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Background Sex is a major source of diversity among patients and a sex-informed approach is becoming a new paradigm in precision medicine. We aimed to describe sex diversity in myelodysplastic syndromes in terms of disease genotype, phenotype, and clinical outcome. Moreover, we sought to incorporate sex information into the clinical decision-making process as a fundamental component of patient individuality. Methods In this multicentre, observational cohort study, we retrospectively analysed 13 284 patients aged 18 years or older with a diagnosis of myelodysplastic syndrome according to 2016 WHO criteria included in the EuroMDS network (n=2025), International Working Group for Prognosis in MDS (IWG-PM; n=2387), the Spanish Group of Myelodysplastic Syndromes registry (GESMD; n=7687), or the Dusseldorf MDS registry (n=1185). Recruitment periods for these cohorts were between 1990 and 2016. The correlation between sex and genomic features was analysed in the EuroMDS cohort and validated in the IWG-PM cohort. The effect of sex on clinical outcome, with overall survival as the main endpoint, was analysed in the EuroMDS population and validated in the other three cohorts. Finally, novel prognostic models incorporating sex and genomic information were built and validated, and compared to the widely used revised International Prognostic Scoring System (IPSS-R). This study is registered with ClinicalTrials.gov, NCT04889729. Findings The study included 7792 (58middot7%) men and 5492 (41middot3%) women. 10 906 (82middot1%) patients were White, and race was not reported for 2378 (17middot9%) patients. Sex biases were observed at the single-gene level with mutations in seven genes enriched in men (ASXL1, SRSF2, and ZRSR2 p

PURPOSE Myelodysplastic syndromes (MDS) are heterogeneous myeloid neoplasms in which a risk-adapted treatment strategy is needed. Recently, a new clinical-molecular prognostic model, the Molecular International Prognostic Scoring System (IPSS-M) was proposed to improve the prediction of clinical outcome of the currently available tool (Revised International Prognostic Scoring System [IPSS-R]). We aimed to provide an extensive validation of IPSS-M. METHODS A total of 2,876 patients with primary MDS from the GenoMed4All consortium were retrospectively analyzed. RESULTS IPSS-M improved prognostic discrimination across all clinical end points with respect to IPSS-R (concordance was 0.81 v 0.74 for overall survival and 0.89 v 0.76 for leukemia-free survival, respectively). This was true even in those patients without detectable gene mutations. Compared with the IPSS-R based stratification, the IPSS-M risk group changed in 46% of patients (23.6% and 22.4% of subjects were upstaged and downstaged, respectively). In patients treated with hematopoietic stem cell transplantation (HSCT), IPSS-M significantly improved the prediction of the risk of disease relapse and the probability of post-transplantation survival versus IPSS-R (concordance was 0.76 v 0.60 for overall survival and 0.89 v 0.70 for probability of relapse, respectively). In high-risk patients treated with hypomethylating agents (HMA), IPSS-M failed to stratify individual probability of response; response duration and probability of survival were inversely related to IPSS-M risk. Finally, we tested the accuracy in predicting IPSS-M when molecular information was missed and we defined a minimum set of 15 relevant genes associated with high performance of the score. CONCLUSION IPSS-M improves MDS prognostication and might result in a more effective selection of candidates to HSCT. Additional factors other than gene mutations can be involved in determining HMA sensitivity. The definition of a minimum set of relevant genes may facilitate the clinical implementation of the score.

Supplementary Data from Valproic Acid at Therapeutic Plasma Levels May Increase 5-Azacytidine Efficacy in Higher Risk Myelodysplastic Syndromes

Acute myeloid leukemia (AML) identifies a heterogeneous group of clonal disorders, both clinically and genetically. A large number of mutations have been described in AML, although only a few are currently employed in clinical practice. Next generation sequencing (NGS) allows for better understanding of the complex genetic background in AML and may direct individualized therapies. In this study, we aim to identify molecular aberrations that are not routinely investigated in AML using an NGS-based panel encompassing 101 genes and to evaluate how their oncogenic potential correlates with survival. Forty consecutive patients with newly diagnosed AML were enrolled between January 2018 and April 2020. We performed targeted NGS and detected 96 mutations in 36 patients (90%), while 14 fusion genes were detected in 13 patients (32%). Each mutation was weighed using OncoScore, a text-mining tool ranking genes according to their oncogenic potential. An OncoScore >= 100 was associated with shorter PFS among our patients (p = 0.05). In 11 patients with no available MRD markers at diagnosis, we were able to perform NGS-based MRD monitoring using targeted deep sequencing. Overall, our study shows that NGS is a powerful tool in AML and should be employed both in routine diagnostic workup and follow up.

Excess iron can be extremely toxic for the body and may cause organ damage in the absence of iron chelation therapy. Preclinical studies on the role of free iron on bone marrow function have shown that iron toxicity leads to the accumulation of reactive oxygen species, affects the expression of genes coding for proteins that regulate hematopoiesis, and disrupts hematopoiesis. These effects could be partially attenuated by iron-chelation treatment with deferasirox, suggesting iron toxicity may have a negative impact on the hematopoietic microenvironment. Iron toxicity is of concern in transfusion-dependent patients. Importantly, iron chelation with deferasirox can cause the loss of transfusion dependency and may induce hematological responses, although the mechanisms through which deferasirox exerts this action are currently unknown. This review will focus on the possible mechanisms of toxicity of free iron at the bone marrow level and in the bone marrow microenvironment.

PURPOSE Recurrently mutated genes and chromosomal abnormalities have been identified in myelodysplastic syndromes (MDS). We aim to integrate these genomic features into disease classification and prognostication. METHODS We retrospectively enrolled 2,043 patients. Using Bayesian networks and Dirichlet processes, we combined mutations in 47 genes with cytogenetic abnormalities to identify genetic associations and subgroups. Random-effects Cox proportional hazards multistate modeling was used for developing prognostic models. An independent validation on 318 cases was performed. RESULTS We identify eight MDS groups (clusters) according to specific genomic features. In five groups, dominant genomic features include splicing gene mutations ( SF3B1, SRSF2, and U2AF1) that occur early in disease history, determine specific phenotypes, and drive disease evolution. These groups display different prognosis (groups with SF3B1 mutations being associated with better survival). Specific co-mutation patterns account for clinical heterogeneity within SF3B1- and SRSF2-related MDS. MDS with complex karyotype and/or TP53 gene abnormalities and MDS with acute leukemia–like mutations show poorest prognosis. MDS with 5q deletion are clustered into two distinct groups according to the number of mutated genes and/or presence of TP53 mutations. By integrating 63 clinical and genomic variables, we define a novel prognostic model that generates personally tailored predictions of survival. The predicted and observed outcomes correlate well in internal cross-validation and in an independent external cohort. This model substantially improves predictive accuracy of currently available prognostic tools. We have created a Web portal that allows outcome predictions to be generated for user-defined constellations of genomic and clinical features. CONCLUSION Genomic landscape in MDS reveals distinct subgroups associated with specific clinical features and discrete patterns of evolution, providing a proof of concept for next-generation disease classification and prognosis.

Chronic Myeloid Leukemia is a clonal disorder characterized by the presence of the Ph-chromosome and the BCR-ABL tyrosine-kinase (TK). Target-therapy with Imatinib has greatly improved its outcome. Deeper and faster responses are reported with the second-generation TKI Nilotinib. Sustained responses may enable TKI discontinuation. However, even in a complete molecular response, some patients experience disease recurrence possibly due to persistence of quiescent leukemic CD34+/lin−Ph+ stem cells (LSCs). Degree and mechanisms of LSCs clearance during TKI treatment are not clearly established. The PhilosoPhi34 study was designed to verify the in-vivo activity and timecourse of first-line Nilotinib therapy on BM CD34+/lin−Ph+ cells clearance. Eighty-seven CP-CML patients were enrolled. BM cells were collected and tested for Ph+ residual cells, at diagnosis, 3, 6 and 12 months of treatment. FISH analysis of unstimulated CD34+/lin− cells in CCyR patients were positive in 8/65 (12.3%), 5/71 (7%), 0/69 (0%) evaluable tests, respectively. Per-Protocol analysis response rates were as follows: CCyR 95% at 12 months, MR4.5 31% and 46% at 12 and 36 months, respectively. An exploratory Gene Expression Profiling (GEP) study of CD34+/lin− cells was performed on 30 patients at diagnosis and after, on 79 patients at diagnosis vs 12 months of nilotinib treatment vs 10 healthy subjects. Data demonstrated some genes significantly different expressed: NFKBIA, many cell cycle genes, ABC transporters, JAK-STAT signaling pathway (JAK2). In addition, a correlation between different expression of some genes (JAK2, OLFM4, ICAM1, NFKBIA) among patients at diagnosis and their achievement of an early and deeper MR was observed.

Background: Although induction chemotherapy results in remission in many older patients with acute myeloid leukemia (AML), relapse is common and overall survival is poor.\ud \ud Methods: We conducted a phase 3, randomized, double-blind, placebo-controlled trial of the oral formulation of azacitidine (CC-486, a hypomethylating agent that is not bioequivalent to injectable azacitidine), as maintenance therapy in patients with AML who were in first remission after intensive chemotherapy. Patients who were 55 years of age or older, were in complete remission with or without complete blood count recovery, and were not candidates for hematopoietic stem-cell transplantation were randomly assigned to receive CC-486 (300 mg) or placebo once daily for 14 days per 28-day cycle. The primary end point was overall survival. Secondary end points included relapse-free survival and health-related quality of life.\ud \ud Results: A total of 472 patients underwent randomization; 238 were assigned to the CC-486 group and 234 were assigned to the placebo group. The median age was 68 years (range, 55 to 86). Median overall survival from the time of randomization was significantly longer with CC-486 than with placebo (24.7 months and 14.8 months, respectively; P

Gaucher disease is characterized by multi-organ infiltration of phospholipid-laden macrophages. Bone involvement is characterized by typical deformities, osteopenia/osteoporosis, pathological fractures, and bone marrow infiltration (avascular osteonecrosis, infarction). Estimation of skeletal disease includes bone quality that contributes substantially to bone strength. We studied 23 type 1 Gaucher patients (median age 22years, range 3-73) on Enzyme Replacement Therapy from 2months to 26years (median 7years); 4 patients had pathological fractures, 10 bone infarctions, 6 avascular osteonecrosis. We noninvasively assessed bone quality by trabecular microarchitecture and macroscopic geometry, using two innovative dual-energy X-ray absorptiometry tools: Trabecular Bone Score (TBS) and Hip Structural Analysis (HSA). Bone quality parameters distinguished the patients with skeletal complications. TBS was significantly lower in patients with avascular osteonecrosis (p=0.049) and pathological fractures (p=0.024), while it could not identify those with bone infarctions. Among HSA parameters, the Cross Sectional Area of the intertrochanteric region and the Buckling Ratio of the narrow neck allowed the distinction of patients with avascular osteonecrosis. BMD was low in 11 patients (50%); neither BMD nor HSA were associated with pathological fractures. The combined evaluation of bone quality and bone quantity is useful to identify GD patients with more severe skeletal involvement.

Background Current prognostic systems for myelodysplastic syndromes (MDS) are based on clinical, pathologic, and laboratory indicators. The objective of the current study was to develop a new patient-centered prognostic index for patients with advanced MDS by including self-reported fatigue severity into a well-established clinical risk classification: the International Prognostic Scoring System (IPSS). Methods A total of 469 patients with advanced (ie, IPSS intermediate-2 or high-risk) MDS were analyzed. Untreated patients (280 patients) were recruited into an international prospective cohort observational study to create the index. The index then was applied to an independent cohort including pretreated patients with MDS from the Dana-Farber Cancer Institute in Boston, Massachusetts (189 patients). At baseline, patients completed the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30). Results A new prognostic index was developed: the FA-IPSS(h), in which FA stands for fatigue and h for higher-risk. This new risk classification enabled the authors to distinguish 3 subgroups of patients with distinct survival outcomes (ie, risk-1, risk-2, and risk-3). Patients classified as FA-IPSS(h) risk-1 had a median overall survival (OS) of 23 months (95% confidence interval [95% CI], 19-29 months), whereas those with risk-2 had a median OS of 16 months (95% CI, 12-17 months) and those with risk-3 had a median OS of 10 months (95% CI, 4-13 months). The predictive accuracy of this new index was higher than that of the IPSS alone in both the development cohort as well as in the independent cohort including pretreated patients. Conclusions The FA-IPSS(h) is a novel patient-centered prognostic index that includes patients' self-reported fatigue severity. The authors believe its use might enhance physicians' ability to predict survival more accurately in patients with advanced MDS. Cancer 2018;124:1251-9. © 2017 American Cancer Society.

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with heterogeneous biological and clinical features. The biomolecular mechanisms of CML response to tyrosine-kinase inhibitors are not fully defined. OBJECTIVE: We undertook a gene expression profiling (GEP) study of selected bone marrow (BM) CD34+/lin-cells of chronic-phase CML patients at diagnosis and after 12 months of TKI nilotinib to investigate molecular signatures characterizing both conditions. METHODS:We selected and counted BM CD34+/lin- cells of 30 CML patients at diagnosis and during 3, 6 and 12 months of first-line nilotinib treatment. GEP was performed between CD34+/lin- cells of patients at diagnosis and the same patients after 12 months of nilotinib. RESULTS: The number of BM CD34+/lin- cells dramatically decreased after 3, 6 and 12 months of nilotinib. GEP detected 264 statistically significant differentially expressed genes at diagnosis vs. 12 months of nilotinib. Functional enrichment analysis revealed groups of genes belonging to 14 pathways differentially active during nilotinib treatment. CONCLUSIONS: In conclusion, lipid, glucose and sphingolipid metabolism, insulin resistance, complement and coagulation, platelet activation, cytoscheleton, cell adhesion, transport, B cell differentiation, RAS-signaling pathway, proliferation, growth factors, and apoptosis were significantly deregulated between CML patients at diagnosis and after 12 months of nilotinib.

Introduction Sex represents a major source of diversity among patients in terms of pathophysiology, clinical presentation, prognosis and response to therapy, and therefore sex (gender)-informed medicine is becoming a new paradigm to refine clinical decision making process in different human diseases. Myelodysplastic syndromes (MDS) are heterogeneous disease characterized by ineffective hematopoiesis and risk of leukemic evolution. We aimed to study clinical effect of sex in MDS as a basis to improve patient prognostication and personalized treatment. Material and Methods We analysed three MDS populations from disease-specific registries (Italian registry n=3015, Dusseldorf registry, n=1185 and Spanish registry, n=7678). Results We first analysed the association of sex with clinical and biological disease-specific features. These analyses were conducted on Italian MDS cohort. Considering WHO categories, female patients showed higher prevalence of single lineage dysplasia and MDS with del(5q), while males were characterized by higher frequency of multilineage dysplasia and excess blasts (P We analysed mutations in 47 MDS-associated genes. Males presented higher prevalence of mutations with respect to females (82.2% vs. 76.2%, P Overall survival was significantly worse for male vs. female patients (P Competitive risk analysis showed higher prevalence of non-leukemic vs. leukemic deaths (P As a final step we aimed to integrate the prognostic value of sex into currently available prognostic systems (IPSS-R). We used random effects Cox proportional-hazards multistate modelling for developing an innovative personalized prognostic model ("Sex and age-adjusted IPSS-R", IPSS-RAS). All the three study populations were included (n=11.878). The predicted and observed outcomes correlate well in internal cross-validation. IPSS-RAS substantially improved predictive accuracy of original IPSS-R (concordance 0.68 vs. 0.62), especially in patients with early disease stage. Interestingly, demographic factors (age and sex) accounted for >30% of whole prognostic power. Conclusion In MDS, sex captures additional prognostic information at individual patient level, possibly reflecting a different molecular background and, most importantly, a sex-specific interaction between disease-related factors and comorbidity. Our results strengthen the rationale to include sex in personalized prognostic assessment in these diseases. Table 1 Disclosures Voso: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Passamonti:Roche: Other: Support of parent study and funding of editorial support; Novartis: Speakers Bureau; BMS: Speakers Bureau. Santoro:Bristol-Myers Squibb, SERVIER, Gilead Sciences, Pfizer, Eisai, Bayer, MSD, Sanofi, ArQule: Consultancy; Takeda, Roche, Abbvie, Amgen, Celgene, AstraZeneca, ArQule, Lilly, Sandoz, Novartis, Bristol-Myers Squibb, Servier, Gilead Sciences, Pfizer, Eisai, Bayer, MSD: Speakers Bureau; Takeda, Roche, Abbvie, Amgen, Celgene, AstraZeneca, ArQule, Lilly, Sandoz, Novartis, Bristol-Myers Squibb, Servier, Gilead Sciences, Pfizer, Eisai, Bayer, MSD: Speakers Bureau; Arqule, Sanofi: Consultancy; Bristol-Myers Squibb, SERVIER, Gilead Sciences, Pfizer, Eisai, Bayer, MSD, Sanofi, ArQule: Consultancy, Speakers Bureau; Bristol-Myers Squibb, SERVIER, Gilead Sciences, Pfizer, Eisai, Bayer, MSD, Sanofi, ArQule: Consultancy; Bristol Myers Squibb, Servier, Gilead, Pfizer, Eisai, Bayer, MSD: Membership on an entity's Board of Directors or advisory committees. Santini:Menarini: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Acceleron: Consultancy; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Johnson & Johnson: Honoraria. Sanz:Takeda Pharmaceutical Ltd.: Membership on an entity's Board of Directors or advisory committees; LaHoffman Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Helsinn: Membership on an entity's Board of Directors or advisory committees. Diez-Campelo:Celgene BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Background:Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder due to the deficiency of beta-glucosidase enzyme. Its prevalence in the non-Ashkenazy Jewish population is 1:40.000-100.000, whereas in Ashkenazy Jewish 1:500-1000. Patients present several hematological symptoms, including splenomegaly (86%), anemia (64%), thrombocytopenia (56%), bleeding, and MGUS, leading them to consult a hematologist on their diagnostic pathway. However, an international survey showed that only 20% of hematologists included GD in the differential diagnosis of a patient with anemia, thrombocytopenia, hepatomegaly, splenomegaly, and bone pain (Mistry PK, Am J Hematol 2007). Indeed, GD is underdiagnosed, patients experience long diagnostic delays, and misdiagnoses, leading to inappropriate procedures, treatments, and complications that often cannot be reversed by treatment. Half of the patients are diagnosed through bone marrow biopsy, although the diagnostic gold standard is the activity of beta-glucosidase on leukocytes or fibroblasts. Among the crucial obstacles to diagnosis, physicians identify the outsourced diagnostic test and, more importantly, the lack of awareness. Thus, ten years ago, a panel of experts published two diagnostic algorithms, one for the Ashkenazi and one for the non-Ashkenazi Jewish population, to facilitate the diagnosis of GD for hematologists (Mistry PK, Am J Hematol. 2011). Newborn screening has been experimented, showing an incidence of 1:22.205 (Burlina AB, J Inherit Metab Dis 2018). However, the large-scale implementation of newborn screening should be carefully evaluated. We hypothesized that an approach that combines the use of a diagnostic algorithm and a simple and cheap test could facilitate the diagnosis. Preliminary results of this study on 196 patients have been previously published, showing a prevalence of 3.6% of GD in a high-risk population (Motta I, Eur J Haem 2016). Aim:The aim of this study was to evaluate the prevalence of GD in a high-risk population presenting to the hematologist for splenomegaly and/or thrombocytopenia associated with other hematological signs or symptoms suggestive for GD. Methods:We designed a multicenter observational study among hematology centers in Italy. The study enrollment started in September 2010 and closed in December 2018. Inclusion and exclusion criteria were based on the published algorithm for the non-Ashkenazi population: Inclusion criteria: Splenomegaly and/or thrombocytopenia plus at least one among bone pain history, anemia, MGUS, polyclonal gammopathy in under 30 yrs, splenectomy;Exclusion criteria: onco-hematological diseases, portal hypertension due to liver diseases, hemoglobinopathies or chronic hemolytic anemias. The beta-glucosidase activity tests on Dried Blood Spot (DBS) were centralized at Ospedale Gaslini, Genoa (Italy). Results:500 subjects have been enrolled. 45 have been excluded because they did not fulfill the inclusion and exclusion criteria. The mean age at enrollment was 46.9±17.4 years, 31.9% (145/455) were females. The majority of enrolled patients had splenomegaly (89.7%), and approximately half (47.9%) thrombocytopenia associated with the other signs/symptoms. Anemia was the most frequent adjunctive sign (23.1%). The prevalence of GD was 3.3% (15/455, IC 95%: 1.9-5.4) in this high-risk population. In 14/15 of these patients, the molecular analysis of GBA gene identified the mutations. GD patients showed a significantly lower PLT count compared to non-GD patients (84.000/mm3 vs. 131.000/mm3, p Interestingly, in the non-GD group, a patient was diagnosed with Acid Sphingomyelinase Deficiency (ASMD), previously known as Niemann Pick type B, which presents with similar signs and symptoms as GD. Conclusions:High-risk population testing is effective in identifying Gaucher disease patients who present to the hematologist because of splenomegaly and/or thrombocytopenia. The use of DBS as a first-level tool is essential to facilitate patient testing. Because of the overlapping features of GD and ASMD, patients fulfilling the criteria applied to this protocol should be tested for both beta-glucosidase and acid-sphingomyelinase activity. Disclosures Motta: Sanofi Genzyme:Honoraria.Barcellini:Agios:Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Novartis:Honoraria, Other: invited speaker , Research Funding;Bioverativ:Membership on an entity's Board of Directors or advisory committees;Incyte:Membership on an entity's Board of Directors or advisory committees;Alexion:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: invited speaker , Research Funding.Cappellini:BMS:Honoraria;Genzyme/Sanofi:Honoraria, Membership on an entity's Board of Directors or advisory committees;CRISPR Therapeutics, Novartis, Vifor Pharma:Membership on an entity's Board of Directors or advisory committees.

Chronic myeloid leukemia (CML) is characterized by the constitutive tyrosine kinase activity of the oncoprotein BCR-ABL1 in myeloid progenitor cells that activates multiple signal transduction pathways leading to the leukemic phenotype. The tyrosine-kinase inhibitor (TKI) nilotinib inhibits the tyrosine kinase activity of BCR-ABL1 in CML patients. Despite the success of nilotinib treatment in patients with chronic-phase (CP) CML, a population of Philadelphia-positive (Ph+) quiescent stem cells escapes the drug activity and can lead to drug resistance. The molecular mechanism by which these quiescent cells remain insensitive is poorly understood. The aim of this study was to compare the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells from CP-CML patients at diagnosis and after 12 months of nilotinib treatment by microarray, in order to identify gene expression changes and the dysregulation of pathways due to nilotinib action. We selected BM CD34+/lin- cells from 78 CP-CML patients at diagnosis and after 12 months of first-line nilotinib therapy and microarray analysis was performed. GEP bioinformatic analyses identified 2,959 differently expressed probes and functional clustering determined some significantly enriched pathways between diagnosis and 12 months of nilotinib treatment. Among these pathways, we observed the under expression of 26 genes encoding proteins belonging to the cell cycle after 12 months of nilotinib treatment which led to the up-regulation of chromosome replication, cell proliferation, DNA replication, and DNA damage checkpoint at diagnosis. We demonstrated the under expression of the ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCD3 encoding proteins which pumped drugs out of the cells after 12 months of nilotinib. Moreover, GEP data demonstrated the deregulation of genes involved in the JAK-STAT signaling pathway. The down-regulation of JAK2, IL7, STAM, PIK3CA, PTPN11, RAF1, and SOS1 key genes after 12 months of nilotinib could demonstrate the up-regulation of cell cycle, proliferation and differentiation via MAPK and PI3K-AKT signaling pathways at diagnosis.

In the PhilosoPhi34 study (EudraCT: 2012-005062-34) on 87 CP-CML patients, we analyzed the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells of 79 patients with chronic-phase chronic myeloid leukemia (CP-CML) patients at diagnosis and after 12 months of nilotinib treatment (Pungolino et al. AM J Hematol. 2018). FISH analyses identified CD34+/lin- Ph+ cells in all 79 CML-CP patients at diagnosis. 78/79 patients achieved at least a complete cytogenetic response after 12 months of nilotinib whereas 1/79 patients relapsed at 12 months. No Ph+ nuclei were detected in 78/79 patients at 12 months. We have demonstrated that genes involved in the JAK-STAT signaling pathway, the cell cycle, and the ATP-binding cassette (ABC) transporters were significantly over expressed in patients at diagnosis compared to 12 months of nilotinib treatment (Trojani et al, PLoS One. 2019). In this preliminary study, we isolated BM CD34+/lin- cells from 9 healthy donors (CTRLs). We investigated the gene expression profiling of 79 CP-CML patients at diagnosis vs. the same patients after 12 months of nilotinib treatment (12 months) vs. 9 CTRLs using Affymetrix HTA 2.0. Data was preprocessed and normalized using RMA and ComBat. Selection of differentially expressed genes (DEg) was performed at diagnosis vs. 12 months of nilotinib vs. CTRLs, using Statistical Analysis for Microarrays (SAM) on 3 groups and a Benjamini Hochberg false discovey rate threshold of 5%, followed, for significance comparisons, by a pair-wise SAM test. We focused on 34 genes of the cell cycle and mitosis, 6 genes belonging to the JAK-STAT signaling pathway, and the ABC transporter gene ABCD3 which were significantly under expressed at diagnosis vs. 12 months of nilotinib vs. CRTLs (Tab.1). In the cell cycle, we found that ORC2, ORC4, ORC5, MCM6, and HDAC2 (G1 phase) were progressively significantly down regulated at diagnosis vs. 12 months vs. CTRLs. We noticed HDAC2 which showed the high fold changes of 2.89 and 4.29 in the comparison between 12 months vs. CTRLs and between diagnosis vs. CTRLs, respectively. This gene plays a crucial role in CML, as HDAC inhibitors treatment represent an effective strategy to target leukemic stem cells in CP-CML patients receiving tyrosine kinase inhibitors. CCNA2, CDK7, CDC6, DBF4 (S phase), WEE1, PRKDC, ATM, MDM2, CCNB1 (G2 phase), and TTK, MAD2L1, BUB1, BUB3, ANAPC4, CDC27, SMC3, YWHAE, and YWHAZ (M phase) were progressively down regulated at diagnosis vs. 12 months vs. CTRLs. Among them, SMC3, YWHAE and YWHAZ showed the following high fold changes in the comparison between 12 months vs. CTRLs and between diagnosis vs. CTRLs: 2.31 and 3.15, 2.59 and 3.94, 2.75 and 4.15, respectively. Notably, the proto-oncogene MDM2 which promotes the development of tumors by targeting p53, was progressively up regulated in CTRLs vs. 12 months vs. diagnosis. In the mitosis, we detected that 10 genes playing a crucial role in mitotic chromosome organization, were progressively under expressed at diagnosis vs. 12 months vs. CTRLs (Tab.1). Notably, CLAPS2, ZW10 and ANLN (hematopoietic cell differentiation) regulate the exit from mitosis. NDC80, SMC4, ZNF207, and NEK2 take part in the mitotic sister chromatid segregation whereas CENPE and SMC2 are mitotic cell cycle check points. In the JAK-STAT signaling pathway, SOS1, PIK3CA, IL7, JAK2, STAM, and PTPN11 were progressively up regulated in CTRLs vs. 12 months vs. diagnosis (Tab.1). ABCD3, encoding a protein which pumped drugs out of the cells, was progressively under expressed at diagnosis vs. 12 months vs. CRTLs as shown in Tab.1. In conclusion, we found progressive gene expression changes in BM CD34+/lin- cells of 79 CP-CML patients at diagnosis vs. 12 months of nilotinib vs. the normal cell counterparts of 9 CTRLs in the cell cycle, JAK-STAT, and the ABC transporter ABCD3. FISH analyses demonstrated that the BM CD34+/lin- cells of 78/79 patients after 12 months of nilotinib were Ph-negative. Despite, our GEP results suggested that BM CD34+/lin- cells after 12 months of nilotinib treatment and the normal cell counterparts differed mostly in the expression of genes regulating the cell cycle and the JAK-STAT signaling pathway. Disclosures Rossi: Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria. Elena:Novartis: Consultancy; Pfizer: Consultancy. Iurlo:Pfizer: Other: Speaker Honoraria; Incyte: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria.

Introduction: Many older patients (pts) with AML respond to intensive induction chemotherapy (IC), but responses are often short-lived and overall survival (OS) is poor. The benefit of post-remission maintenance treatment (Tx) for pts with AML is unclear, as no therapy has shown to significantly improve OS. CC-486 is an oral hypomethylating agent that allows for prolonged drug exposure during each Tx cycle to sustain therapeutic activity. We hypothesized that prolonged Tx with CC-486 could be effective as post-remission maintenance in AML. Herein, we report the primary results of QUAZAR AML-001 (NCT01757535), a phase III international, randomized, double-blind, placebo (PBO)-controlled study evaluating CC-486 as maintenance therapy in pts aged ≥55 years with AML in first remission following IC. Methods: Eligible pts had de novo or secondary AML, intermediate- or poor-risk cytogenetics, and Eastern Cooperative Oncology Group performance status (ECOG PS) scores of ≤3; had achieved first complete remission (CR) or CR with incomplete count recovery (CRi) after IC, with or without consolidation chemotherapy; and were not candidates for hematopoietic stem-cell transplant (HSCT). Within 4 months of attaining CR/CRi, pts were randomized 1:1 to receive CC-486 300 mg or PBO once-daily on days 1-14 of repeated 28-day Tx cycles. A 21-day dosing schedule was permitted for pts who experienced AML relapse with 5-15% blasts in blood or bone marrow while on-study. Tx could continue indefinitely until the presence of >15% blasts, unacceptable toxicity, or HSCT. The primary endpoint was OS. Secondary endpoints included relapse-free survival (RFS), health-related quality of life (HRQoL), and safety. Samples were collected for exploratory translational endpoints, including measurable residual disease (MRD). Kaplan-Meier estimates of OS and RFS were compared for CC-486 vs. PBO by stratified log-rank test. Hazard ratios (HRs) and 95% confidence intervals (CIs) were generated using a stratified Cox proportional hazards model. Results: Between May 2013 and October 2017, 472 pts were randomized to receive CC-486 (n=238) or PBO (n=234). Baseline characteristics were balanced between Tx arms. Median age was 68 years (range 55-86), 91% of pts had de novo AML, and 86% and 14% of pts, respectively, had intermediate-risk or poor-risk cytogenetics. Following induction, 81% of pts achieved a CR and 19% achieved CRi; 80% of pts had received consolidation chemotherapy (45% received 1 consolidation cycle and 31% received 2 consolidation cycles). At a median follow-up of 41.2 months, OS was significantly improved with CC-486 vs. PBO: median OS was 24.7 months vs. 14.8 months from time of randomization, respectively (P=0.0009; HR 0.69 [95%CI 0.55, 0.86]) (FigureA). RFS was also significantly prolonged: median RFS was 10.2 months in the CC-486 arm, compared with 4.8 months in the PBO arm (P=0.0001; HR 0.65 [95%CI 0.52, 0.81]) (Figure B). OS and RFS benefits of CC-486 were demonstrated regardless of baseline cytogenetic risk, the number of prior consolidation cycles received, and CR/CRi status. CC-486 did not adversely impact overall HRQoL vs. PBO, as assessed by mean changes from baseline in HRQoL measures during Tx. CC-486 had a manageable safety profile generally consistent with that of injectable azacitidine. Median exposure to CC-486 was 12 cycles (range 1-80) and to PBO was 6 cycles (1-73). The most frequently reported adverse events (AEs) with CC-486 and PBO were grade 1 or 2 gastrointestinal (GI) events, including nausea (64% and 23%, respectively), vomiting (59% and 10%), and diarrhea (49% and 21%). The most common grade 3-4 AEs were neutropenia (CC-486, 41%; PBO, 24%), thrombocytopenia (23% and 22%), and anemia (14% and 13%). Serious AEs were infrequent, mainly infections, which occurred in 17% of pts in the CC-486 arm and 8% of pts in the PBO arm. Few AEs led to Tx discontinuation, most often GI events (CC-486, 5%; PBO, 0.4%). Conclusions: CC-486 is the first therapy used in the maintenance setting to provide statistically significant and clinically meaningful improvements in both OS and RFS in pts with AML in remission following induction chemotherapy, with or without consolidation. Oral CC-486 has a manageable safety profile and represents a new therapeutic standard for pts with AML in remission. Disclosures Wei: Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Montesinos:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: Research support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Teva: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau. Ravandi:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Orsenix: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Xencor: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Macrogenix: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sayar:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Porkka:Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Selleslag:Celyad: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy; Daichii Sankyo: Consultancy; Janssen-Cilag: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Astex Otsuka: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau. Sandhu:Takeda: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Bioverativ: Consultancy; Celgene Corporation: Consultancy; Pfizer: Consultancy; Janssen: Consultancy. Giai:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Beltrami:Ospedale Policlinico San Martino: Employment. Ossenkoppele:Celgene Corporation: Consultancy, Honoraria, Research Funding. La Torre:Celgene Corporation: Employment, Equity Ownership. Skikne:Celgene Corporation: Employment, Equity Ownership. Kumar:Celgene Corporation: Employment, Equity Ownership. Dong:Celgene Corporation: Employment, Equity Ownership. Beach:Celgene Corporation: Employment, Equity Ownership. Roboz:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Introduction Chronic myeloid leukemia (CML) is a stem cell disease characterized by the constitutive activity of the oncoprotein BCR-ABL that activates multiple signal transduction pathways. Tyrosine-kinase inhibitor (TKI) nilotinib successfully inhibits the activation and the proliferative function of BCR-ABL in patients with CP-CML. Despite the success of nilotinib, some patients become refractory suggesting the presence of a population of Philadelphia positive (Ph+) quiescent stem cells escaping the drug activity. Thus, the molecular mechanisms underlying CML remain poorly understood. In this study, we enrolled 87 CP-CML patients (Pungolino et. al. Am J Hematol. 2018). Samples were collected on the behalf of the Rete Ematologica Lombarda (REL) the PhilosoPhi34 study (EudraCT: 2012-005062-34), which included 15 centers from Italy. We undertook gene expression profiling (GEP) of selected bone marrow (BM) CD34+/lin- cells of 80 patients at diagnosis vs. the same patients after 12 months of nilotinib to investigate gene expression changes induced by the treatment. Methods We isolated CD34+/lin- cells from BM samples in 87 patients at diagnosis whereas the same cells were also selected from 80/87 patients after 3, 6 and 12 months of nilotinib (Trojani et. al. Cancer Biomark. 2017). BM mononuclear cells (MNCs) as well as BM CD34+/lin- cells of all 80 CML patients were counted at diagnosis and during the treatment with nilotinib (at 3, 6, and 12 months, respectively). Standard FISH tested isolated BM CD34/lin- cells for the 87 patients at diagnosis, and for 80/87 patients after 3, 6 and 12 months of nilotinib treatment, respectively. Therefore, we performed GEP analyses of selected BM CD34+/lin- cells of 80/87 patients at diagnosis vs. the same patients after 12 months of nilotinib treatment. Then, we executed bioinformatic preprocessing and correction for batch effects on raw microarray data. Finally, we conducted differential expression analysis and significantly perturbed genes were subjected to functional clustering. Results We observed a wide variability of the number of BM MNCs as well as the number of the BM CD34+/lin- cells among the 80 CP-CML patients at diagnosis and after 3, 6 and 12 months of nilotinib for each patient (Table 1). Figure 1 showed that the number of the BM CD34+/lin- cells dramatically decreased between the diagnosis and after 3 as well as 6 months of nilotinib. We noticed that the BM CD34+/lin- cells slightly increased between 6 and 12 months of nilotinib which might be caused by the gradual repopulation of the normal CD34+/lin- cells in the bone marrow as FISH results suggested. FISH analysis detected CD34+/lin- Ph+ cells in 87 CP-CML patients at diagnosis. No positive Ph+ nuclei were detected on CD34+/lin- cells of 79/80 patients after 12 months of treatment (to categorize a sample as negative, at least 200 nuclei were examined). All of these 79 patients achieved at least complete cytogenetic response. 1/80 patient relapsed at 12 months. We conducted GEP analyses on 78 subjects because, due to experimental issues, two patients were not considered for differential expression analyses, as the microarray CEL files of the 12 months' samples were corrupted and missed probe intensities for most of the probes. GEP analyses determined 2,959 significantly differently expressed probes between diagnosis and after 12 months of nilotinib treatment. Functional clustering identified some pathways significantly enriched between diagnosis and 12 months of nilotinib. Among these pathways, we found that ABCC4, ABCC5, and ABCD3 genes associated with ATP-binding cassette (ABC) transporters were up regulated at diagnosis. GEP results highlighted that 26 genes belonging to cell cycle, mitosis, DNA damage and repair were over expressed at diagnosis. Moreover, GEP data demonstrated that JAK-STAT signaling pathway was deregulated: JAK2, IL7, STAM, PIK3CA, PTPN11, RAF1, SOS1 were over expressed whereas IL22RA1 was under expressed at diagnosis vs. 12 months of nilotinib, respectively. Conclusions In summary, we reported that BM CD34+/lin- cells from CP-CML patients after 12 months of nilotinib were characterized by changes of expression of genes involved in cell cycle checkpoints and mitosis, ABC transporters genes that pump drugs outside form the cells, and JAK-STAT signaling pathway genes responsible for the proliferation, differentiation and cell survival in CML. Disclosures Rossi: Teva: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) represents the only curative treatment for patients with myelodysplastic syndrome (MDS), but involves non-negligible morbidity and mortality. Crucial questions in clinical decision-making include the definition of optimal timing of the procedure and the benefit of cytoreduction before transplant in high-risk patients. We carried out a decision analysis on 1728 MDS who received supportive care, transplantation or hypomethylating agents (HMAs). Risk assessment was based on the revised International Prognostic Scoring System (IPSS-R). We used a continuous-time multistate Markov model to describe the natural history of disease and evaluate the effect of different treatment policies on survival. Life expectancy increased when transplantation was delayed from the initial stages to intermediate IPSS-R risk (gain-of-life expectancy 5.3, 4.7 and 2.8 years for patients aged ⩽55, 60 and 65 years, respectively), and then decreased for higher risks. Modeling decision analysis on IPSS-R versus original IPSS changed transplantation policy in 29% of patients, resulting in a 2-year gain in life expectancy. In advanced stages, HMAs given before transplant is associated with a 2-year gain-of-life expectancy, especially in older patients. These results provide a preliminary evidence to maximize the effectiveness of allo-SCT in MDS.

The primary objective of this study was to evaluate the health-related quality of life (HRQOL) in lower-risk, transfusion-dependent patients with myelodysplastic syndromes (MDS) treated with deferasirox. A secondary objective was to investigate the relationship between HRQOL, serum ferritin levels and transfusion dependency.This was a prospective multicentre study enrolling 159 patients, of whom 152 received at least one dose of deferasirox. HRQOL was assessed with the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) at baseline and then at 3, 6, 9 and 12 months. Primary analysis was performed estimating mean HRQOL scores over time by a linear mixed model on selected scales.The median age of treated patients was 72 years (range 24-87 years). No statistically significant changes over time were found in mean scores for global health status/quality of life (p=0.564), physical functioning (p=0.409) and fatigue (p=0.471) scales. Also, no significant changes were found for constipation (p=0.292), diarrhoea (p=0.815) and nausea and vomiting (p=0.643). Serum ferritin levels were not associated with HRQOL outcomes. A higher patient-reported baseline pain severity was an independent predictive factor of an earlier achievement of transfusion independence with a HR of 1.032 (99% CI 1.004 to 1.060; p=0.003).HRQOL of transfusion-dependent patients with MDS receiving deferasirox therapy remains stable over time. HRQOL assessment might also provide important predictive information on treatment outcomes.NCT00469560.

Background Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder which molecular base is represented by the bcr-abl fusion gene, encoding for the constitutionally activated BCR-ABL tirosine-kinase. Three Tirosin-Kinase Inhibitors (TKI) are approved for first line treatment: Imatinib (IM) and the second generation (2G) TKI Nilotinib (NIL) and Dasatinib. 2G TKI are known to provide faster and deeper molecular responses (MR) compared to Imatinib, but serious toxicities may hamper long term treatment with these molecules. Furthermore, 2G TKI were usually employed as second line after IM failure, while the inverse sequence from second to first generation TKI (like an induction-maintenance model) has not been explored yet. We used this schedule in a small group of patients in the PhilosoPhi34 study (EudraCT: 2012-005062-34), a clinical trial designed by the REL (Rete Ematologica Lombarda) cooperative group. This study was composed by three consecutive phases: a Recruitment Phase, a Core Phase (CP) in which patients received NIL 300 mg BID for 12 month (mos), and an Observational Phase (OP), restricted for patients who obtained at least complete cytogenetic response at the end of the CP. During OP, treatment choice was up to the physician and any TKI approved for first line treatment could be used, including IM. In 2017 we presented preliminary data showing that a 12-mos-NIL treatment followed by IM appears as a safe and effective choice for first line therapy in chronic phase CML. Fluctuations in BCR/ABL ratio were similar between IM and NIL treated pts, and the probability of loss of MR4 or MR3 was the same in the two groups; furthermore, despite fluctuations, MR was maintained or improved over time in IM subgroup. Our purpose is to verify these data after 24 mos follow up (FU) at the end of OP. Methods We analyze PhilosoPhi34 database; MR is reported at 3, 6 and 12 mos during the CP and every 6 mos during the OP. The last pt completed the 24 mos of OP in June 2018. Database is still open, evaluations ongoing, and some data can be missing yet: our preliminary observations concern pts with available data of 24 mos OP. Results Seventy-nine pts started the OP. Fourteen pts switched to IM during the OP (Table 1) due to high cardiovascular risk or grade 1-2 chronic AEs . Only 11 pts started IM since the beginning of OP, and we consider these pts in our analysis. Sokal score was high in 2 pts (18%), intermediate in 5 (45.5%), low in 4 (36.5%). At the beginning of OP, 6 pts had a MR ≥ 4 (54.5%), 5 had MR3 (45.5%). At 12 mos of the OP, 7 had MR ≥ 4, 3 had MR3 and 1 had lost MR3 with PCR 0.192%IS (1/5, 20%). At 24 mos of the OP, 9 had MR ≥ 4 (81,8%), and 2 had MR3. Notably, none of pts lost MMR; 2/3 pts(66%) improved response from MR 3 to MR 4 and the pt who transiently lost MMR at 12 mos, recovered it at 24. Sixty-four pts maintained 2G TKI: 62 NIL, 2 other TKI (not considered for analysis). Of them, 4 were lost during this phase: 2 within the first year of OP, other 2 within 12 and 24 mos of OP. In the NIL group, Sokal score was high in 10 pts (16.6%), intermediate in 19 (31.6%) and low in 31 (51.6%). At the beginning of OP, 32 pts had MR ≥ 4 (51.6%), 21 had MR3 (33.8%) and 9 less than MR 3 (14.5%). Responses were improved over time: at 12 mos, 36 pts had MR ≥ 4 (60%), 20 had MR3 (33%) and 4 less than MR3 (6%). At 24 mos 46 pts had MR ≥ 4 (78%), 8 MR3 (13.5%) and 4 less than MR3 (8,5%), Among them, 1 pt experienced disease progression due to a mutation. In particular, during the second year of OP, 11 pts improved response from MR3 to MR ≥ 4(11/20, 55%). Discussion Our data show progressive MR improvement in both IM and NIL group. In particular, risk of loss of MMR is not increased in IM group. More data, more balanced groups and a longer FU are necessary to further confirmations, but after three years of FU, we consider this combination of NIL-followed-by-IM a possible strategy for first line treatment in chronic phase CML, in particular for pts with cardiovascular risk factors. Disclosures Rossi: Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Introduction The diagnosis of a hematological neoplastic disease (HND) bears a great impact on the patient family, which suffers abrupt changes in living patterns because of prognosis, prolonged hospitalization and therapy related adverse events. Parents often believe that the best way to protect their children from suffering is to avoid communications about the disease, as "they wouldn't understand". Many hospitals offer family support, usually managed by psychologists; we hypothesized that the hematologist can play a key role in this process, being the one who primarily takes care of the patient, possesses the scientific skills necessary to explain the disease and is viewed by the family members as the key player. Since 2010, patients admitted to the Hematology Division at San Gerardo Hospital in Monza - Italy, who have minors in their family can participate in the "Emanuela Project" (see below). Methods The aim of this pilot study is to evaluate the impact of this intervention on children health status as perceived by their parents either affected or unaffected by HND. Ten hospitalized patients with a HND disease diagnosed between November 2017 and May 2018, with at least one child aged 0-18 years were recruited after signing an informed consent. Intervention: children can visit their parents in a dedicated hospital room in the days after diagnosis and an informal talk with a hematologist and a psychologist is organized; the hematologist, using simple images and metaphors (e.g. "flowered garden" to represent the normal bone marrow), explains the illness and answers questions, while the psychologist helps children to express emotions about the situation. A questionnaire, administered 30-60 days after the intervention to all parents, explores their perceptions about changes in each child; itconsists of 18 multiple choice questions and 15 open questions. Data were analyzed with statistical software STATA. Open questions were fully read and interpreted by authors; T-LAB software was used to evaluate relevant recurring words . Results All 10 patients that were offered the intervention consented to it; 9 out of 10 patients have returned their questionnaires by July 2018 Five of them were fathers (55.56%) and 4 mothers (44.44%), with a mean age 50.22+/- 9.19 (SD). Diagnoses were Acute Leukemia (5), Lymphoma (2) and Multiple Myeloma (2). Mean duration of hospital stay was 26.2 days (+/- 12.8 SD) . The study included 16 children, aged 4 to 18 (mean 10 +/- 5 SD). We analyzed 28 questionnaires. Data from multiple choice questions exploring changes in children behavior suggest that, according to both parents there was no substantial worsening in school performance, appetite, sleep patterns (see table 1). These findings suggest that talking to children about the disease didn't traumatize them, and gave some concrete and reassuring answers to unexpressed fears. An interesting finding is that 44% of children increased their need to be in contact with the ill parent, showing a strengthening of relationships inside the family. One relevant finding concerns the possibility to talk about HND inside the family: 93% of parents gave a score of 3 (=often) or 4(=always) to this question. 100% of parents stated that it was never necessary to keep clinic visits or hospital admissions hidden from their children; 80% never had to hide side effects of therapies. All healthy parents and 87.5% of sick ones found that communicating with their children was a correct strategy, and that this intervention by hematologist and psychologist was useful. According to 88% of them, such a difficult task is responsibility of parents and of a specific professional figure, the hematologist. The use of simple images and metaphors helped 85.7% of healthy parents and 62.5% of ill ones to understand the illness better. According to 75% of parents, the intervention also played a key role in improving their relationship with doctors. The T-Lab analysis of recurring words is presented in Tab 2. Conclusions Data indicate that for parent it is important to be supported by their hematologist in the difficult task of explaining their illness to their children. The Emanuela Project allows parents to retain a parental role despite their illness; communication contributes also to increase trust in the medical staff and compliance to physically and emotionally demanding treatments. These data will be compared to those obtained in nearby hospitals who do not offer such a service. Disclosures Gambacorti-Passerini: BMS: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding.

5-Azacytidine is an effective therapy in high risk MDS and oligoblastic AML. This "real life" analysis was made on 185 patients treated with 5-azacytidine in 10 centers afferent to REL ("Rete Ematologica Lombarda"), a network in Lombardia region. The aim was to assess the influence of disease and comorbidity risk assessments on the survival. The results confirm the utility of 5-azacitidine in prolonging OS regardless of advanced age and the presence of comorbidities. They also encourage an early treatment since patients with IPSS-R High risk MDS have better outcome with respect to Very High risk ones. According to the IPSS cytogenetic risk, there was no difference in the outcome between Intermediate and High risk patients. Nevertheless, a poorer cytogenetic risk, according to the IPSS-R cytogenetic stratification, negatively influenced the outcome.

Clofarabine has been shown to be effective in AML patients, either as single agent or, mainly, in association with intermediate dose cytarabine. Based on these reports, we conducted a preliminary study combining clofarabine and intermediate dose cytarabine in AML patients who relapsed or failed to respond to at least two induction therapies. We treated 47 patients affected by relapsed/refractory AML with a regimen including clofarabine at 22.5 mg/m(2) daily on days 1-5, followed after 3 hr by cytarabine at 1 g/m(2) daily on days 1-5. Ten patients received a further consolidation cycle with clofarabine at 22.5 mg/m(2) and cytarabine at 1 g/m(2) day 1-4. Among the 47 patients, 24/47 (51%) achieved a complete remission, 5/47 (10.5%) a partial response, 10/47 (21%) had a resistant disease, and 6/47 (13%) died of complications during the aplastic phase. The most frequent nonhematologic adverse events were vomiting, diarrhea, transient liver toxicity, febrile neutropenia, and infections microbiologically documented. Among the 24 patients who obtained a CR 13 underwent allogeneic bone marrow transplantation. In 14 patients, complete remission duration was shorter than 12 months, whereas 10 patients experienced longer complete remission duration. These very preliminary results suggest that clofarabine-cytarabine regimen is effective in this particularly poor prognosis category of patients, representing a potential "bridge" toward bone marrow transplant procedures. Safety data were consistent with previously reported salvage therapies. Further studies and a longer follow up are warranted.

Purpose: Epigenetic changes play a role and cooperate with genetic alterations in the pathogenesis of myelodysplastic syndromes (MDS). We conducted a phase II multicenter study on the combination of the DNA-methyltransferase inhibitor 5-azacytidine (5-AZA) and the histone deacetylase inhibitor valproic acid (VPA) in patients with higher risk MDS. Experimental Design: We enrolled 62 patients with MDS (refractory anemia with excess blasts, 39 patients; refractory anemia with excess blasts in transformation, 19 patients; and chronic myelomanocytic leukemia (CMML), 4 patients) and an International Prognostic Scoring System (IPSS) rating of Intermediate-2 (42 patients) or high (20 patients). VPA was given to reach a plasma concentration of >50 μg/mL, then 5-AZA was added s.c. at 75 mg/m2 for 7 days in eight monthly cycles. Results: The median overall survival was 14.4 months. At a median follow-up of 12 months (range, 0.7-21.0), the disease progressed in 20 patients, with 21% cumulative incidence of progression. Of 26 patients who completed eight cycles, 30.7% obtained complete or partial remission, 15.4% had a major hematologic improvement, whereas 38.5% showed stable disease. Drug-related toxicity was mild. Favorable prognostic factors for survival were IPSS Intermediate-2 and plasma VPA of ≥50 μg/mL (log rank = 0.013 and 0.007, respectively). Analysis of polymorphisms important for the metabolism of the drugs used in the trial showed that carriers of the CYP2C19*2 variant of cytochrome P450 required higher VPA doses to achieve the target VPA plasma concentration of 50 μg/mL on day 1 of 5-AZA treatment (P = 0.0021). Conclusion: Our data show that the 5-AZA/VPA combination is active and safe in patients with MDS with a poor prognosis. Achievement of VPA therapeutic levels may indeed increase 5-AZA efficacy.

Background. Chronic Myeloid Leukaemia (CML) can be effectively treated with the first generation Tyrosine Kinase Inhibitor (TKI) Imatinib, and more effectively with the second generation TKI, like Nilotinib. However, despite the deeper and faster responses induced by nilotinib in a large proportion of patients, the possible eradication of the pathological stem cells is not yet clearly elucidated. In fact, in vitro data suggest that quiescent stem cells are not sensitive to Bcr/Abl inhibition (Corbin AS, et al 2011; Hamilton A, et al 2012). A preliminary in-vivo study (Defina M, et al 2012) shows that in patients in CCyR even after short-term of nilotinib therapy, residual leukemic progenitors are rarely detected. Methods. On behalf of Rete Ematologica Lombarda (REL), Italy, we designed a single arm prospectic study, PhilosoPhi34 (EudraCT: 2012-005062-34), with the aim to investigate the efficacy of nilotinib 300 mg BID in obtaining the disappearance of Bone Marrow (BM) leukemic stem cells (CD34+/lin-Ph+) in newly diagnosed CP-CML. Primary objective of the study: to enumerate the BM CD34+/lin-Ph+ cells at the end of 6 months of treatment. Secondary objectives: to enumerate the BM CD34+/lin-Ph+ cells at 3 and 12 months; to assess the percentage of patients showing MR ≤10% IS at 3 months and MR ≤1% IS at 6 months and the MMR IS and MR4.5 IS by 3, 6 and 12 months of treatment. BM blood samples (range of 5-20 ml) were collected at diagnosis and after 3, 6 and 12 months of nilotinib treatment. BM mononuclear cells were purified by density gradient centrifugation and then CD34+/lin- cells were isolated using Diamond CD34 Isolation Kit (Miltenyi Biotec). The purity of CD34+/lin- cells was about 97% as determined by flow cytometry. BM CD34+/lin- cells were counted and a range of 100,000-800,000 has been noted at diagnosis. After the treatment we observed that the number of CD34+/lin- cells dramatically decreased after 3 (1,000-600,000), 6 (1,000-260,000) and 12 months (100-130,000). In particular, CD34+/lin- cells were even less than 1000 at 12 month of treatment. In order to verify the disappearance of leukemic stem cells, isolated CD34+/lin- cells were tested by standard FISH (i.e. to categorize a sample as negative at least 200 nucleus were examined). From April 2013 and June 2015 we enrolled 87 pts, as for protocol. We report here the preliminary results. Results. Of 56 patients in CCyR after 6 months of treatment, FISH performed on BM CD34+/lin- cells nuclei was evaluable in 51 cases (5 negative cases were excluded because of less than 200 nucleus were analysed). In 4 out of 51 patients (7.8%), Ph+ nuclei were detected. The Sokal score of these 4 patients was 1 low, 2 intermediate and 1 high risk with a ratio (positive nuclei/total nuclei) of 295/300, 1/200, 2/92, 3/300, respectively. Among 58 patients tested at 3 months and 44 tested at 12 months of treatment, the number of evaluable patients was 48 and 37, respectively; 8/48 (16.6%) and 0/37 (0%) patients showed Ph+ nuclei. Only 2 out of 8 positive patients had a high Sokal score. Regarding efficacy of treatment, Table 1 summarizes the MRs IS observed after 3, 6 and 12 months of treatment in 71, 57 and 41 patients, respectively. Conclusion. Data of this prospective study confirms that nilotinib 300 mg BID, rapidly and progressively induces the clearance of BM CD34+/lin-Ph+ cells in CP-CML patients. In particular, on CD34+/lin- cells, after 6 months of treatment, only 7.8% of patients showed positive nuclei. On 37 patients after 12 months of treatment, no positive nuclei were detected. So far, the kinetic of reduction of such cells seems not influenced by Sokal score. According to international studies, PhilosoPhi34 shows a very high efficacy of Nilotinib to induce MRs in CP-CML patients, at the standard time points. Table 1. Molecular Response (MR) in CP-CML patients treated with Nilotinib 300mg BID from diagnosis. MR IS 3 months 6 months 12 months ≤10% 67/71 94% 57/57 100% 40/41 97.50% ≤1% 57/71 80% 55/57 96.50% 40/41 97.50% ≤0.1% 17/71 24% 41/57 72% 35/41 85% ≤0.01% 3/71 4% 19/57 33% 20/41 48.70% MR4.5(UD) 2/71 2.80% 10 (6)/57 17.5% (10.5%) 15 (9)/41 36.5% (22%) UD: undetectable We acknowledge all REL Colleagues for their collaboration and Novartis SpA for the partial financial support to the study. Disclosures No relevant conflicts of interest to declare.

We analyzed appearance of non transferrin bound iron (NTBI) in 30 transplant eligible patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) during conventional chemotherapy treatment program and evaluated possible relationship with transfusional body iron intake, iron parameters and clinical complications. For each course, serum samples for NTBI detection were taken prior to chemotherapy, during treatment and during subsequent bone marrow myelosuppression: NTBI was assessed by HPLC. Appearance of NTBI was observed from the start of induction treatment and was still detectable during bone marrow myelosuppression; the recovery of the bone marrow function coincided with the disappearance of NTBI. This kinetic was observed in all subsequent high doses chemotherapy courses, independently from confounding variables such as transfusional iron intake and transferrin saturation. NTBI seems to be a consequence of chemotherapy induced lysis of bone marrow cells and, partly, of hepatocytes after cytotoxic injury. The subsequent persistence of NTBI throughout bone marrow myelosuppression is related to the transient suspension of erythropoietic activity. Moreover, NTBI levels >2μM at the beginning of iatrogenic myelosuppression were associated with higher risk of sepsis caused by Gram negative Bacilli (RR 2.571), also compared with other infectious complications (RR 1.954).

Background In the absence of randomized, controlled trial data to support iron chelation therapy in transfusion-dependent patients with myelodysplastic syndromes (MDS), continued evidence from large prospective clinical trials evaluating the efficacy and safety of iron chelation therapy in this patient population is warranted. Methods The safety and efficacy of deferasirox was examined in a prospective, open-label, single-arm, multicenter trial of transfusion-dependent patients with International Prognostic Scoring System low- or intermediate-1-risk MDS and evidence of transfusion-related iron overload. The effects of deferasirox therapy on hematological response and disease progression were also examined. Results Of 159 participants enrolled from 37 Italian centers, 152 received ≥1 dose of deferasirox (initiated at 10–20 mg/kg/day and titrated as appropriate), and 68 completed the study. Of 84 patients who discontinued deferasirox therapy, 22 died during the trial, and 28 withdrew due to an adverse event (AE). Fourteen treatment-related grade 3 AEs occurred in 11 patients, whereas no grade 4 or 5 drug-related AEs were reported. Significant risks for dropout were a higher serum ferritin level at baseline, a higher MDS-Specific Comorbidity Index, and a shorter diagnosis–enrollment interval. Median serum ferritin level fell from 1966 ng/mL to 1475 ng/mL (P

Background CML is a clonal disorder characterized by the presence of the Philadelphia (Ph) chromosome which encodes for the bcr-abl tyrosine-kinase (TK). Target therapy with the TK inhibitors (TKIs)) has greatly improved its outcome. Treatment with second generation TKIs - e.g. nilotinib (NIL) - results in deeper and faster responses and prevents disease progression. Sustained responses may enable TKI discontinuation. However, even in the event of qPCR negativity, a fraction of patients (pts) experience disease recurrence possibly due to persistence of quiescent leukemic stem cells (LSCs). Degree and mechanisms of LSCs clearance during TKI treatment are not established yet and conflicting results are reported in the literature. Work from the group of Bocchia (Bocchia 2008; Defina 2012) showed reduction of LSCs during long term imatinib (IM) therapy; moreover, in CCyR pts residual LSCs are more rarely detected after NIL compared to IM therapy and, in a small fraction of pts this occurs after very short-term NIL therapy. This data conflicts with in vitro evidence that NIL is not superior to IM in inducing growth suppression in CML LSCs (Konig, 2008). To verify the in vivo activity and time-course of first-line NIL therapy on bone marrow (BM) Ph+ stem cells (CD34+/lin-) clearance, on behalf of the Rete Ematologica Lombarda (REL) the PhilosoPhi34 study (EudraCT: 2012-005062-34) was designed. Primary efficacy endpoint was to measure the rate of BM residual CD34+/lin-Ph+ cells in CCyR pts at 6 months of treatment. Methods BM cells were collected and stored at diagnosis and at 3,6 and 12 mos of treatment. CD34+/lin- cells were purified using a Diamond CD34 Isolation Kit Miltenyi (97% of purity). FISH analysis of selected unstimulated CD34+/lin- cells was performed according to standard procedures; considering the low sensitivity of the test, in order to define the test as negative at least 200 nuclei were examined. The A'Hern single stage design was chosen for the present study; considering the CCyR results obtained in the ENESTnd study and the anticipated number of un-evaluable tests, a minimun of 87 pts were required. Results Enrolment of the 87 pts was completed by June 2015. Table 1 summarises pts' characteristics and response to treatment. FISH results are as follows: at 3 mos, 8/65 (12,3%; CI 95%: 2,3%-15,7%) evaluable FISH tested positive (10 negative tests not evaluable); at 6 mos 5/71 (7%; CI 95% :2,3-15,7%) evaluable FISH tested positive (7 negative tests not evaluable); at 12 mos, 0/68 (0%; CI95%:0,0-5,2%) evaluable FISH tested positive (9 negative tests not evaluable). At any time point, Sokal score did not predict for FISH results. However, as outlined in Table 2, H-Sokal score pts are less prevalent among pts who achieve a CCyR, a requirement for FISH analysis. Of the 4 pts who failed the treatments' objectives by 12 mos, 1 was in CCyR with detectable residual CD34+/lin-Ph+ cells at 3 mos; 2 were not in CCyR and with residual CD34+/lin-Ph+ cells at 3mos; 1 was in CCyR and with CD34+/lin-Ph- cells at 3 and 6 mos but with increasing qPCR. Only 1 pt with CD34+/lin-Ph- cells at all time points and with optimal molecular response harboured a NIL-resistant mutation at 26 mos of treatment. None of the 22 pts (including 4 H-Sokal score pts = equal proportion of study cohort) in Molecular Response (MR) 3.0 at 3 mos had a positive FISH at 3 and 6 mos or failed treatment at follow-up. Conclusion. Our final results on the whole cohort of pts confirm our preliminary data on the efficacy of NIL 300 g BID in early clearance of BM LSCs (CD34+/lin-Ph+) in newly diagnosed CP-CML patients tested at 3, 6 and 12 mos of treatment. Moreover, according to our data, fast disease debulking seems crucial for obtaining BM LSCs clearance and it can be speculated that the same mechanism responsible for this early MR 3.0 achievement is also capable of preventing H-Sokal risk pts from failing treatment. Disclosures Orlandi: Ariad: Honoraria; BMS: Honoraria; Novartis: Honoraria.

To assess the economic outcomes produced when a conventional antibiotic treatment regimen requiring three administrations per day was replaced with a treatment regimen requiring only one daily administration, the efficacy, tolerability and cost of ceftazidime was compared with that of ceftriaxone (both drugs in combination with amikacin) for the empirical treatment of febrile granulocytopenic patients with haematological malignancy. 102 febrile patient-episodes were randomly assigned to receive ceftazidime (6g in three divided doses) or ceftriaxone (2g as a single daily dose), both in combination with amikacin. The response was evaluable in 94 patients (47 in each group). 75 (80%) patients had an absolute granulocyte count lower than 100/mm(3) at the onset of fever or during the first week of antibiotic therapy. 61 (64.9%) were affected by acute leukaemia. Multiple daily ceftazidime plus amikacin was effective in 33 of 47 (70.2%) patients, and single daily ceftriaxone plus amikacin in 31 of 47 (66%) patients (p > 0.2). Among patients successfully treated, median time to defervescence was 3.3 days (range 1 to 11) for ceftazidime plus amikacin and 4.5 days for ceftriaxone plus amikacin (range 1 to 15) [p = 0.14]; study drugs were continued for 12 (range 7 to 26) and 12.3 days (range 7 to 28), respectively. Our study demonstrated that single daily administration of ceftriaxone was as effective and well tolerated as multiple daily administration of ceftazidime when both were administered in combination with amikacin. Cost analysis showed that compared with the thrice daily regimen, administration of single daily doses of ceftriaxone for a 12-day treatment period would result in a net cost saving of $US392 (626 940 Italian lire).

Hypomethylating drugs are useful and have been approved for the treatment of myelodysplastic syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML). However, phase 2 and 3 studies that assessed these agents in MDS, have included only a small number of patients with CMML, and there are just a few specific reports on CMML patients. The Azacitidine is actually authorised for the treatment of CMML patients with 10–29% marrow blasts without myeloproliferative disorder, who are not eligible for haematopoietic stem cell transplantation. This hypomethylating agent in MDS is known for causing transient cytopenias, most often occurring during the first 2 cycles. Here we report a case of an atypical delayed and prolonged hematologic toxicity during Azacitidine treatment in a CMML patient; furthermore we also reviewed the literature regarding the efficacy of the drug and the management of hematologic adverse effects, in term of dose adjustments or alternative schedule of administration, in specific CMML setting.

The ability of recombinant interleukin 2 (rIL2) activated lymphocytes (LAK) to purge BM samples contaminated by tumour cells was evaluated. Human BM mononuclear cells were contaminated with 10% of the lymphoma line CA46 and then cultured in liquid medium containing 1000 U/ml of rIL2 and/or LAK autologous to the used BM. At the end of coculture the growth of residual tumour cells and of CFU-GM were evaluated by clonogenic assay. No tumour cell growth was observed in 5/5 independent experiments after 18 h of coculture with LAK. No significant inhibition of CFU-GM growth was also noted. Subsequently, the effect of LAK on BM obtained from four leukaemic patients and contaminated with 20-50% of their own AML and ALL cells was studied using MAb as a tool for identifying leukaemic cells. LAK eliminated 24-78% of contaminating cryopreserved uncultured autologous leukaemic cells. In five cases the BM was contaminated by a low (2%) amount of ALL cells. In these patients the monoclonal heavy chain rearrangement typical of ALL was no longer visible after coculture with LAK. Evidence for selective tumour cytotoxicity by LAK was confirmed by using autologous BM cells as hot and cold targets in a 51Cr release assay. Finally, successful haematologic reconstitution of lethally irradiated BALB/c mice was obtained using syngeneic BM cocultured with LAK. These results support the investigational use of rIL2 and LAK in the treatment of human leukaemia.

orrigendum to ‘Hematological improvement during iron-chelation herapy in myelodysplastic syndromes: The experience of the “Rete matologica Lombarda”’ [Leuk Res 37 (2013) 1233–1240] lfredo Moltenia,∗, Marta Rivaa, Annamaria Pellizzarib, Lorenza Borinc, lessandra Freyried, Rosa Grecoa, Marta Ubezioe, Massimo Bernardi f, lessio Fariciotti g, Guido Nadorh, Michele Nichelatti i, manuele Ravanoa, Enrica Morraa

Abstract 2877 The Anaplastic Lymphoma Kinase (ALK) gene is fused to several partner genes (mainly NPM) in the majority of Anaplastic Large Cell lymphoma (ALCL) patients (pts). Deregulated ALK tyrosine kinase activity represents the driver alteration in this disease, through the phosphorylation of proteins belonging to signal transduction pathways involved in cellular proliferation, apoptosis and differentiation. Although ALK+ ALCL pts are responsive to cytotoxic drugs, relapses occur frequently and bear a dismal prognosis. Crizotinib is a competitive small-molecule inhibitor of the ALK and c-Met/HGFR receptor tyrosine kinases with cellular IC50 values in NPM-ALK expressing cells comprised between 24 and 60 nM. A dose of 250 mg BID orally was previously established as the recommended dose in a phase I study. We report here on the safety and activity profile of crizotinib in two ALK+ ALCL pts resistant to cytotoxic therapy who received crizotinib 250 mg BID as part of a compassionate use named patient protocol. No steroids or drugs with antineoplastic activity were allowed; potent CYP3A4 inhibitors/inducers were also excluded. Pt # 1 is a 26 year old female who received 7 cycles of CHOP-15 with a partial response that lasted only 1 month. Subsequently she was treated with the DHAP and ICE regimens in an attempt to collect stem cells for an autologous BMT. However, even with these two salvage regimens, the pts relapsed within 2–3 weeks after each one. Pretreatment evaluation included fever (>38 C), cervical and inguinal adenopathies, positive PET and CT scans of para-aortic and iliac adenopathies; a bone marrow (BM) aspirate showed 3% of cells with positivity using an ALK break-apart probe by FISH. Fever disappeared within 48 hours after starting crizotinib; by day 7 all superficial adenopathies were no longer present. PET, CT and BM aspirate performed at day 28 days showed regression of previous lesions persisting so far for 2 months. Side effects included transient ocular flashes and grade I LFT elevation. Pt # 2 is a 21 year old male diagnosed with ALK+ ALCL in August 2009. The pt was treated with 6 cycles of CHOP obtaining a CR that was lost in February 2010. The pt underwent re-induction/mobilization chemotherapy with MAD and received an Autologous Bone Marrow Transplantation in May 2010 after conditioning with the BEAM regimen; he obtained a PR which was lost again after 1 month. Pretreatment evaluation included fever (>39 C, requiring 60 mg prednisone and 3 g paracetamol daily), axillary and inguinal adenopathies, positive PET and CT scans of all internal nodal stations; BM showed 8% of positive cells using an ALK break-apart probe. Within 8 days of treatment constitutional symptoms subsided, all superficial adenopathies disappeared and PET scan shows complete regression of all lesions. Adverse events reported so far include grade I dizziness. Longer follow-up data along with results from a third ALCL patient will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Epigenetic changes have been shown to play a role and to cooperate with genetic alterations in the pathogenesis of myelodysplastic syndromes (MDS). The potential reversibility of DNA and chromatin modifications makes chromatin remodeling enzymes attractive targets for therapeutic intervention in this disease. We conducted a phase II study on the combination of the DNMT inhibitor 5-azacitidine (5-AZA), the histone deacetylase inhibitor valproic acid (VPA), and all-trans retinoic acid (ATRA) in patients with intermediate-2/high-risk myelodysplastic syndromes. Bone marrow morphology was centrally reviewed before enrolment. VPA was given at 600–1500 mg daily to reach a final plasma concentration above 50 microg/ml, then 5-AZA was added at a standard dose of 75 mg/sqm daily, subcutaneously, 7 days for 8 cycles. In case of minor response, stable disease or failure after 4 cycles, ATRA was added at 30 mg/sqm orally daily, on days 8–27 for 4 cycles. Treatment was continued in responding patients until response persisted. The protocol included 62 patients (43 males, 19 females, median age 67 years, range 53–83 yrs). Diagnosis was RAEB for 37 patients (60.7%), RAEB-t for 21 (32.8%), and CMML for 4 patients (6.5%). The IPSS was int-2 (1.5) for 46 patients and High (≥2) for 16 patients. A valproic acid concentration between 45 and 55 microg/ml was reached in a median of 7 days (range 4–28 days). Three patients died before start of treatment, while 58.7% of patients (95% C.I.: 51.3–67.1) were alive at 12 months. Out of 27 patients who completed 8 treatment cycles, 8 patients (29.6%) obtained complete and partial remission, 3 patients (11.1%) major hematological improvement while 10 patients (37.4%) showed a stable disease. Transformation into AML or progression occurred in 20 patients. RBC transfusion needs decreased significantly from a median of 3 units (range 0–16) before start of treatment to 0 (range 0–7) after 8 cycles. Neurological toxicity occurred in 6 patients. Our data show that the 5-AZA/VPA/ATRA combination is safe and feasible in poor prognosis MDS patients.

The clinical usefulness of our assay can be demonstrated by two typical cases: Fig. 1 gives the course of a patient who was investigated three times during CR. While the proportion of leukaemic clones was below 20% during maintenance chemotherapy it had increased to 60% 10 months later during CR. 3 months later, the patient relapsed clinically. In contrast, another patient maintained a proportion of 5-30% of phenotypically leukaemic clones at repeated investigations over a period of nearly 3 years without relapsing clinically (Fig. 2). Our data clearly demonstrate that “complete remission” of adult AML represents a balance of leukaemic and normal hematopoiesis rather than eradication of leukaemia. They argue for the action of mechanisms which suppress the outgrowth of leukaemic progenitor cells in viva. Our in vitro culture system is applicable to all cases of AML and not restricted to certain immunological constellations as the investigation of uncultured bm cells in ALL [5] and much more sensitive than the Southern blot methodology in those cases which have a gene rearrangement as a clonal marker [4, 61. Since it is relatively easy to perform it can be a valuable tool in clinical trials of postremission therapy including alternative approaches (e.g. cytokines like interleukin-2 or autologous bone marrow transplantation) as well as for testing the effectivity of in vitro purging methods.

Background Anemia is a common symptom in patients with Myelodysplastic Syndromes (MDS) and although erythropoietic agents are often active, it is frequently treated with red blood cell (RBC) transfusions. A substantial proportion of patients might also eventually become transfusion-dependent and, Iron-chelating therapies might be important to minimize complications of iron overload. Objectives To investigate the impact of deferasirox therapy on health-related quality of life (HRQOL) of lower risk transfusion-dependent MDS patients over a one year period. Secondary objectives were to investigate relationships between HRQOL and ferritin levels and to explore the prognostic value of baseline HRQOL on the probability of achieving transfusion independence. Patients and Methods This was a prospective study whose clinical findings (i.e., primary endpoint was safety and tolerability) were previously reported. HRQOL was a secondary endpoint of the study and we herein report, for the first time, HRQOL prospective findings. Eligible patients included: MDS patients 18 years or older, International Prognostic Scoring System (IPSS) low or intermediate-1 risk and diagnosed with transfusional siderosis following a minimum of 20 blood transfusions. Patients received daily oral deferasirox at a dose between 10 and 30 mg/kg of body weight for a period of 1 year. HRQOL was assessed with the EORTC QLQ-C30. HRQOL at baseline and at 3, 6, 9 and 12 months after treatment start. The EORTC QLQ-C30 consists of 30 items and includes five functional scales (physical, role, emotional, social, and cognitive), three symptom (fatigue, nausea and vomiting and pain) and a global health status/QOL scale and six single items (dyspnea, insomnia, appetite loss, constipation, diarrhea and financial difficulties). The mean trend of HRQOL over time was estimated via a linear mixed model with a one-step autoregressive covariance structure. Such covariance structure provided the best model fit among those investigated. Results Overall, 159 patients were screened at 37 centers. The median duration of disease at enrollment was 32 months and median number of units of packed RBC received was 37. Seven patients did not start treatment at all and thus there were 152 expected HRQOL forms at baseline assessment. Out of these, 146 patients returned the questionnaire yielding a baseline compliance of 96%. No statistically significant differences over time were found for any scale of the EORTC QLQ-C30. Figure 1 depicts mean scores over time for selected scales of: fatigue, physical functioning, pain and global HRQOL. No HRQOL differences were found between patients with serum ferritin levels lower or higher than 2000 μg/L (pretreatment median value) at baseline. Also, the possible impact of ferritin level on HRQoL over time was estimated via a linear mixed model with a one-step autoregressive covariance structure. Coefficients and p values are reported in table 1. The prognostic impact of baseline HRQOL on the probability of achieving transfusion independence (i.e., defined as freedom from transfusion for 3 consecutive months) was investigated. Higher severity of pain (P=0.007) was associated with a greater likelihood of achieving transfusion independence. Multivariate analysis, controlling for age, IPSS risk score, time from diagnosis, number of previous blood transfusions and baseline ferritin level confirmed the independent value of pain (P=0.003). Conclusion Current findings suggest that Deferasirox therapy does not decrease HRQOL in lower risk transfusion-dependent MDS patients. Patients with higher baseline pain severity seems more likely to achieve transfusion independence and further analysis is needed to understand underlying reasons. Disclosures: No relevant conflicts of interest to declare.

Summary The NK and LAK activity of peripheral blood lymphocytes of leukemic patients as well as the susceptibility of their acute myeloid (AML) and lymphoblastic (ALL) leukemia cells to autologous and allogeneic LAKs were examined. In addition, neoplastic cells at diagnosis and at relapse were compared in the same patients for several features, including in vitro susceptibility to LAKs and to the drugs used in the induction phase, expression of MDR phenotype and of adhesion molecules, and differentiation markers. The NK activity of patients' LAK cells on K562 was significantly lower than that of a group of healthy donors whereas no differences were found in LAK activity as evaluated on Daudi cells. Three of 5 AML and 3 of 4 ALL were significantly more susceptible to autologous and allogeneic LAK lysis when blasts obtained at relapse were compared with leukemic cells of the same patients at diagnosis. This different lysability was not associated with in vitro modified sensitivity to drugs used in induction treatment. Moreover, no elevation in the expression of the multidrug-resistance (MDR)-related P170 glycoprotein was noted in relapsing leukemic cells. Even the expression of adhesion molecules and differentiation markers did not correlate with lysability of leukemic cells. These data demonstrate that relapsing leukemic blasts can be significantly lysed by LAK cells and suggest a rationale for adoptive immunotherapy with IL-2 and LAK cells in the treatment of acute leukemic patients.