ALBERT

Description: Abstract 4941 Since the multidrug resistance phenotype was first observed 30 years ago, other 48 proteins, belonging to an evolutionary highly conserved family of transport proteins, named ABC proteins, has been identified and grouped in eight subclasses, according to divergent evolution. In eukaryotes, ABC proteins are involved in transport across the membrane to extracellular space or into intracellular organelles playing an important role in cell physiology. Almost three distinct subfamilies (B, C and G) have been recognized to transport a wide variety of amphipatic or hydrophobic molecules, including most of anticancer drugs compounds, affecting drug absorption, disposition, metabolism, excretion and toxicity (ADMEtox) and has been associated to chemotherapy failure in solid and hematologic neoplasia. In particular the role of ABCG2 has been pinpointed over the past years for its high expression on primitive hematopoietic and on progenitors cells and for its ability to confer to the cancer cell the properties of cancer stem cell, with increased survival capacity. At present over 40 single nucleotide polymorphisms of ABCG2 protein have been identified. Among them the 421C〉A (Q141K) variant has been shown able to alter protein function and to modify in vitro sensitivity to many anticancer drugs, compared to the wild type protein. In the present paper we have evaluated the incidence and the impact on therapy outcome in a series of 100 caucasic patients with acute myeloid leukemia, receiving the same chemotherapy program including idarubicin. Q141K polymorphism was detected in 19/100 (19%) patients, without correlation with any clinical-biological characteristic at diagnosis (such as sex, age, peripheral blast count, FAB subtype, karyotytpe, CD34 expression). ABCG2 protein was overexpressed in 10/19 (52.6%) of mutated patients and in 37/83 (44.5%) of patients expressing the wt form of ABCG2 and no difference in intensity of ABCG2 expression was observed in the two groups. Again no difference in ABCG2 mRNA transcription was detected between patients carrying wt or polymorphic protein. Complete remission rate was comparable in wt and mutated patients (38/81, 45,7% vs 10/19, 52,6%). However patients with Q141K polymorphism (irrespective to the intensity of expression of ABCG2) protein) had a leukemia free survival comparable to patients overxpressing wt protein, and significantly shorter than patients without wt ABCG2 overepxression (chi square 12,2, p=0,002, logrank test). In conclusion our data demonstrated Q141K polymorphism of ABCG2 transporter protein identified a subset of patients with high probability of relapse when treated with idarubicin suggesting that other drugs should be considered in consolidation/maintenance treatment to improve patients outcome. Finally the impact of Q141K polymorphism on treatment toxicity is under investigation. Work supported by PRIN 2007WEYB34-004 Disclosures: No relevant conflicts of interest to declare.

Description: The aims of our study were to verify whether it was possible to generate in vitro, from different adult human tissues, a population of cells that behaved, in culture, as multipotent stem cells and if these latter shared common properties. To this purpose, we grew and cloned finite cell lines obtained from adult human liver, heart, and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs, obtained from the 3 different tissues, expressed the pluripotent state–specific transcription factors Oct-4, NANOG, and REX1, displayed telomerase activity, and exhibited a wide range of differentiation potential, as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content, and shared a common gene expression signature, compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular, the pathways regulating stem cell self-renewal/maintenance, such as Wnt, Hedgehog, and Notch, were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and functional features of mature cells even embryologically not related to the tissue of origin.

Description: Abstract 4848 Objectives: the prognosis of patients with cytogenetically normal acute myeloid leukemia (CN-AML) is highly variable and can be influenced by several clinical and biological variables. Nevertheless, some biological data may be conflicting and difficult to combine with the clinical ones. Methods: in order to propose a simple scoring system, we retrospectively analysed the clinical data of 337 patients newly diagnosed with CN-AMLs, aged less than 65 years, consecutively treated in eleven hematological Italian Centres from 1990 to 2005. Two hundred nineteen patients (65%) received a fludarabine-based induction regimen. All the other patients received a conventional induction regimen, including cytarabine, one anthracycline with or without etoposide. Univariate and multivariate analysis on event free survival and overall survival (EFS and OS) were performed. Patients addressed to allogeneic stem cell transplantation were censored at the time of transplant. Factors found to be significant in univariate analysis were tested in multivariate analysis. A numerical score was derived from the regression coefficients of each independent prognostic variable. The Prognostic Index Score (PIS) for each patient was then calculated by totalling up the score of each independent variable. Patients could thus be stratified into low-risk (score = 0–1), intermediate-risk (score = 2) and high-risk group (score grater than 3). The score obtained in this group of patients (training set) was then tested on 193 patients with newly diagnosed with CN-AMLs, aged less than 65 years, enrolled in the GIMEMA LAM99p clinical trial (validation set). Results: the clinical variables that were independent prognostic factors on EFS in the training set of patients were: age 〉 50 yrs (regression coefficient: 0.39, HR 1.5, score = 1), secondary AML (regression coefficient: 0.90, HR 2.5, score = 2) and WBC 〉 20 × 10^9/L (regression coefficient: 0.83, HR 2.3, score = 2). For what concerns the OS, the same variables showed the followings statistical data: age 〉 50 yrs (regression coefficient: 0.48, HR 1.6, score = 1), secondary AML (regression coefficient: 0.99, HR 2.7, score = 2) and WBC 〉 20 × 10^ 9/L (regression coefficient: 0.87, HR 2.4, score = 2). In the training set of patients, the median EFS was 22, 12 and 8 months in the low, intermediate and high-risk group (p

Description: Introduction Evolution to myelofibrosis (MF) represents a relatively rare but always severe event in patients with essential thrombocythemia (ET) and polycythemia vera (PV). Few reports have focused on the clinical and biological features at diagnosis of ET and PV that correlate with progression to MF. Aims and Methods We retrospectively studied a series of patients with post-ET and post-PV MF and compared with a group of ET and PV patients with a long follow-up without myelofibrotic evolution, with the aim to identify prognostic factors for MF. Forty-three patients with post-ET (n=29) and post-PV (n=14) MF followed at our institution were compared with 125 ET and 75 PV patients with at least 9 years of follow-up without evolution. Diagnosis of ET and PV was confirmed according to WHO criteria (including JAK2 analysis, performed since 2006 and study), evolution to MF was defined according to IWG-MRT proposed criteria. The following parameters, available for all patients at diagnosis of ET or ET, were taken into consideration to find prognostic risk factors for myelofibrosis: age, platelet (PLT) count, hemoglobin (Hb) and hematocrit (Hct) levels, white blood cell (WBC) count. Statistical analyses were conducted using Student t test. Results Median time from diagnosis of ET/PV and progression to MF was 156 months (range: 29-314). Comparing baseline characteristics of patients who evolved to MF and those who did not, we did not found any significant correlation. Mean data at diagnosis for patients with (n = 43) or without (n=200) subsequent evolution to MF were as follow: age 52.1 vs 53.1 years (p=0.79), Hb 15.4 vs 15.7 g/dl (p=0.59), Hct 47.2 vs 47.1% (p=0.67), WBC 9.8 vs 9.1 x 109/l (p=0.11), PLT 713 vs 689 x 109/l (p=0.87). Also when considering only the 29 post-ET MF and the 125 ET patients, there was no clinical feature present at diagnosis that could foresee a future myelofibrotic evolution. Conversely, in the 14 post-PV MF and 75 PV patients, progression to MF was predicted only by higher WBC count (11.4 vs 9.3 x 109/l, p=0.046), while no correlation was found with age, Hb, Hct or PLT [Table 1]. Conclusions Concordant with some previous reports, our data suggest a possible role of leucocytosis as an adverse risk factor for progression to MF in patients with PV, though not in ET. Other clinical characteristics present at diagnosis, such as advanced age, anemia or polycythemia and thrombocytosis do not seem to be associated with higher risk of fibrotic evolution in patients with myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

Description: Background .Chronic GVHD (cGVHD) is the most common and severe late complication after allogeneic stem cell transplantation (SCT), because of increasing use of matched unrelated donors, peripheral blood stem cells and reduced intensity conditioning (RIC). Impaired immune reconstitution and unbalanced Th1-type or Th2-type immune response with cytokines dysregulation seem to play a key role in the pathogenesis of chronic GVHD (cGVHD). Aims. In order to study the immunologic mechanisms involved in cGVHD, we prospectively evaluated the Th1 (TNF-alpha, IFN-gamma) and Th2 (IL-4, IL-6, IL-10) cytokine patterns and lymphocyte subsets in the setting of allogeneic SCT-RIC. Patients and methods. We assessed by ELISA the serum levels of TNF-alpha, IFN-gamma, IL-4, IL-6, IL-10 and soluble tumor necrosis factor receptors I and II (sTNF-R) in 8 healthy donors and in 19 patients undergoing allogeneic SCT-RIC (13 patients with related and 6 patients with unrelated donor). Serum levels were assessed before transplantation and monthly from second to twelfth month after SCT. Total lymphocytes and their subsets with different co-stimulatory molecules (CD4, CD8, CD19, CD28/3,CD25/4, CD134/4, CD152/3, CD16/56, CD4/45ro, CD4/45ra, CD8/45ro, CD8/45ra) were evaluated by flow cytometry in peripheral blood (PB) monthly after SCT. Cyclosporine A was used as GVHD prophylaxis in all patients from third month until its tapering or until the beginning of the therapy against extensive cGVHD. Wilcoxon test was used for statistical analysis of the data. Results. Twelve out of 19 patients developed cGVHD at a median time of 7 months (range, 6–10); cGVHD was extensive in 8 patients at a median time of 8 months (range, 6–12). The levels of cytokines did not differ significantly in patients pre-SCT and healthy donors. Patients with cGVHD differed from those without cGVHD because of: significantly higher levels of TNF-alpha from third to sixth month after SCT (3rd, p=0.003; 4th p=0.001; 5th, p=0.0005; 6th, p=0.01); significantly higher levels of sTNF-R II at 6th (p=0.01); significantly higher levels of IL-10 (p= 0.004) at 4th month; significantly lower number of NK cells in PB from third to sixth month after SCT (3rd, p=0.004; 4th p=0.009; 5th, p=0.0007; 6th, p=0.0003); significantly lower number of CD 152/3 cells in PB from third to sixth month after SCT (3rd, p=0.009; 4th p=0.0004; 5th, p=0.0004; 6th, p=0.007); significantly lower number of CD 4/25 cells in PB at 4th (p=0.0004) and 5th (p=0.01) Discussion. Our sequential study showed: increased levels of Th1 (TNF-alpha) and Th2 (IL-10) cytokines with different kinetics after SCT and before the onset of cGVHD; a decrease of NK and T cells with regulatory molecules such as CD152 after SCT and before the onset of cGVHD. These results suggest an overall prevalence of a TNF-alpha oriented response in patients with cGVHD. Defects of immunoregulatory cells could be related to these fluctuating and unbalanced cytokine patterns. However, further studies with more patients are required to support these preliminary results.

Description: Abstract 967 Human genome sequence variation, in the form of single nucleotide polymorphisms (SNPs) as well as more complex structural variation (such as insertions, duplications and deletions) in drug-metabolizing enzyme and transporter genes (DMETs), affects each individual's response to anticancer agents and thus the likelihood of experiencing an adverse drug reaction. Therefore, understanding relationships between genetic variation and differential drug responses is a challenge to improve both the safety and efficacy of drugs. Accordingly, in order to investigate a potential association between SNPs and clinical response and toxicity, we comprehensively assessed the allele frequencies of 1931 genetic variations of 225 absorption, distribution, metabolism and excretion genes in 59 AML patients younger than 65 years, using the new Affymetrix drug-metabolizing enzyme and transport (DMET Plus) genotyping platform (Affymetrix, Inc. Santa Clara, CA, USA). All patients were enrolled in a phase III multicenter clinical trial combining low dose of Gemtuzumab-ozogamicin (GO) with FLAI regimen (Fludarabine, Cytarabine, Idarubicin) as Induction chemotherapy i (eudract: 2007–005248-26; ClinicalTrials.gov NCT00909168). The induction regimen (GO-FLAI) included fludarabine (25 mg/sqm) and Ara-C (2 g/sqm) on days 1–5, idarubicin (10 mg/sqm) on days 1, 3, and 5 and GO (3 mg/sqm) on day 6. Hematopoietic stem cell transplant was planned for all high risk AML patients in first complete remission (CR) after consolidation with intermediate doses of Ara-C and idarubicin (ID-AC and IDA). Cytogenetic, multidrug-resistance phenotype, FLT3 and NPM mutation status, WT1 quantitative expression analyses, were performed at diagnosis in all patients. Furthermore, high-resolution SNP array analysis by GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0 (Affymetrix) was also performed. DNA processing and genotype identifications were performed according to the Affymetrix DMET platform's instructions. All statistical analyses were performed using the R package 2.11.1. Allele frequencies estimation and principal component analyses (PCAs) were performed using the adegenet library, whereas an ad hoc R function was developed to compute two-side Fisher's exact test p-values for each SNP in order to test their association with clinical response and toxicity.Results of potential interest were limited to those in which the p-value was

Description: In the last year, the issue of cardiotoxicity of imatinib mesylate (IM) was on focus. Emerging data seem to deny an increased risk of cardiac events in patients treated with IM, which is the frontline therapy in chronic myeloid leukemia (CML). B-type natriuretic peptide (BNP) is released by the heart in response to myocardial tension and is considered an accurate test for the diagnosis of heart failure. The measurement of BNP in the serum is a rapid and easy tool for evaluation of ventricular function, also in asymptomatic patients. We have measured BNP level in 50 consecutive patients (33 males and 17 females) with IM treated chronic phase CML. Patients were enrolled, after informed consent was obtained, as they presented for a follow-up visit at our Institution. BNP was measured using a direct chemiluminescent sandwich immunoassay: the analytical range extends from 0 to 5000 pg/ml, with a sensitivity 22 pg/ml, compared to only 1/25 (4%) in the cohort

Description: The inhibition of the c-Kit signal transduction pathway by imatinib mesylate (STI571) and the inhibition of the post-translational modification of the N-K Ras proteins by farnesyl transferase inhibitors (FTIs) have shown potential efficacy in treating acute myeloid leukemias. We investigated the activity of (STI571) and of two distinct FTIs, R115777 and SCH66336, on two pairs of acute leukemia (AL) human tumor cell lines, each pair consisting of the parental cell line, HL60 and CCRF-CEM, and of its drug-selected multidrug resistant (MDR) Pgp-positive subline, HL60-DNR and CEM-VLB. The effectiveness of STI571, R115777 and SCH66336 in inhibiting cell proliferation of AL cell lines have been evaluated by colorimetric MTT assay. Cell growth was evaluated after a 7-day incubation at 37°C and 5% CO2 by using 50 microl per well of the MTT solution (5 mg/mL). The inhibition dose 50 (ID50) was defined as the drug dose that inhibited cell growth to 50% of the control. The results were correlated with the MDR phenotype and c-Kit expression. The toxic effect of STI 571 on all the acute leukemias derived cell lines was very low for both parental and MDR-Pgp+ sublines, ranging from 0.9 and more than 5 microM. STI571 activity was influenced by the c-Kit (CD117) expression and not by the MDR expression of cell lines. In fact, among these acute leukemia derived cell lines the higher toxic effect (ID50=0.9 microM) was obtained in HL60 DNR, the only cell line with a CD117 positivity. R115777 was more toxic than SCH66336 on all the tested AL cell lines. The Inhibition Dose 50 (ID50) of the two farnesyl transferase inhibitors ranged from 0.0065 to 0.21 microM for R115777 and from 0.3 to 6.5 microM for SCH66336. These results suggest that a potential synergistic effects of STI571 and R155777 combination could be explored in c-Kit positive acute myeloid leukemias.

Description: Although a large amount of data is available on the effects of filgrastim (granulocyte colony-stimulating factor [G-CSF]) on the mobilization of stem cells in the circulation, data concerning its effects on bone marrow (BM) harvesting is scarce and controversial. We have designed a randomized trial comparing filgrastim-mobilized peripheral blood stem cell (PBSC) transplantation with filgrastim-primed autologous bone marrow transplantation (ABMT). Fifty-five patients affected by non-Hodgkin's (n = 38) or Hodgkin's (n = 17) lymphoma, selected for autologous transplantation over a 12-month period in a single institution, were randomized 2:1 to undergo BM or PB harvest/collection after priming for 3 days with filgrastim, 16 μg/kg body weight daily subcutaneously. BM priming with G-CSF allowed the harvest of a significantly higher number of mononuclear cells (MNC) (0.53 × 108/kg, range, 0.32 to 1.40), as compared with a historical control of unprimed BM harvests (0.43 × 108 MNC/kg, range, 0.15 to 0.72, P = .001). After high-dose ablative therapy, median time to neutrophil recovery above 0.5 × 109/L was 12 days for BM and 11 days for PB (P = .219); median time to platelet recovery above 20 × 109/L was 13 days for BM and 11 days for PB (P = .242). The same number of red blood cells, platelet transfusions, and posttransplant G-CSF doses were required in the two groups of patients. Less patients (50% v 70%) became febrile in the group transplanted with mobilized PB, but days of fever/patient and days on antibiotics were overlapping. The median time spent in the hospital after reinfusion was 16.5 and 15.5 days after primed BM and primed PB, respectively (P = .134). These data suggest that in patients with lymphoma submitted to autologous transplantation, the reinfusion of filgrastim-primed BM or filgrastim-mobilized PB leads to similar results, with an advantage of only 1 day in the neutrophil recovery and 1 day on the time spent in the hospital in favor of primed PB. Either option can be chosen on the basis of the availability of a surgery room or cell separator facilities and considering the patients' characteristics and wishes.

Description: INTRODUCTION. The addition of Gemtuzumab-ozogamicin (GO) to an induction regimen including synergistic drugs, such as intermediate dose of cytarabine (Ara-C), idarubicin and fludarabine (FLAI), could reduce treatment failure in AML patients. Nevertheless, the role and safety of this antibody target-therapy in first-line chemotherapy in patients younger than 65 years has not yet been defined. PATIENTS and METHODS. The primary goal of this multicenter prospective trial (EUDRACT Number 2007-005248-26) was to evaluate the efficacy and the safety profile of FLAI plus GO as induction regimen. Sixty-eight consecutive AML patients were included from five Italian hematological centres. All patients were younger than 65 with a median age of 48 years and CD33 expression exceeded 20% in all cases. The M/F ratio was 37/31, and 54/68 (79%) of patients were poor-risk at diagnosis. The induction regimen (FLAI-GO) included fludarabine (30 mg/sqm) and Ara-C (2 g/sqm) on days 1–5, idarubicin (10 mg/sqm) on days 1, 3, and 5 and GO (3 mg/sqm) on day 6. Hematopoietic stem cell transplant (HSCT) was planned for all high risk AML patients in first complete remission (CR) after consolidation with intermediate doses of Ara-C and idarubicin (IDAC-IDA). Cytogenetic, multidrug-resistance phenotype, FLT3 mutation status, and WT1 quantitative expression analyses were performed at diagnosis in all patients. WT1 expression and cytogenetic (in positive cases) analyses were performed after induction to detect and follow Minimal Residual Disease. RESULTS. Patients were evaluated for response rate, treatment-related adverse events, overall survival and relapse free survival. After induction with FLAI-GO, CR rate was 86% (54 of 63 evaluable pts); one patient achieved partial remission and eight were resistant. There were only two cases of death during induction (DDI). After FLAI-GO, the mean value of WT1 dropped from 4540±2342 copies/104ABL to 180±277 copies/104ABL. The toxicity of FLAI-GO was acceptable; 58% of patients experienced transient and reversible GO infusion-related adverse events (especially fever and chills), but no cases of veno-occlusive disease occurred during CHT or after HSCT. After a median follow-up of 9 months (range 1–32), 60/68 (88%) patients are alive (57/60 in CR). The probability of 12 and 18 mths OS a was 91% and 78%, respectively. The probability of 12 and 18 mths RFS was 87 % and 78%, respectively. Allogeneic and autologus HSCT was performed in 30 (44%) and 8 (12%) patients. CONCLUSIONS. The preliminary results of this multicentric trial confirm that FLAI-GO is an effective and well tolerated induction regimen for CD33 positive AML patients younger than 65 years, with a high complete response rate, favourable safety profile and low DDI.