit. In idiopathic myelofibrosis (IMF) PDGF is considered to be one of the growth factors responsible for the development of bone marrow fibrosis. Recently, it has been shown that imatinib has antifibrogenic effect on bone marrow fibrosis in chronic myelogenous leukemia. Treatment with imatinib alone in IMF has been associated with significant side effects. In this study, the safety and efficacy of imatinib therapy in IMF, either administered as a single agent or in combination with hydroxyurea (HU) and/or alpha-interferon (IFN-alpha) are evaluated. Eleven patients (median age, 63 years; range, 33-82 years) with IMF (n = 8) or postpolycythemic myelofibrosis (PPMF) (n = 3) were studied All patients had been treated with HU (n = 9) and/or IFN (n = 7) before study entry. In all but one patient, treatment with these agents was discontinued when imatinib therapy was instituted. One patient continued IFN when treatment with imatinib was started. Imatinib was given at a dose of 400 mg/day. Nine patients were in an advanced disease phase. The patients have been followed for a median period of 2 months (range, 0.5-12 months). Treatment with imatinib has been stopped in six patients (55%), because of overt side effects (n = 4), recurrence of transitory dizziness and visual defects owing to a rising platelet count (n = 1), or the occurrence of an acute subdural hemorrhage that was evacuated without neurological deficits (n = 1). In nine patients imatinib treatment was followed by a rise in leukocyte and platelet counts that required combination with HU or IFN. The combined treatment modalities were followed by a rapid decrease in cell counts and were well tolerated apart from IFN side effects. A beneficial effect of imatinib was documented in three patients. It is concluded that leukocytosis and thrombocytosis are seen in most patients with myelofibrosis during treatment with imatinib. Combination therapy with HU or IFN seems safe and well tolerated and followed by a decrease in disease activity. A subgroup of patients in an early disease phase might benefit from imatinib therapy alone.

Imatinib mesylate (IM), 400 mg daily, is the standard treatment of Philadelphia-positive (Ph+) chronic myeloid leukemia (CML). Preclinical data and results of single-arm studies raised the suggestion that better results could be achieved with a higher dose. To investigate whether the systematic use of a higher dose of IM could lead to better results, 216 patients with Ph+ CML at high risk (HR) according to the Sokal index were randomly assigned to receive IM 800 mg or 400 mg daily, as front-line therapy, for at least 1 year. The CCgR rate at 1 year was 64% and 58% for the high-dose arm and for the standard-dose arm, respectively (P = .435). No differences were detectable in the CgR at 3 and 6 months, in the molecular response rate at any time, as well as in the rate of other events. Twenty-four (94%) of 25 patients who could tolerate the full 800-mg dose achieved a CCgR, and only 4 (23%) of 17 patients who could tolerate less than 350 mg achieved a CCgR. This study does not support the extensive use of high-dose IM (800 mg daily) front-line in all CML HR patients. This trial was registered at www.clinicaltrials.gov as #NCT00514488.

Abstract 667 Background: Targeted tyrosine kinase inhibitor (TKI) therapy efficiently induces rapid hematologic and cytogenetic responses in most CML patients. In vitro studies have suggested that CML stem cells are resistant to TKIs and therefore the treatment may need to be life-long. However, the in vivo effects of TKIs on the leukemic stem cell pool in a patient population have not been prospectively assessed. In addition, the biological impact and prognostic value of leukemic stem cell burden at diagnosis is unknown. Aim: To analyze the proportions of Ph+ cells in the stem cell compartment in newly diagnosed CP CML patients at diagnosis, and correlate the initial leukemic stem cell burden to biological variables and hematological toxicity during first 3 months of TKI therapy. Patients and Methods: 42 newly diagnosed CP CML patients within the Nordic countries were randomized to receive either dasatinib 100 mg (n=21) or imatinib 400 mg (n=21) once daily. Stem cell assays were performed at diagnosis and at 1, 3, and 6 months from start of TKI therapy. After pre-selection of CD34+ cells from large volume bone marrow (BM) aspirates with paramagnetic beads, the CD34+ cells were fractionated into CD38 positive and negative pools using a sorting flow cytometer. The proportion of Ph+ cells in the stem cell fractions was assayed by counting 1000 cells with interphase FISH for BCR-ABL1. Results: Measurement of Ph+ stem cells was feasible in most patients at diagnosis and results from 36 evaluable patients will be presented. The median volume of BM aspirate was 28, 37, 40 and 36 ml at diagnosis, 1, 3, and 6 months after therapy start, respectively. The median yield of BM mononuclear cells was 1000, 110, 93 and 79 ×106, respectively. The median proportion of Ph+ cells was significantly lower in the more primitive CD34+CD38neg fraction when compared to the CD34+CD38+ fraction or to unfractionated BM (79%, range 0.6–100%; 96%, 50–100%; and 96%, 57–100%, respectively, p=0.0001). The proportion of Ph+ cells in the CD34+CD38neg fraction at diagnosis correlated with high leukocyte count (r=0.59, p Conclusions: The proportion of Ph+ stem cells at the time of diagnosis varied from 1 to 100% between individual CML patients. It was correlated with hemoglobin concentration, leukocyte count, blast percentage and spleen size at diagnosis and with hematological toxicity during early course of treatment, mirroring paucity of healthy hematopoietic stem cell reservoir. The size of the leukemic stem cell pool at diagnosis may be a powerful prognostic marker and a major biological determinant for the high Sokal risk group. The effect of TKI therapy on the malignant stem cell pool size and correlation to therapy responses will be evaluated when all patients have reached the primary study endpoint of 6 months. Disclosures: Mustjoki: BMS, Novartis: Honoraria. Richter:BMS, Novartis: Consultancy, Honoraria. Simonsson:BMS, Novartis: Consultancy, Honoraria, Research Funding. Porkka:BMS, Novartis: Consultancy, Honoraria, Research Funding. Hjorth-Hansen:BMS, Novartis: Consultancy, Honoraria, Research Funding.

Abstract 3280 Poster Board III-1 Background: Imatinib mesylate (IM) 400 mg once daily (OD) is the current standard first-line therapy for CML. Several biological and clinical observations suggest that combining IM with interferon alpha (IFNa) may improve the outcome of treatment. Aim: To compare the effects of standard-dose IM to combination of IM and IFNa in newly diagnosed chronic phase CML patients with an intermediate or low Sokal risk score. The primary end point was to compare the major molecular response (MMR) rate after 12 months between the treatment arms (intention-to-treat analysis). Patients and therapies: In a Nordic CML Study Group (NMCLSG: Denmark, Finland, Norway and Sweden) and Israel multicenter study we randomized 114 newly diagnosed CML patients in complete hematological remission following 3 months of IM 400 mg OD induction therapy. The study arms were IM (Glivec, Novartis) and the combination of IM and IFNa 2b (PegIntron, Schering-Plough). IM dose was fixed at 400 mg OD. IFNa was started at 30 μg/week but could be escalated to 50 μg/week or reduced down to 15 μg/week depending on tolerability. Molecular response was evaluated by blood RQ-PCR for BCR-ABL1 and was expressed on the international scale (IS). Results: As of August 17, 2009, 79 patients were evaluable for primary endpoint and 47 of these were in MMR (59%) The rate of complete cytogenetic response (CCgR) was 68/79 (86%). Nineteen patients (17%) were considered as treatment failures (grade 4 nonhematological adverse event, refusal, loss of CCgR, progression to advanced phase, protocol violation or other reason). Conclusions: A final analysis will be performed when we have complete data on all randomized patients. The comparison by treatment arm on molecular and cytogenetic responses as well as other relevant data (treatment failures, patients off-treatment for protocol violations, refusal or toxicity) will be presented on site. Supported by European LeukemiaNet, WP 4. Disclosures: Simonsson: Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding. BjÖreman:BMS: Consultancy, Honoraria. Mustjoki:BMS: Honoraria. StrÖmberg:Novartis: Honoraria; BMS: Honoraria. Weiss Bjerrum:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Gruber:Novartis: Research Funding. Nagler:Novartis: Consultancy. Porkka:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding.

Abstract 784 Background: In vitro studies have suggested that CML stem cells are resistant to tyrosine kinase inhibitors (TKIs), but in vivo effects in patients have not been prospectively assessed. Furthermore, the inter-individual variation of the leukemic stem cell pool at diagnosis and its possible prognostic value is unknown. Patients: 46 newly diagnosed CML-CP patients were randomized 1:1 to receive either dasatinib 100 mg or imatinib 400 mg QD. The primary endpoint was a comparison of the proportion of Ph+ cells in CD34+CD38− and CD34+CD38+ compartment at 6 months between the study arms. Key secondary endpoints were the fraction of Ph+ cells in the stem cell compartments at 1 and 3 months, and molecular and cytogenetic responses at 3, 6, 12 and 18 months. Experimental endpoints included the percentage of Ph+ cells in the stem cell compartment at diagnosis and its correlation with therapeutic response. Results: One patient in the imatinib arm and none in the dasatinib arm progressed to blast crisis within first 12 months. 4/22 of dasatinib patients have discontinued the treatment due to side-effects (mainly pleural effusion) and 1 patient due to insufficient response. 3/24 imatinib patients have discontinued the therapy (1 blast crisis, 1 side-effects, 1 other malignancy). Early cytogenetic responses were superior in the dasatinib arm: the median percentage of Ph+ cells in the bone marrow was 81% (imatinib) vs. 70% (dasatinib) at 1 month (p=0.15) and 5% vs. 0% at 3 months (p=0.0085). At 12 months all dasatinib (n=20) and 19/20 imatinib patients were in CCyR (results based on patients on treatment at 12 months). MMR rate was significantly higher in the dasatinib arm already at 6 months (70% vs. 20%, p=0.002) and similarly at 9 (75 vs. 26%, p=0.004) and 12 months (88% vs. 40%, p=0.009). Undetectable BCR-ABL1 transcripts (at least CMR4) were observed in 20% of the dasatinib patients at 6 months compared to none in the imatinib arm (p=0.11) and 44% in the dasatinib arm at 12 months compared to 7% in the imatinib arm (p=0.037). The median percentage of Ph+ cells, as measured by FISH (1000 cells analyzed), in the CD34+CD38− fraction at diagnosis was 79% (range 1–100%) compared to 96% (range 50–100%) in CD34+CD38+ fraction. The proportion of Ph+ cells in CD34+CD38− fraction correlated with WBC count (r=0.50, p79% of Ph+ cells in CD34+CD38− fraction at diagnosis. During TKI therapy, the proportion of Ph+ cells decreased rapidly in the stem cell fractions. At 1 month, the median proportion of Ph+ cells was 14% and 56% in CD34+CD38− and CD34+CD38+ fractions compared to 69% in whole BM (p Dasatinib-treated patients had significantly lower proportion of Ph+ cells in CD34+CD38+ fraction at 3 months than imatinib patients (0.05% vs. 0.68%, p=0.0318). A similar trend was also observed at 1 month (24% vs. 69%, p=0.05), but no difference existed at 6 months. In CD34+CD38− fraction the proportions of Ph+ cells did not differ significantly at 1 (11 vs. 17%, p=0.91), 3 (0.2 vs. 0.3%, p=0.44) or 6 months (0 vs. 0%, p=0.75). Conclusions: In comparison to imatinib, dasatinib induced superior therapeutic responses with remarkably high MMR and CMR rates. This was associated with a faster reduction of Ph+ cells at the progenitor CD34+CD38+ cell level. Surprisingly, both drugs rapidly depleted Ph+ cells from the more primitive CD34+CD38− compartment. The proportion of Ph+ stem cells at diagnosis varied significantly among individual patients and bore prognostic value. Patients with a low proportion of Ph+ leukemic stem cells at diagnosis achieved faster and better cytogenetic and molecular responses. The leukemic stem cell burden at diagnosis may be a biomarker predicting treatment outcome and reflecting key biological factors in CML. Disclosures: Mustjoki: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Richter:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Dybedal:Novartis: Travel support. Fioretos:Cantargia AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Qlucore AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weiss Bjerrum:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Simonsson:Novartis, BMS, Merck, Pfizer: Consultancy, Honoraria. Porkka:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Hjorth-Hansen:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.