Search

Dasatinib and nilotinib are tyrosine kinase inhibitors (TKIs) developed to overcome imatinib resistance in Philadelphia-positive leukemias. To assess how Bcr-Abl kinase domain mutation status evolves during sequential therapy with these TKIs and which mutations may further develop and impair their efficacy, we monitored the mutation status of 95 imatinib-resistant patients before and during treatment with dasatinib and/or nilotinib as second or third TKI. We found that 83% of cases of relapse after an initial response are associated with emergence of newly acquired mutations. However, the spectra of mutants conferring resistance to dasatinib or nilotinib are small and nonoverlapping, except for T315I. Patients already harboring mutations had higher likelihood of relapse associated with development of further mutations compared with patients who did not harbor mutations (23 of 51 vs 8 of 44, respectively, for patients who relapsed on second TKI; 13 of 20 vs 1 of 6, respectively, for patients who relapsed on third TKI).

Resistance to imatinib in Philadelphia-positive (Ph+) leukemia patients is often associated with selection of point mutations in the Bcr-Abl kinase domain (KD). Dasatinib and nilotinib are second-generation tyrosine kinase inhibitors (TKIs) with different binding modes with respect to imatinib, that have been shown to confer in vitro and in vivo activity against many Bcr-Abl mutated forms. However, both dasatinib and nilotinib have been shown to retain some ‘Achilles heels’, and they include both imatinib-resistant mutations (e.g., T315I) and some novel, inhibitor-specific ones. Selection of either type of KD mutations has frequently been observed in patients (pts) who relapse after an initial response to dasatinib or nilotinib and represents one of the major hurdles on the road to successful treatment of imatinib-resistant pts. We have monitored Abl KD mutation status in a total of 121 pts who received dasatinib (n= 78) or nilotinib (n=43) as 2nd TKI after imatinib failure since February 2005. Fifty-eight (48%) pts had chronic phase (CP) chronic myelogenous leukemia (CML), 63 pts (52%) had accelerated phase (AP) or blast crisis (BC) CML or Ph+ acute lymphoblastic leukemia (ALL). Median age was 55 years (range, 18–76); median time from diagnosis was 49 months (range, 4–181); median time on imatinib was 32 months (range, 4–66). Median follow-up of all pts who received a 2nd TKI is 7 months (range, 1–38). Median follow-up of pts who are still on 2nd TKI treatment is 32 months (range, 28–38). Relapses after an initial response have so far been observed in 46/121 pts. Thirty-eight out of these 46 pts had AP/BC CML or Ph+ ALL at the time 2nd TKI was started. Forty-one out of 121 (34%) pts have experienced relapse after an initial response during the first 12 months of 2nd TKI treatment (median time to relapse, 6,5 months; range 4–12 months), while only five of the 45 (11%) pts who were still on 2nd TKI treatment after >12 months have relapsed (at 13, 15, 18, 20 and 33 months, respectively). Interestingly, none of these 5 pts had never achieved more than a minor cytogenetic response (CgR), and 4/5 pts were receiving a reduced TKI dose because of toxicity. In 36/46 (78%) cases, relapse was associated with newly acquired Abl KD mutations. In particular 26/30 (87%) pts who relapsed on dasatinib and 10/16 (63%) pts who relapsed on nilotinib had evidence of a newly acquired KD mutation presumably responsible for treatment failure. Newly acquired mutations in pts who relapsed on dasatinib as 2nd TKI were T315I (n= 12 pts) F317L (n= 8 pts) T315A (n=3 pts); V299L (n=3 pts); F317I (n=2 pts); 2 pts had multiple mutations. Newly acquired mutations in pts who relapsed on nilotinib as 2nd TKI were E255K (n=3); E255V (n=2); Y253H (n=2); T315I (n=1); F359V (n=1); F359C (n=1). Sixteen pts (but none of those harboring the T315I) switched to dasatinib or nilotinib or high-dose imatinib as 3rd TKI and this rescued hematologic or even cytogenetic responses in a proportion of cases. Our observations suggest that: newly acquired mutations leading to relapse in Ph+ leukemia pts receiving dasatinib or nilotinib as 2nd TKI usually arise rapidly; the likelihood of mutation selection consistently decreases over time, and seems mainly confined to advanced phase pts and to pts with no or minor CgR; almost all (87%) cases who developed resistance to dasatinib had newly acquired KD mutations - suggesting that the higher potency with respect to imatinib can overcome Bcr-Abl gene amplification and that Src kinase inhibition may turn off Bcr- Abl-independent resistance mechanisms; a lower incidence (63%) of newly acquired KD mutations was observed in pts who developed resistance to nilotinib; with the exception of T315I, there is little if no overlap between dasatinib and nilotinib-resistant mutants, which may allow to regain responses by switching TKIs.

5-azacytidine (AZA) significantly prolonged overall survival in higher-risk patients with myelodysplastic syndromes (MDS) in a large, international, randomized, phase III trial (AZA-001). However, data about efficacy and safety of AZA in lower risk MDS are less consistent and only few small studies have addressed this topic. Among a total of 246 MDS treated with AZA in 31 different Italian Institutions since 2005 within to a national patient named program, we evaluated 82 patients scored as low/int-1 IPSS risk MDS. Median age was 68 years (range 34–85), male/female ratio 50/32. According to WHO classification, there were 21 RA/RARS, 4 5q-syndromes, 20 RCMD, 24 RAEB-1, 5 RAEB-2, 4 CMMoL, and 4 MDS unclassified. Median time from diagnosis was 27 months (range 1–132). Sixty-eight patients (82.9%) were transfusion-dependent, sixty (74%) had received a prior treatment, mostly with erythropoiesis stimulating agents. AZA was administered as single drug in 61 patients (74.4%), while in the remaining subjects it was variously combined with growth factors, valproic acid or other agents. Forty-eight patients (58.5%) received a “standard” AZA dose of 75 mg/sqm/d s.c., thirty-four (41.5%) a fixed dose of 100 mg/d s.c. Single cycle treatment duration was 7 days in 45 patients (54.9%), < 7 days in 32 patients (39%), > 7 days in 3 patients (3.7%), unknown in 2 patients (2.4%). The median number of monthly cycles was 6 (range 1–21), and 63 patients (76.8%) completed at least 4 cycles. The most relevant toxicities observed (grade 3–4) were represented by myelosuppression (22%) and infections (6%). According to 2006-updated IWG criteria, overall response rate was 39% (47.5% in patients who had completed at least 4 cycles). In particular, complete response, partial response and hematological improvement occurred in 12.2%, 8.5% and 18.3% of patients (15.8%, 11.1% and 20.6% in those who were treated with at least 4 cycles), respectively. Stable or progressive disease was observed in 29.3%/25.6% and 30.2%/22.2% of patients receiving less than or at least 4 cycles, respectively. Response duration ranged from 1 to +21 months. There were no significant differences in response rate according to dose and schedule employed, although a slight trend in favour of 75 mg/sqm vs 100 mg fixed dose was seen (45.8% vs 29.4%, respectively). There was also no difference in the percentages of response according to age, previous treatment and transfusion dependence. Overall survival at 2 years was 62%. A survival benefit emerged for responding patients, compared to non responders (82% vs 57%) (p=0.015). A favourable trend was also observed for transfusion-independent patients, while age, pre-treatment and AZA dose did not influence survival. These data indicate that AZA may be safe and effective for a subset of patients with low/int-1 IPSS risk MDS, resistant or not suitable for alternative treatments. The efficacy may improve if at least 4 cycles are administered.

We analysed the in vitro effects of a new hydroxamate derivative, ITF2357, on AML cells. ITF2357 potently induced histone acetylation. ITF2357 0.1 microM blocked proliferation and induced apoptosis in AML1/ETO-positive Kasumi-1 cells, while AML1/ETO-negative HL60, THP1 and NB4 cell lines were sensitive only to 1 microM ITF2357. Apoptosis was induced by 0.1 microM ITF2357 in AML1/ETO-positive primary blasts and U937-A/E cells induced to express AML1/ETO, but not in U937-A/E cells non-expressing AML1/ETO. In Kasumi-1 cells 0.1 microM ITF2357 induced AML1/ETO degradation through a caspase-dependent mechanism. ITF2357 0.1 microM also determined DNMT1 efflux from, and p300 influx to, the nucleus. Moreover, 0.1 microM ITF2357 determined local H4 acetylation and release of DNMT1, HDAC1 and AML1/ETO, paralleled by recruitment of p300 to the IL-3 gene promoter. ITF2357 treatment, however, did not induce re-expression of IL-3 gene. Accordingly, the methylation level of IL-3 promoter, as well as of several other genes, was unmodified. In conclusion, ITF2357 emerged as an anti-leukaemic agent very potent on AML cells, and on AML1/ETO-positive cells in particular. More relevantly, clearly emerged from our results that ITF2357 could be an ideal agent to treat AML subtypes presenting AML1/ETO fusion protein which determine HDAC involvement in leukaemogenesis.

Background: The risk of venous thromboembolism (VTE) is high in newly diagnosed myeloma (MM) patients who receive thalidomide-containing regimens. Anticoagulant prophylaxis is recommended but it’s not clear which is more appropriate. In this prospective, multicenter phase III trial we evaluated the safety and the efficacy of low-molecular weight heparin (LMWH) or low-dose aspirin (ASA) or low-fixed dose warfarin (WAR) as anticoagulant prophylaxis. Methods: In a GIMEMA study, newly diagnosed MM patients were randomized to VTD (Velcade 1.3 mg/m² d 1,4,8,11; Thalidomide 200 mg/d; high-dose Dexamethasone 320 mg/21 d) or TD (Thalidomide 200 mg/d; high-dose Dexamethasone 320 mg/21 d) or VMPT (Velcade 1.3 mg/m² d 1,8,15,22; Melphalan 9 mg/m² d 1–4; Prednisone 60 mg/m² d 1–4; Talidomide 50 mg/d) or VMP (Velcade 1.3 mg/m² d 1,8,15,22; Melphalan 9 mg/m² d 1–4; Prednisone 60 mg/m² d 1–4). In a sub-study, patients treated with VTD or TD or VMPT were randomly assigned to receive LMWH (Enoxaparin 40 mg/d) or ASA (Aspirin 100 mg/d) or WAR (Warfarin 1.25 mg/d) for the duration of the induction therapy. Patients treated with VMP did not receive any prophylaxis and were used as controls. End-points were incidence of VTE, acute cardiovascular events, sudden death, bleeding and any other serious adverse events. A total of 950 patients will be included in this study. An interim analysis was performed after the first 200 patients were enrolled. Results: Eighty-two patients received VTD, 84 TD, 34 VMPT and 35 VMP. Two-hundred patients (117 males, median age 58 years) were analyzed: 65 patients were randomized to LMWH, 66 to ASA and 69 to WAR. Patient characteristics were similar in all groups. All patients completed at least the first 3 cycles of therapy. The incidence of VTE was 2/65 (3%) in the LMWH group, 6/66 (9%) in the ASA group and 2/69 (3%) in the WAR group, but differences did not reach statistical significance. VTEs were 2/35 (6%) in the VMP group who did not received any prophylaxis. The cumulative incidence of VTE was 4/116 (3%) in patients treated with Velcade plus Thalidomide, and 6/84 (7%) in those treated with TD (p=0.33). No acute cardiovascular events or sudden deaths were reported. The incidence of bleeding was 0/65 (0%) in the LMWH group, 2/66 (3%) in the ASA group and 2/69 (3%) in the WAR group. Conclusion: The overall incidence of VTE was less than 10% in all groups. ASA patients had higher frequency of VTE; LMWH patients had lower risk of bleeding; patients who received Velcade had lower frequency of VTE. An update of these data and an analysis of risk factors will be presented at the meeting.

We recently showed that the histone deacetylase inhibitor D1 induced apoptosis in the t(8;21) Kasumi 1 acute myeloid leukaemia (AML) cell line and activated caspase 9. The present study characterised the effects of the combined administration of D1 with PD98059, SB203580 or SP600125, specific inhibitors of mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 or Jun N-terminal kinase (JNK), respectively. Among these inhibitors, SP600125 was the only one to markedly induce apoptosis and decrease cell proliferation. These experiments showed that SP600125 activated caspase 8 and confirmed that D1 activated the intrinsic pathway of apoptosis, as caspase 8 was not affected while Bcl-2 was down-regulated following D1 administration. The combination of the two drugs enhanced caspase-8 activation and induced apoptosis in an additive fashion. JNK was constitutively activated in the Kasumi 1, NB4, HL60 and THP-1 human AML cell lines, as well as in primary blasts from a t(8;21) AML patient. In all these cells, the pro-apoptotic effect of the two drugs alone was increased when they were combined. On this basis, the combined administration of D1 with SP600125 seems to be very promising as a potential anti-leukaemic tool in AML.

Background. Nilotinib (NIL) is a potent and selective BCR-ABL inhibitor approved for the frontline treatment of chronic myeloid leukemia (CML) based on the results of the ENESTnd study. The sustained superiority of NIL vs. imatinib (IM) was confirmed after 5 years of follow-up (Hughes et al, abs. 677, EHA 2014). However, few data are available on patients (pts) treated frontline with NIL outside of Company-initiated trials. Objectives. To analyze the long-term outcome in a large, independent cohort of newly diagnosed CML pts treated frontline with NIL-based regimens. Methods. We analyzed 215 pts, enrolled in 2 multicenter phase II studies conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00481052 and NCT00769327) or treated at the Bologna University Hospital, with NIL 300 mg or 400 mg BID as initial treatment; 123 pts received a sequential treatment with NIL and IM (NIL-IM), with a 3-months (mos) rotation period (all patients received NIL in the first 3 mos). The median age was 53 years (range 18–86). Ten out of 215 pts (5%) had a high EUTOS score. The median follow-up was 57 mos (range 36–81 mos). We assessed: the rates of Complete Cytogenetic Response (CCyR) and Major Molecular Response (MMR); the rates of optimal responders at each milestone according to ELN 2013 recommendations; the overall survival (OS; any death included), progression-free survival (PFS; progression to accelerated/blast phase [AP/BP] and deaths for any cause), failure-free survival (FFS; failures according to ELN 2013 recommendations and deaths for any cause), and event-free survival (EFS; events: failures, permanent discontinuation of NIL for any cause, including deaths). All analysis was made according to the intention-to-treat principle. Results. The cumulative rates of CCyR and MMR were 93% and 88%, respectively. At 3 mos, 82% of the pts were in Partial Cytogenetic Response and 90% had a BCR-ABL/ABL (IS) < 10%; at 6 mos, 86% were in CCyR and 83% had a BCR-ABL/ABL (IS) < 1%; at 12 mos, 72% were in MMR; all these pts were optimal responders according to ELN 2013 recommendations. Overall, 80 (37%) pts permanently discontinued NIL: 45 (21%) for adverse events or intolerance; 25 (12%) for failures; 7 (3%) while in stable MR4; 3 (1%) for other reasons. Cardiovascular adverse events (CVAE) were cause of permanent NIL discontinuation, after a median time of 37 mos, in 13 (6%) pts, and included 4 peripheral arterial occlusive diseases and 3 ischemic coronary diseases; only one pt died for CVAE. Nine (4.1%) pts progressed to AP/BP, 8/9 during the 1st year of therapy and one after 25 mos; all pts subsequently died (after a median of 13 mos, range 1-34 mos). NIL-resistant mutations were identified in 6 of these pts (4 T315I; 1 Y253H; 1 F359V); 7/9 progressions occurred in patients receiving NIL-IM. In addition, 6 pts were classified as failures at 3,6, or 12 mos according to ELN 2013 recommendations; afterwards, 10 pts developed a secondary resistance (3 loss of CHR, 3 loss of CCyR, and 4 confirmed loss of MMR). Overall, 17 (8%) pts died, in 7 cases for reasons unrelated to CML progression. The estimated 6-year OS, PFS, FFS, and EFS were 91%, 91%, 83%, and 59%, respectively. Conclusions. Our National experience showed that most pts treated frontline with NIL-based regimens were optimal responders according to ELN recommendations and that 91% of the patients were estimated to be alive and progression-free at 6 years. In particular, NIL alone was highly effective in the prevention of AP/BP. Considering that AP/BP had in most cases an early onset and an extremely poor prognosis, its prevention should be the priority of CML treatment, especially in the firsts 2-3 years. However, afterwards, the relatively high number of CVAE observed, suggests to focus, at least in selected patients, on strategies aimed at the prevention of CVAE (NIL dose reduction? switch to IM?). Acknowledgments. European LeukemiaNet, COFIN, Bologna University, BolognAIL Disclosures Gugliotta: Novartis: Consultancy; Bristol-Myers-Squibb: Consultancy. Castagnetti:Novartis: Consultancy; Bristol-Myers-Squibb: Consultancy. Breccia:novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Abruzzese:Novartis: Consultancy. Martinelli:ARIAD: Consultancy; Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Saglio:BMS: Consultancy, Fees for occasional speeches Other; Novartis: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other; Pfizer: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other; ARIAD: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other. Baccarani:Novartis, BMS, Pfizer, Ariad: Consultancy, Honoraria, Speakers Bureau. Rosti:Novartis: Consultancy; Bristol-Myers-Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy.

Introduction. Chronic myeloid leukemia (CML) patients (pts) treated with imatinib first line achieve complete cytogenetic response (CCyR) in > 70% of cases and major molecular response (MMR) in 18-58%. These pts have a life expectancy similar to the general population. However even undetectable BCR-ABL may not equate to eradication of the disease because of the sensitivity of Q-RT-PCR. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, has been recently developed (Goh HG et al., 2011). dPCR corresponds to a 100 fold increase in sensitivity as compared to Q-RT-PCR. Therefore, dPCR by assessing the presence of minimal residual disease with higher sensitivity, could potentially identify pts in whom CML has been eradicated. Aims. The Imatinib Suspension And Validation (ISAV) study is aimed at validating the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results. Methods. This study involves 15 sites, 10 in Italy and 1 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. CML pts (Chronic or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible for this study. Patients had to be in CMR for at least 18 months (mts), with a minimum of 3 Q-RT-PCR performed at their own sites. After signing the informed consent, blood samples are obtained for dPCR and the pts discontinue imatinib therapy. Standard Q-RT-PCR is performed monthly (mts 1-6) and then bimonthly for 36 mts to assess the maintenance of the molecular remission. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resume imatinib treatment at the same dosage used before interruption. Patients’ quality of life during imatinib discontinuation/resumption is evaluated through the EORTC – C30 Quality of Life questionnaire. Results. The enrolment in ISAV began in November 2011 and ended in July 2013. The study enrolled 112 pts: Italy 69.6%, Germany 21.4%, Canada 5.3%, Spain 2.6% and Israel 0.9%. Among the 112 pts, 59.3% were male and 37.0% were aged 65 or older; median duration of imatinib treatment was 103.1 mts with median duration of CMR of 25.8 mts before imatinib discontinuation. To date, the median follow-up (FUP) time is 16.6 mts [95% CI: 14.9-18.2]. Forty-seven pts (43.5%, 95% CI: 34.0-53.4) of the 108 eligible pts relapsed and resumed imatinib; 38/47 (80.9%) of them relapsed in the first 9 mts and the last relapse occurred 19.6 mts after imatinib discontinuation. A loss of CCyR occurred in 11 pts (23.4%): 10/11 CCyR losses were recovered; 1 patient withdrew the consent shortly after obtaining a partial cytogenetic response. No case of CML progression was observed. After the resumption of imatinib the median time to either MMR or CMR was 1.9 [95% CI: 1.2-2.4] mts. Of the 61 not-relapsed pts, 43 (39.8% of the total) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity was 3.6 mts [95% CI: 3.0-4.8] and the range of duration of Q-RT-PCR positivity (below 0.1%) was between 5.7 and 29.2 mts. No significant correlation between relapse and previous duration of imatinib treatment, use of interferon, time to CCyR or duration of CMR was identified. An inverse relationship between pts age and risk of relapse is evident: 90% of pts < 45 years relapsed vs 37.5% in the class ≥ 45 - < 65 years and 27.5% of pts ≥ 65 years, p(χ2) Conclusions. After 32 mts from the beginning of the study, with a median FUP of 16.6 mts, 43.5% of pts relapsed; the majority of relapses developed in the first 9 months after imatinib discontinuation. Age < 45 years and dPCR positivity are significantly associated with relapses. Funded by Regione Lombardia. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Interleukin-6 (IL-6) protects multiple myeloma cells against apoptosis induced by glucocorticoids. Here, we investigated whether inhibition of the IL-6 signaling pathway by the IL-6 receptor superantagonist Sant7 enhances the in vivo antitumor effects of dexamethasone on the IL-6–dependent multiple myeloma cell line INA-6. For this purpose, we used a novel murine model of human multiple myeloma in which IL-6–dependent INA-6 multiple myeloma cells were directly injected into human bone marrow implants in severe combined immunodeficient (SCID) mice (SCID-hu). The effect of in vivo drug treatments on multiple myeloma cell growth was monitored by serial determinations of serum levels of soluble IL-6 receptor (shuIL-6R), which is released by INA-6 cells and served as a marker of tumor growth. In SCID-hu mice engrafted with INA-6 cells, treatment with either Sant7 or dexamethasone alone did not induce significant reduction in serum shuIL-6R levels. In contrast, the combination of Sant7 with dexamethasone resulted in a synergistic reduction in serum shuIL-6R levels after 6 consecutive days of treatment. Gene expression profiling of INA-6 cells showed down-regulation of proliferation/maintenance and cell cycle control genes, as well as up-regulation of apoptotic genes in multiple myeloma cells triggered by Sant7 and dexamethasone combination. In vitro colony assays showed inhibition of myeloid and erythroid colonies from normal human CD34+ progenitors in response to dexamethasone, whereas Sant7 neither inhibited colony growth nor potentiated the inhibitory effect of dexamethasone. Taken together, these results indicate that inhibition of IL-6 signaling by Sant7 significantly potentiates the therapeutic action of dexamethasone against multiple myeloma cells, providing the preclinical rationale for clinical trials of Sant7 in combination with dexamethasone to improve patient outcome in multiple myeloma.

HuN901 is a humanized monoclonal antibody that binds with high affinity to CD56, the neuronal cell adhesion molecule. HuN901 conjugated with the maytansinoid N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1), a potent antimicrotubular cytotoxic agent, may provide targeted delivery of the drug to CD56 expressing tumors. Based on gene expression profiles of primary multiple myeloma (MM) cells showing expression of CD56 in 10 out of 15 patients (66.6%) and flow cytometric profiles of MM (CD38brightCD45lo) cells showing CD56 expression in 22 out of 28 patients (79%), we assessed the efficacy of huN901-DM1 for the treatment of MM. We first examined the in vitro cytotoxicity and specificity of huN901-DM1 on a panel of CD56+ and CD56− MM cell lines, as well as a CD56− Waldenstrom’s macroglobulinemia cell line. HuN901-DM1 treatment selectively decreased survival of CD56+ MM cell lines and depleted CD56+ MM cells from mixed cultures with a CD56− cell line or adherent bone marrow stromal cells. In vivo antitumor activity of huN901-DM1 was then studied in a tumor xenograft model using a CD56+ OPM2 human MM cell line in SCID mice. We observed inhibition of serum paraprotein secretion, inhibition of tumor growth, and increase in survival of mice treated with huN901-DM1. Our data therefore demonstrate that huN901-DM1 has significant in vitro and in vivo antimyeloma activity at doses that are well tolerated in a murine model. Taken together, these data provide the framework for clinical trials of this agent to improve patient outcome in MM.

B1647 is a cell line derived from bone marrow cells of a patient with acute myeloid leukemia (M2) with a complete erythro-megakaryocytic phenotype and bears both k and p isoforms of c-mpl. Interestingly, spontaneous B1647 cell proliferation is significantly potentiated by thrombopoietin (TPO).We aimed to evaluate the proliferative signal transduction events following the activation of c-mpl and we stimulated B1647 cells with TPO 40 ng/mL for 3, 7, 15 and 30 minutes; cells were then lysed and whole lysates were immunoprecipitated with anti-phosphotyrosine antibodies.In our hands, TPO stimulation induced phosphorylation of several substrate proteins in B1647 cells. The increase in tyrosine phosphorylation from background spontaneous activation was transient, maximal after 10 minutes and declined to reach constitutive levels after 30 minutes. In particular, protein substrates between 50 and 140 kDa appeared to be selectively phosphorylated by TPO. We demonstrated that Jak2, Stat3 and Shc were activated in B1647 cells after TPO, as already shown for different cell lines by other authors. Moreover, Lyn kinase activation was detected. Grb2 co-immunoprecipitated with phosphorylated proteins. The phosphorylation of Syk kinase was not demonstrated, whereas Vav was activated by TPO.The pattern of protein phosphorylation determined in B1647 cells by TPO testifies the role of this cytokine in sustaining cell growth and indicates Lyn tyrosine kinase as a possible target protein in transduction of the TPO proliferative signal.

Regiospecific synthesis of 12 novel n-butyric and phenylalkylcarboxylic monoesters of mannose and xylitol was achieved. The strategy adopted, avoided a tedious intramolecular transesterification step, previously described for the synthesis of analogous compounds and permitted the facile synthesis of a new generation of stable derivatives. The general tolerance of the drugs has been assayed after intravenous administration of a bolus dose into mice. Monobutyric esters showed a low toxicity commensurate with the requirements for future development. A relationship was observed between chain length and toxicity. In contrast, phenylacetic, 3-phenylpropionic and 4-phenylbutyric esters were found to be toxic. Phenylbutyric esters induced marked and specific neuromuscular damage. Preliminary biological investigations of the new series of monobutyric esters showed them to retain the benificial biological properties of butyric acid whilst remaining relatively non toxic. They induced an inhibition of in vitro proliferation of 10 human cases of de novo acute myeloid leukemia (AML) primary cultures and AML established cell lines. AML blasts growth appeared to be blocked and cell differentiation was established. Transcription and expression of maturation markers and finally apoptosis were observed. Moreover, human gamma-chain hemoglobin (HbF) synthesis in erythroleukemia cells was stimulated by monobutyric esters. Mannose and xylitol butyric derivatives would appear to have exciting potential in treatment of beta-Hemoglobinopathies, sickle cell anemia and cancer.

Background Nilotinib is a potent and selective BCR-ABL inhibitor approved for the frontline treatment of CML. The latest update (4-yr follow-up) of the ENESTnd study demonstrated sustained superiority of nilotinib vs. imatinib (Hochhaus et al, EHA 2013, abstract 712). The CML Italian Registry of Nilotinib is the largest series of patients treated frontline with nilotinib-based regimens, outside of Company-sponsored trials. Therefore, it represents an important resource for an independent evaluation of the outcome of such patients. Aims to analyze the response rates and outcome in an independent cohort of patients treated frontline with nilotinib-based regimens in Italy. Methods The CML Italian Registry of Nilotinib includes 215 patients, enrolled in 2 multicenter phase II studies conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00481052 and NCT00769327) with nilotinib 300 mg or 400 mg BID as initial treatment; 123 patients received a sequential treatment with nilotinib and imatinib, with a 3-mo rotation period. The median age was 53 years (range 18–86). Ten out of 215 patients (5%) had a high EUTOS score. The median follow-up was 43 months (range 18–69 months). We analyzed: the rates of Complete Cytogenetic Response (CCyR) and Major Molecular Response (MMR); the overall survival (OS; any death included), progression-free survival (PFS; progression to accelerated/blast phase [AP/BP] and deaths for any cause), failure-free survival (FFS; failures according to ELN 2013 recommendations and deaths for any cause), and event-free survival (EFS; events: failures, permanent discontinuation of nilotinib for any cause, including deaths). Results Rates of CCyR were 72% and 92% by 3 months and 12 months, respectively. Rates of MMR were 56% and 83% by 3 months and 12 months, respectively. The cumulative rates of CCyR and MMR were 93% and 92%, respectively. Overall, events were recorded in 64 (30%) patients: 31 (14%) patients permanently discontinued nilotinib for adverse events or intolerance; 22 (10%) patients failed therapy according to ELN 2013 recommendations, including 8 (3.7%) patients that progressed to AP/BP; 11 (5%) patients permanently discontinued nilotinib for other reasons. All progressions to AP/BP occurred within the first year of therapy, and all patients subsequently died. Nilotinib-resistant mutations were identified in 5 of these patients (4 T315I; 1 Y253H). No difference in the rate of progression to AP/BP was observed between patients receiving nilotinib alone or nilotinib and imatinib in sequential schedule. Overall, 15 (7%) patients died, in 7 cases for reasons unrelated to CML progression. The 4-year OS, PFS, FFS, and EFS were 93%, 93%, 86%, and 69%, respectively. Conclusion These Italian nilotinib registry data provide an independent and unbiased overview of the therapeutic effects of nilotinib, with high and early rates of complete cytogenetic and major molecular response. All progressions (3.7%) to AP or BP occurred within the 1st year of therapy; however, all cases were fatal, emphasizing how crucial is the prevention of AP/BP. With a median follow-up of 43 months, 69% of patients were still on nilotinib, and 93% were alive and progression-free. Acknowledgments European LeukemiaNet, COFIN, Bologna University, BolognAIL Disclosures: Gugliotta: Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Castagnetti:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Bocchia:Novartis and Bristol Mayer Squibb: Honoraria. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Saglio:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Cavo:Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Baccarani:Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Pfizer: Honoraria; Ariad: Honoraria. Rosti:Novartis: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Ariad: Consultancy; Roche: Speakers Bureau.

Introduction Imatinib induces complete cytogenetic response (CCyR) in up to 80% of chronic myeloid leukemia (CML) patients (pts) and major molecular response (MMR) in 33-60% of them. These patients enjoy life expectancy similar to general population. However even undetectable BCR-ABL may not equate to eradication of the disease because the sensitivity of the standard diagnostic method, the Q-RT-PCR, is limited. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, corresponding to a 100 times increased sensitivity as compared to conventional Q-RT-PCR, was developed (Goh HG et al., Leuk Lymphoma 52(5): 896-904. 2011). Therefore dPCR, assessing with more sensitivity the presence of minimal residual disease, could potentially identify pts in whom CML is eradicated. The Imatinib Suspension And Validation (ISAV) study is aimed at assessing the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results. Methods This study involves 15 sites, 10 in Italy and 5 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. In this study CML patients (Chronic Phase or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible. Patients had to be in CMR for at least 18 months, with a minimum of 3 Q-RT-PCR performed in their own centers. After signing the informed consent, pts were tested for dPCR and discontinued imatinib therapy. They are being monitored by standard Q-RT-PCR for 36 months to assess the maintenance of the molecular remission. At the end of this period, a peripheral blood sample for dPCR analysis will be obtained from those pts who will still have undetectable BCR-ABL transcripts by Q-RT-PCR, to verify CML eradication. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resumed imatinib treatment at the same dosage used before interruption. Patients’ quality of life during imatinib discontinuation/resumption is being evaluated trough the EORTC – C30 Quality of Life questionnaire. Results The enrollment in the ISAV study began in November 2011 and ended in June 2013. The study enrolled 112 pts: Italy 69.6%, Berlin 21.4%, Montreal 5.3%, Zaragoza 2.6% and Tel Hashomer 0.9%. Sixty-one percent of the pts were male and 38% were aged 65 or older; median duration of imatinib treatment is 102 months with median duration of CMR of 32 months before imatinib discontinuation. To date, the median follow-up (FUP) time is 4.6 months [95% CI: 4.1-5.8] and 92 pts out of 112 (82%) had at least one Q-RT-PCR performed after imatinib discontinuation. The following analysis is restricted to 48 pts with a minimum of 6 months of FUP. Of these 48 pts, 20 remained Q-RT-PCR negative (42%, 95% CI:29-56%, median duration of negativity after imatinib discontinuation: 10.3 months). Nineteen pts (40%, 95% CI:27-53%) relapsed and resumed imatinib. All relapses occurred in the first 10 months and all but 3 of them in the first 6 months. A loss of CCyR happened in 5 pts out of 19 (26%): 1 pt regained CCyR after 3 months of re-treatment and is now in CMR, 1 pt died shortly after the diagnosis of relapse because of lung adenocarcinoma and 3 pts are now being monitored after imatinib resumption. No case of progression of CML was observed. After the resumption of imatinib the median time to either MMR or CMR, whichever came first, was 2.1 [95% CI: 0.9-5.8] months. Finally, nine pts (18%, 95% CI:10-31%) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity in this group of pts was 2.92 months (range 1-5 months), and the range of duration of Q-RT-PCR positivity (below 0.1%) is between 2 and 14 months. No significant correlation between relapse and previous duration of imatinib treatment, time to CCyR or duration of CMR was present. Patients previously treated with interferon showed a trend toward lower risk of relapse which is not significant so far. Finally, 19% of pts complained of musculoskeletal/articular pain after imatinib discontinuation. Conclusions After 21 months from the beginning of the study with a median follow-up of 4.6 months, 40% of pts relapsed; the majority of relapses happened in the first 6 months after imatinib discontinuation. The correlation of dPCR results with clinical outcomes will be presented at the meeting. Disclosures: le Coutre: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Research Funding. Gozzini:Novartis: Consultancy; Bristol Myers Squibb: Consultancy.

Abstract 4615 Introduction. Alterations in chromatin organization are a common mechanism of leukemogenesis. Drugs affecting epigenetics have been proposed as therapy of hematopoietic neoplasms. We have previously shown selective activity of histone deacetylase (HDAC) inhibitors on AML1/ETO positive cells (Barbetti, et al- Oncogene, 2007). The AML1/ETO multiprotein complex induces and stabilizes the local DNA methylation pattern by recruiting HDACs and DNA methyl-transferase-1 (DNMT1), resulting in the stable silencing of AML1-controlled genes, like IL3. Our hypothesis is that DNMT inhibitors (DNMTi) 5-Azacytidine (AZA) and Decitabine (DAC) exert a targeted action on AML1/ETO positive leukemic cells. Methods. We used U937-A/E-9/14/18 cell line, carrying a ponasterone-A inducible construct of AML1/ETO (kindly provided by M. Luebbert). We analyzed the effects of AZA and DAC at different doses (0.01, 0.1, 1 and 10μM), evaluating cell viability and apoptosis (by Annexin V assay, and by WB caspase 9, 8, 3 -cleavage). Cells were then treated with AZA 0.1–1μM or DAC 0.1–0.01μM for 24h. We determined by chromatin immunoprecitation (ChIP) assay the association of the IL3 promoter with H4acetylated, H3K4me3, H3K9me2 and H3K27me3 and the quantitative expression of IL3 by Q-RT PCR, calculated by a comparative threshold cycle method, using the formula 2̂(−ΔΔCt). Other promoters controlled by AML1/ETO, like those of GM-CSF, MPO and CEBPalpha were also evaluated. Methylation of IL3 promoter was evaluated by Methylation Specific PCR (MSP). Confirmatory experiments were carried out in the human leukemic cell line Kasumi-1 naturally expressing AML1/ETO. Results. Although both DNMTi were active on leukemic cells, irrespective of the presence of AML1/ETO expression, the dose at which this effect was evident was differing of one logarithm. AZA 0.1μM significantly increased the percentage of apoptotic cells only in U937 AML1/ETO positive cell line. We observed that untreated apoptotic cells percentage was 4.72% (±0.55 Standard Deviation: SD) while after AZA 0.1μM was 5.70% (± 0.24 SD) (P0.05). These data were confirmed by the induction of cleavage of caspase 9, 8 and 3. DAC 0.01μM significantly induced apoptosis cells only in U937 AML1/ETO positive cells (NT= 6.27% ±0.24 SD; DAC 0.01μM = 7.50% ±0.57 SD; P Given these results, we investigated whether this different response was correlated with chromatin rearrangements directly involving the AML1/ETO gene. We evaluated IL3 expression after AZA and DAC treatments, which induced a significant increase, higher in AML1/ETO-expressing than in non-expressing U937 cells. In fact, in AML1/ETO positive cells the amount of IL3 mRNA 2̂(−ΔΔCt) after AZA and DAC respect to NT was 10.5 ± 0.3 DS and 6.1± 0.5 SD, respectively. In AML1/ETO negative cells, after AZA was 5.70 ±2.7 SD (P>0.05) and after DAC 2.28 ± 0.4 SD (P>0.05), both respect to NT. By ChIP assay we showed that in untreated leukemic cells, both in U937 AML1/ETO-positive and negative cells, at baseline H3K4me3 was not associated with the IL3 promoter, while H3K9me2 was associated with it. H4-acetylated became associated to the IL3 promoter after AZA and DAC treatments but exclusively in U937 cells expressing AML1/ETO. On the other hand, H3K27me3 was co-immunoprecipitated with the IL3 promoter in U937 AML1/ETO negative and in U937 AML1/ETO positive untreated cells. AZA and DAC treatments led to the dissociation of this modified histone from the IL-3 promoter indicating the modification of chromatin rearrangement (Fig 1). Conclusions. Our observations demonstrate that AML1/ETO positive cells are more sensitive to DNMTi than cells not carrying such chimeric gene, in terms of biological effects like cytotoxicity and induction of apoptosis. The dissociation of the IL3 promoter from H3K27me3 and its association with H4acetylated upon DNMTi exposure is observed exclusively in U937 AML1/ETO-positive cells and clearly indicates that the epigenetic mechanism of action of these drugs is exerted selectively on cells harbouring the chimeric protein. This result could support a working hypothesis to develop a treatment for AML1/ETO-positive leukaemias including DNMTi in the therapeutic armamentarium. Disclosures: Santini: Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria.

Abstract 2810 Azacitidine is a pyrimidine nucleoside analogue of cytidine and it is at present the standard treatment for myelodysplastic syndromes (MDS). A large number of studies performed to evaluate the mechanism of resistance/response to nucleoside chemotherapeutics like cytosine arabinoside or gemcitabine, have demonstrated the importance of the levels of expression of the enzymes involved in their metabolism. Despite the high clinical interest in azacitidine, little is known about its intracellular metabolism and scanty data are available in literature. Recently, differences in the expression of these enzymes has been reported in cell lines differentially sensitive to hypomethylating agents. Clinical responses to azacitidine are heterogeneous, mainly due to clinical characteristics of MDS patients, so there is a need to identify molecular markers that could predict and/or monitor the efficacy of this therapy. A better understanding of the biological mechanisms involved in the activation and DNA uptake of azacitidine is therefore necessary to possibly identify responsive MDS patients. Objectives: To measure the expression levels of the 5 enzymes involved in azacitidine metabolism and to correlate their gene expression with the clinical response to the drug. To test possible causes of differences in gene expression. Material and Methods: DNA and RNA was extracted from mononuclear cells of 39 IPSS high risk MDS patients treated with at least 6 cycles of azacitidine 75mg/m2/7 days every 28 days. Of them, 14 patients were responders and 25 non-responders to azacitidine according to IWG 2006 criteria. Gene expression was assessed with quantitative PCR, using an ABI GeneAmp® 5700. The specific oligonucleotides and TaqMan® probes of every selected gene will be acquired among the Assay-on-Demand® Gene Expression Products (Applied Biosystems). Promoter methylation of UCK1 was evaluated by methylation specific PCR using specific primers. The UCK1 coding region was sequenced by Sanger method. Results: Gene expression of hENT1, UCK1, DCK, RNR1 and RNR2 was compared between the groups of responders and non-responders. Individually, none of the genes was differentially expressed in responders versus non-responders. Nevertheless, a trend for lower expression in non-responders was shown for the UCK1 gene (median responders: 12.4 versus median non responders: 10.7; P = 0.095). In order to verify whether the presence of aberrant methylation could impact in UCK1 gene expression, we measured the methylation status of a putative CpG island in the UCK1 gene promoter region. The CpG island was unmethylated in all the samples analyzed, including responders and non-responder cases, as well as healthy donors. Moreover, to verify whether the difference in gene expression could be due to nucleotide variants in the gene sequence, we sequenced the UCK1 sequence. We found one polymorphic locus (rs2296957) in exon 6 and additional five intronic SNPs (rs3904060 and rs7867616, Intron 1; rs2296956 and rs189473964, Intron 3; rs150900763, 5' UTR) at the intron/exon boundaries. The presence of an individual genotype at these loci was not associated with therapy response and UCK1 gene expression. Conclusions: Our results show that absence of clinical response to azacitidine could be partly correlated with lower expression of UCK1. This is most likely causing a scarce azacitidine monophosphorylation by UCK1 in non-responders patients. The methylation status of the UCK1 gene promoter is not affecting gene expression. In addition, the genetic polymorphisms found in the UCK1 sequence do not associate with therapy response nor with gene expression. Further studies on larger series of MDS cases are needed to confirm these results which could help driving therapeutic decisions. Disclosures: No relevant conflicts of interest to declare.

Abstract 2797 Background: Nilotinib is a second-generation tyrosine kinase inhibitor that demonstrated higher and faster responses and lower progression rates compared to imatinib in ECP CML patients. Early cytogenetic and molecular responses have been associated with a better outcome in patients treated with imatinib, dasatinib, or nilotinib frontline. In particular, BCR-ABL transcript level < 10% at 3 months has been associated with: increased 2-year cumulative incidence of complete cytogenetic response (CCyR), major molecular response (MMR), and MR4.5 (>4.5 log reduction in BCR-ABL transcript level) with dasatinib (Marin D et al, Blood 2012); increased CCyR rate by 1 year, MMR rate by 2 years, and better progression-free survival (PFS) at 2 years, with dasatinib (DASISION; Hochhaus A et al, ASH 2011, abs. 2767); increased MMR rate by 1 year, and better PFS and overall survival (OS), with nilotinib (ENESTnd; Hochhaus A et al, EHA 2012, abs. 584). Here we report a landmark analyses based on BCR-ABL transcript level and cytogenetic response at 3 months to evaluate their impact on subsequent response and outcome in an independent cohort of patients treated with nilotinib-based regimens in Italy (CML Italian Registry of Nilotinib). Methods: The CML Italian Registry of Nilotinib includes 215 patients, enrolled in 2 multicenter phase II studies conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00481052 and NCT00769327) or treated at the Dpt. of Hematology and Oncology, Bologna University Hospital, with nilotinib 300 mg BID or 400 mg BID as initial treatment. The median age was 53 years (range 18–86). The median follow-up was 29 months (range: 18–47 months). At 3 months 196/215 (91%) and 189/215 (88%) patients were evaluable for the molecular and cytogenetic response, respectively. BCR-ABL transcript levels were: 21% in 88%; > 1% to 2 10% in 11%; > 10% in 1%; given the very low proportion of patients with a BCR-ABL > 10% (n. 2), to the purpose of these analyses, patients have been divided into 2 groups, with a transcript level 21% (n. 173, 88%), or > 1% (n. 23, 12%). Cytogenetic response was: CCyR 84%; partial cytogenetic response (PCyR) 9%; less than PCyR 7%. We analyzed the rate of MMR at 1 year, and the failure-free survival (FFS, according to ELN 2009 definitions), PFS (transformation to accelerated or blastic phase), and OS (any death included) according to the BCR-ABL transcript levels and to the cytogenetic response at 3 months. Results: Of the 2 patients with a BCR-ABL transcript level > 10% at 3 months, 1 progressed to blastic phase at 1 year, while the other one obtained a CCyR at 6 months and a MMR at 24 months. Patients with BCR-ABL 2 1% at 3 months had a higher rate of MMR at 12 months with respect to those with a transcript level > 1% (79% vs. 35%, p Conclusion: In our national experience, where 42 centers are represented with at least 1 patient, a very small proportion of patients treated frontline with nilotinib failed to achieve a reduction to < 10% level of BCR-ABL transcript (2/196 or 1%) if compared to other reports: in the frame of the ENESTnd trial, 24/258 or 9% (Hochhaus et al, EHA 2012, abs. 584). The cut-off of 1% of BCR-ABL at 3 months in our experience is a reliable surrogate marker of response at 1 year (MMR) and outcome (PFS and FFS), along with the level of cytogenetic response achieved. These patients with > 1% of BCR-ABL at 3 months are characterized by a higher propensity to fail the treatment and to progress. Consequently, they deserve a close monitoring and, for those not achieving the expected cytogenetic and molecular responses later on (at 6–12 months), an optimization of the dose of nilotinib or a treatment modification is advised. Acknowledgments: European LeukemiaNet, University of Bologna, BolognAIL, COFIN. Disclosures: Gugliotta: Novartis: Consultancy, Honoraria; Bristol-Myers-Squibb: Consultancy, Honoraria. Castagnetti:Novartis: Consultancy, Honoraria; Bristol-Myers-Squibb: Consultancy, Honoraria. Breccia:Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Abruzzese:Bristol Myers-Squibb and Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Saglio:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy. Martinelli:Novartis: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers-Squibb: Consultancy, Honoraria, Speakers Bureau. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Rosti:Novartis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau.

Abstract 1686 The Eutos score has recently been published as a new prognostic score in CML because of its prognostic ability and its simplicity in the imatinib era. It maintains the percent of basophils and the spleen size and it eliminates three other laboratory values and the variable of the age, that is usually important in the prognosis of almost all the diseases. This score was able to predict the probability of achieving a complete cytogenetic response (CCgR) within 18 months, which is the most solid and confirmed surrogate marker of survival in CML treated with imatinib. Thus we reviewed the cases of CML affecting individuals > 75 years and we apply the Eutos score and compare results with classic Sokal risk. We collected 221 patients from 29 of the major Italian hematological centers for diagnosis and treatment of CML. Because of the lack of data for the calculation of the scores in 84 patients, 137 patients were evaluable. The M: F ratio was 70: 67, the median age was of 78 years (range 75–89). Patients aged >75 years as expected showed frequently comorbidities (cardiovascular, respiratory, renal, oncologic ones). The distribution according to the Sokal score was: 3 patients in the low risk group, 95 patients in the intermediate risk group and 39 patients in the high risk group. As expected, there were only few patients in the low Sokal risk group aged >75 years in demonstration of the importance of the age in this score. Calculating Eutos score we have identified only 6 patients with a high score, whereas the remaining 131 patients were in the low risk group. The group with high Eutos risk score included 5 males and 1 female; their median age was 77 years (range 75–78), at the onset their median white blood cell count was 33.840/mmc (range 29.500–300.000), platelet count was 1.443.000/mmc (range 453.000–4.849.000), their median level of hemoglobin was 8.8 g/dl (range 7.7–13.9). All of them showed percent of basophils>12%, whereas at physical examination splenomegaly was usually moderate with no hepatomegaly. Due to the small sample of patients in the high Eutos score statistical analysis was not feasible but we tried to correlate baseline informations to the outcome. Not all the high Eutos risk patients were in the high Sokal risk group (3 patients were in the high Sokal risk group and 3 were in the intermediate one) none was in the low Sokal risk group. All but one patients in the high risk Eutos score were treated with imatinib in early chronic phase of CML. Then we considered the complete cytogenetic response (CCyR) at 18 months, performed through conventional cytogenetic analysis on bone marrow cells by G-banding technique. This has been chosen for the validation of the Eutos score as a solid early surrogate marker of outcome. The overall rate of CCyR was 100% in the low Sokal risk group, 63% in the intermediate Sokal risk group and 69% in the high Sokal risk group; the same type of response was 67% in the low Eutos risk group and 50% in the high Eutos risk group (Table 1). In the high Eutos risk group, 2 patients achieved CCyR, 1 patient achieved MMR, 2 patients did not achieve it and 1 patient was not evaluable. Two patients died because of comorbidities (relapse of colon cancer and cardio-respiratory insufficiency) at 19 and 39 months of follow up. Resistance to imatinib was registered in 17 out of 131 patients (13%) in the low Eutos risk and in 3 out of 6 patients (50%) in the high Eutos risk. In our series of CML patients >75 years, high Eutos score was infrequent (4.38%). Moving from Sokal to Eutos score, patients were largely reallocated as low risk Eutos score. It is not clear whether the low frequency of high risk Eutos might be attributed to a early diagnosis of CML in this subset of patients with age-related comorbidities requiring frequent laboratory exams. In view of the ability to predict CCyR at 18 months, Eutos score validation in the setting of elderly patients with CML could be relevant to clinical practice.Table 1Distribution of patients according Sokal and Eutos scoreN° patients% CCyRSOKALLow risk3100%Intermediate risk9563%High risk3969%EUTOSLow risk13167%High risk650% Disclosures: Russo Rossi: Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

Abstract 5052 Background: 5-azacytidine (Vidaza, AZA) and 5-aza-2'-deoxycytidine (Decitabine, DAC) are active in therapy of myelodysplastic syndromes (MDS) probably with different mechanisms of action. Azacitidine is approved by FDA and EMEA and is routinely used in therapy of higher risk MDS in Europe and in the US. Decitabine is a demethylating agent approved for treatment of IPSS INT-2 and high risk MDS by FDA but not yet from EMEA and it seems particularly active in Chronic myelomonocytic leukemia (CMML) and AML of the elderly. Difference in mechanisms of action of these 2 agents have been determined in vitro, involving nuclear acid incorporation, cell cycle and apoptosis, gene and protein expression. Patients: Nine patients (4 RAEB 2, 4 AML, 1 CMML-1) were treated with decitabine 20mg/m2 × 5 days every 28 days as single agent or in association with Gemtuzumab ozogamicin 3 mg/m2 in day 5 and 9. IPSS score at diagnosis was: 1 low, 4 intermediate 1, 4 intermediate 2. All patients received at least 4 cycles of azacitidine 75 mg/mq per seven days every 28, 44% of them reached at least Hematological improvement and received DAC as second line after relapse. Median age was 70 (62–84) and 8/9 were male. Methods: Medical records of 14 patients who received decitabine treatment were reviewed and survival was calculated by the Kaplan Meier method and differences in survival calculated by the log-rank test using SPSS software. Complete remission was defined by normo-cellular bone marrow with 1.5 mmol/L, WBC count by >1000, and platelet count by >50,000. Results: Median overall survival (OS) was 121 days, range 27–586 days. Overall response rate (CR+PR+HI) was 33.3% with mean duration of response was 3.33 months. Mean number of cycles was 4 (range 1–20). Median OS of responder patients was 12 months vs 1 month of non responder patients. OS of patients treated with DAC after AZA-resistance was 2 months vs 4 months of patients relapsed, but who reached at least HI with AZA (p>0.05). Longer history of MDS disease seemed not relevant for outcome. Therapy was well tolerated and only two patients required a long hospitalization (42 and 77 days) for sepsis due to relevant myelosuppression. Conclusions: Decitabine treatment has some activity in MDS, CMML and AML patients refractory to AZA or who relapsed after initial response to AZA, suggesting that the two supposed hypomethylating agents act via partially different molecular mechanisms. Disclosures: No relevant conflicts of interest to declare.

Abstract 2799 Background. In IPSS lower-risk Myelodysplastic Syndromes (MDS), anemia is the main therapeutic challenge. Erythropoietin stimulating agents (ESAs) can frequently correct anemia, but not all patients respond, and median response duration to ESAs is around 2 years (Park, Blood 2008;111:574). Patients with deletion of chromosome 5q are usually considered refractory to ESAs. Endogenous serum erythropoietin levels have been demonstrated in only one study to be higher in patients carrying del5q than in patients with Lower-Risk MDS and different abnormalities in karyotypes. Aims. We wanted to further analyze differences in management, efficacy and tolerability of ESAs therapy between MDS with and without del5q. Patients and Methods. We studied 239 MDS patients affected by IPSS low and intermediate 1 risk disease. 82 patients carried deletion of 5q, 12/82 in association with other anomalies. MDS patients with del 5q were WHO diagnosed as 9 RA, 9 RCMD, 13 RAEB-1, 47 5q minus syndrome and 2 unclassifiable MDS and divided by IPSS risk: 49 low risk and 33 INT-1. Patients without del5q were diagnosed as follows: 59 RA, 24 RARS, 43 RCMD, 17 RAEB-1, 4 CMML and 10 unclassifiable MDS. When subdivide per IPSS risk: 90 low risk and 67 INT-1. 75% of patients with del5q were female against 37% of patients without del5q. Medical record of patient were collected and reviewed, statistical analysis were perform with SPSS software. IWG criteria for response to ESA treatment were applied. Results. Endogenous erythropoietin (EPO) level at diagnosis were significant higher in patients with del 5q (mean 599,6 mU/ml) than in others (mean 99,3 mU/ml)(p=0.002). There was no significant difference, in terms of endogenous EPO levels, either stratifying by WHO classification (p=0.996) or by IPSS score (INT-1 325.8 mU/ml, low 138.6 mU/ml, p=0.120), or blast % (p>0.05). Serum creatinine levels were correlated with endogenous EPO levels (p=0.003 Spearman's rho −0.258). Serum Epo was also correlated to age (p=0.026 Spearman's rho −0.183). No correlation was seen between number of transfusions at diagnosis and serum EPO levels and between hemoglobin levels and serum EPO levels. 18 patients (22%) with deletion of 5 q responded to ESAs treatment versus 56,8% of responding patients without del 5q (p Disclosures: No relevant conflicts of interest to declare.

Abstract 2770 In the “real world” of clinical practice, often physicians choose to treat very elderly CML patients with imatinib (IM) at lower than standard (400 mg/day) dose, but there are no published data on the results. To highlight this issue, we retrospectively revised 200 > 75 years old CML patients in chronic phase treated with IM 29 haematological Italian Institutions. We compared 58 patients (29%) who received low-dose IM (≤ 300 mg/day) according to physician decision (LD group) with the remaining 142 patients (71%) who received standard dose IM (SD group). In the SD group, there were 73 males and 69 females with a median age at IM start of 77.9 years (IR 76.0–80.3), Sokal Risk at diagnosis was low in 3 patients, intermediate in 86, high in 39 and not evaluable in 12. Two or more concomitant diseases requiring specific treatments were present in 93/142 patients (65.4%), with 85 patients (59.8%) taking 3 or more concomitant drugs. Twenty-seven patients (19.0%) were in late chronic phase (≥ 12 months from diagnosis before starting IM); on the whole, median time from diagnosis to IM was 1.1 months (IR 0.5–3.0). In the LD group, there were 31 males and 27 females with a median age of 80.2 years (IR 77.9–84.5) at IM start, Sokal Risk at diagnosis was intermediate in 34 patients, high in 17 and not evaluable in 7. Two or more concomitant diseases requiring specific treatments were present in 43/58 patients (74.1%), with 43 patients (74.1%) taking 3 or more concomitant drugs. Fifteen patients (25.8%) were in late chronic phase; on the whole, median time from diagnosis to IM was 1.8 months (IR 0.7–10.4). Starting dose of IM was 300 mg/day in 44 patients (75.8%) and < 300 mg/day in 14 patients (24.2%). According to CTC-AE, grade 3–4 hematological and extra-hematological toxicities were observed in 29 (20.4%) and 30 (21.1%) patients in the SD group compared with 10 (17.2%) and 14 (24.1%) patients in the LD group, respectively. Overall, 63 patients in the SD group (44.3%) required a dose reduction compared to 13 (22.4%) in the LD group (p=0.004): eleven (7.7%) patients in the SD group discontinued IM for toxicity compared to 13 (22.4%) in the LD group (p=0.004). Response to IM in the 2 groups is detailed in the table.SD groupLD grouppN° patients evaluable for response13656Early discontinuation8 (5.8%)8 (14.3%)0.054Resistant disease3 (2.2%)1 (1.8%)0.859Complete haematological response only24 (17.6%)12 (21.4%)0.527Partial cytogenetic response9 (6.6%)6 (10.7%)0.329Complete cytogenetic response92 (67.6%)29 (51.8%)0.052Major molecular response69 (50.7%)17 (30.3%)0.012 After a median follow-up of 33.7 months (IR 18.1–64.7), in the SD group 35 patients died (5 from disease progression and 30 from unrelated causes), 5 patients were lost to follow-up and 102 are still alive: in the LD group, 15 patients died (3 from disease progression and 12 from unrelated causes), 3 patients were lost to follow-up and 40 are still alive. In the SD group, 2-year and 5-year overall survival were 93.2% (CI95% 88.6–97.2) and 65.7% (CI95% 55.0–76.3), respectively; in the LD group, 2-year and 5-year overall survival were 89.7% (CI95% 80.4–98.9) and 67.0% (CI95% 49.6–84.4), respectively. In conclusion, in very elderly CML patients even reduced IM dose appears to be safe and effective enough to achieve sustained cytogenetic and molecular responses with prolonged overall survival. Therefore, also very elderly patients with co-morbidities should have this chance of cure without no upper age limit. Disclosures: Russo Rossi: Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

Abstract 1689 Background: Approximately half of patients with chronic-phase chronic myelogenous leukemia (CP-CML) fail imatinib treatment, both in interventional (Deininger et al., Blood 2009 114: abstract 1126) and observational trials (Michallet et al., Curr Med Res Opin 2010;26(2): 307–17), primarily due to the development of resistance and/or toxicity (Kantarjian and Cortes. J Clin Oncol 2011; 29(12): 1512–16). Clinical evidence suggests that optimal outcomes are achieved when dasatinib is administered early after imatinib failure (Quintas-Cardama et al., Cancer 2009;115(13): 2912–21); however, limited data are available regarding outcome of patients intolerant to imatinib in the real-life setting. Aims: FORTE (Factors impacting On Response To dasatinib in Europe) is a European observational study that aims to estimate the relationship between time elapsed from first detection of imatinib resistance/intolerance until initiation of dasatinib and best response to dasatinib, adjusted for other potentially explanatory factors. Methods: Data were collected retrospectively and prospectively from medical records. Best response to dasatinib, as recorded in patient's charts, was reported by an ordered categorical variable, combining all types of response achieved by a patient and selecting the “highest” level. A predefined list of covariates (sex; age at dasatinib initiation; time from diagnosis to dasatinib initiation; best response to prior imatinib and last imatinib dose) were entered/removed in a Proportional Odds model to identify factors potentially influencing dasatinib best response over a 12-month observation period. Results presented here focus on the imatinib-intolerant subpopulation. Results: FORTE enrolled 549 adult patients with imatinib-resistant/intolerant CP-CML at 124 sites across 12 European countries. Of 457 eligible patients, 176 (39%) were imatinib intolerant, including 71 (16%) who were both resistant and intolerant to imatinib. Approximately half (47%) of intolerant patients were male. Median age at dasatinib initiation was 59 years and median times from CML diagnosis to imatinib and dasatinib initiation were 1.5 and 39.8 months, respectively. Sokal and Hasford scores were intermediate/high in 74% and 72% of assessed patients, respectively. Overall, 54% of 171 imatinib-intolerant patients had achieved a complete cytogenetic response (CCyR) or major molecular response (MMR) on imatinib. During the 12 month observation period, 72% of imatinib-intolerant patients achieved a CCyR or MMR with dasatinib. Adjusting for the effect of best imatinib response, an analysis of 161 evaluable patients showed strong statistical evidence (p Conclusions: Results from the imatinib-intolerant population of the FORTE study suggest that an early switch to dasatinib provides a clinical advantage to CP-CML patients intolerant to imatinib. Findings from this real-life observation are consistent with data from interventional trials. Disclosures: Marin: Bristol-Myers Squibb: Research Funding. Morra:Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Janssen Cilag: Speakers Bureau. Niederwieser:Bristol-Myers Squibb: Speakers Bureau. Schloegl:AOP Orphan: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Ho:Bristol-Myers Squibb: Consultancy, Honoraria, Speakers Bureau. Ossenkoppele:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Guerra:Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Mori:Bristol-Myers Squibb: Employment. Pans:Bristol-Myers Squibb: Employment. van Baardewijk:Bristol-Myers Squibb: Employment. Steegmann:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau.

Abstract 4537 INTRODUCTION. Autologous HSCT is currently under investigation for the treatment of refractory, progressive MS. A better selection of patients and the use of less aggressive regimens resulted in a significant drop of Transplant-related Mortality (TRM), which is mostly related to immunosuppression. CMV and EBV tend to reactivate in ATG-treated allogeneic HSCT. We investigated the frequency of CMV/EBV viral load in a consecutive cohort of MS patients who underwent HSCT in our Center following BEAM and Anti-T Lymphocyte Globulin (ATG) conditioning regimen. PATIENTS AND METHODS. Thirty-one patients underwent auto-HSCT for severe MS in our Institution from 1999 to 2010. All patients were mobilized by Cyclophosphamide 4 g/sqm + G-CSF and conditioned with BEAM plus rabbit ATG (Thymoglobulin©) at a dose ranging between 7.5 and 10 mg/Kg. Monitoring of CMV/EBV DNA by quantitative PCR on either circulating Mononuclear Cells (MNC) or Whole Blood (WB) was performed in 29 pts. In 18 patients ≥2 tests were available for both CMV and EBV from +20 and +120 days. In 2006, following a case of severe CMV infection, a specific protocol was developed, aimed at monitoring on a weekly basis the viral load in case reactivation until complete negativisation. Pre-emptive treatment was administered in case of viral DNA load >1×103 copies/105MNC or >1×1044 copies/ml WB in two subsequent determinations. RESULTS. Overall 84 and 95 tests were carried out for CMV and EBV, respectively. 10/29 patients (34.5%) were shown positive for CMV at a median of 33,5 days from HSCT (28-47), whereas 11/29 patients (37.9%) were positive for EBV at a median of 29 days from HSCT (20-109). Five patients resulted positive for both viruses. According to the ongoing protocols, 20% of CMV+ patients and 18% of EBV+ patients received a pre-emptive treatment with a fast drop of viral load. In the other patients the reactivation was self-limiting in 2–3 weeks from the first positive determination. One patient developed a severe CMV infection at + 47 days successfully treated the iv administration of Gancyclovir and Foscarnet. One EBV+ patient developed fever which responded promptly after treatment with rituximab. Moreover, in 16/29 patients a sporadic assessment of CMV/EBV was performed at early (between +5 and + 20) and late phases (beyond 120 days) without any evidence of viral reactivation. CONCLUSIONS. Autologous HSCT with BEAM/ATG can be safely performed in MS patients. A significant reactivation of both CMV and EBV can occur in the 3 months following HSCT, possibly related to the ATG administration. Monitoring of viral load and, whenever necessary, pre-emptive anti-viral treatment should be performed in order to improve safety of HSCT in this subset of non-neoplastic patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 606 Myelodysplastic syndromes (MDS) are affecting mainly elderly patients, and age is considered per se a negative prognostic factor. Most of the elderly patients have comorbidity that presumably negatively impact the overall survival and quality of life and may influence response to therapy. A subanalysis of a recently concluded international trial demonstrated that MDS patients aged > 75 yrs treated with azacitidine have a significantly longer overall survival respect to best supportive care treated patients (Seymour, 2010). We analyzed whether presence and number of comorbidities could have an impact on response and management of azacitidine treatment in elderly and very elderly patients treated in our Center, outside clinical trials. We evaluated outcome, survival and type of response, as well as adverse events in all patients. We analyzed 59 elderly MDS patients (IPSS INT-1 15/59, INT-2 32/59 and High 12/59) treated with Azacitidine 75 mg/kg/day sc for 5 days every 28. Mean number of cycles was 9 (range:2-42). Mean age was 69 yrs (50-82); 30% of patients (18/59) were >= 75 yrs and 39% of the latter (7/18) >= 80 yrs. We also evaluated Charlson comorbidity index (CCI) (10 patients scored == 4), the Cumulative Illness Rating Scale (CIRS) (25 patients scored == 4) and the Adult Comorbidity Evaluation-27 (ACE-27) (13 patients scored ==5). Overall response rate (HI, CR and PR) according IWG criteria 2006 was 49.1%, stable disease was obtained in 37.1 % of MDS patients. We demonstrated by Fisher test that these IWG responses did not correlate with age. We also evaluated CCI, CIRS, and the ACE-27 in relation to age and to hematological response and no correlation was showed. Hematological or non hematological adverse events were presented by 34% and 36% of patients, respectively. Adverse events were uniformly distributed independently from age. Median overall survival (OS) of our patient cohort was 21 months. Median OS in patients < 75yrs (20 monthts) and >= 75 yrs (16 months) was not significantly different (p value > 0.65). OS in patients with higher CCI, CIRS, and the ACE-27 was respectly 10 months, 6,5 months and 6 months. 5 patients presented renal failure, but treatment with standard dose aza did not worsen creatinine clearance. Very elderly patients with comorbidities may be treated with success with azacitidine, without any substantial increase in AE. Nevertheless comorbidities negatively influence overall survival. Evaluation of comorbidities with validated indexes is an useful and easily applicable tool to refine prognostic evaluation. ACE-27, although more complex, seems to give best prognostic evaluation. Disclosures: No relevant conflicts of interest to declare.

Abstract 2270 An update of the European LeukemiaNet criteria for monitoring response of chronic myeloid leukemia patients was recently published and provisional criteria to evaluate patients during second generation TKI therapy after resistance to imatinib were proposed. In our study we retrospectively tested these criteria in a large series of CML patients resistant to imatinib further treated with second generation TKIs with the aim to analyze the outcome of suboptimal response and failure patients compared to those with optimal response and to validate the provisional criteria for monitoring response. One hundred twenty-seven CML patients resistant to imatinib were collected from 6 different Italian hematologic centers. There were 66 males and 61 females, median age 54 years (range 25–80). Twenty-seven patients were in late chronic phase after IFN resistance. Ninety-seven patients received second-generation TKI after acquired resistance, whereas 30 patients had primary resistance. We found that at different time points (3, 6 and 12 months), patients classified as failure showed significantly worse 2-year overall survival (OS), progression-free survival (PFS) and event-free survival (EFS) than sub-optimal and optimal response patients. At 3 months, “failure” patients, had an OS of 83% compared to 86% of sub-optimal and 97% of optimal response patients (p=0.001); PFS was 77% for failure patients compared to 92% and 99% for sub-optimal and optimal response patients, respectively (p=0.001), whereas EFS was 41% for failure vs 59% for sub-optimal (p=0.001) and 85% and optimal response patients, respectively (sub-optimal vs optimal p Disclosures: No relevant conflicts of interest to declare.

Abstract 1229 In the “real world” of clinical practice, many very elderly CML patients have been treated with imatinib (IM), but there are few data on the results and the best initial dosage in such patients. To highlight peculiar aspects of toxicity and efficacy of IM in this subset which accounts for at least 10–15% of all CML cases, we retrospectively revised 156 CML patients in chronic phase treated with IM when aged > 75 years from 23 haematological Institutions in Italy; there were 85 males and 71 females, median age at IM start was 78.4 years (IR 76.1 – 81.4), Sokal Risk at diagnosis was low in 2 patients, intermediate in 90, high in 50 and not evaluable in 14. One or more concomitant diseases requiring specific treatments were present in 144/156 patients (92.3%), with 94 patients (60.2%) assuming 3 or more concomitant drugs. Thirty patients (19.2%) were in late chronic phase (≥ 12 months from diagnosis) and pretreated (25 with HU and 5 with IFN) before starting IM; on the whole, median time from diagnosis to IM was 1.2 months (IR 0.5 – 3.6). Starting dose of IM was 400 mg/day in 117 patients (75.0%) and 300 mg/day or less in 39 patients (25.0%); overall, 59 patients (37.8%) (52/117 at 400 mg starting dose and 7/39 at 3 300 mg starting dose) needed a dose reduction and 18 (11.5%) discontinued IM for toxicity (early toxicity in 13 and late toxicity in 5). Excluding the 13 patients who discontinued IM due to early toxicity, maximum tolerated daily dose during treatment was 400 mg in 63 patients, 300 mg in 51 patients and < 300 mg in 29 patients. According to CTC-AE, grade 3 – 4 hematological and extra-hematological toxicities were observed in 34 (21.7%) and 34 (21.7%) patients, respectively; 5 patients (3.2%) presented a pleural effusion during IM treatment. After a median treatment period of 29.4 months (IR 7.9 – 54.4), 8 patients (5.1%) are still too early (< 6 months of treatment), 13 (8.3%) discontinued IM due to early toxicity, 3 (1.9%) were resistant and 1 (0.7%) died from unrelated cause early after IM initiation: the remaining 131 patients (84.6%) achieved a complete haematological response (CHR). Among these 131 patients in CHR, 11 refused any other karyotipic or molecular evaluation (1 lost CHR and shifted to hydroxyurea, 4 are still alive in CHR, 6 died in CHR from unrelated causes), 17 achieved CHR only and 103 (66.0% of all 156 patients) achieved a cytogenetic response (CyR), which was major in 11 patients and complete (CCyR) in 92 (58.9% of all 156 patients). In addition, among the 92 patients in CCyR, 62 (39.7% of all 156 patients) achieved a molecular response (major molecular response in 40 patients and complete molecular response with an undetectable BCR/ABL hybrid gene at qualitative nested PCR in 22 patients). After a median follow-up of 34.0 months (IR 12.9 – 60.0), 36 patients have died (5 from disease progression and 31 from unrelated causes), 4 patients were lost to follow-up and 116 are still alive: 2-year and 4-year overall survival were 90.2% (CI95% 84.8 – 95.6) and 76.8% (CI95% 68.6 – 85.0), respectively. In conclusion, results from this large unselected cohort of patients show that should be definitely considered unethical to avoid IM therapy to any elderly patient; no upper age limit should be given but also very elderly (and with concomitant severe diseases) patients should have this chance of cure. The role of a reduced starting dose of IM warrants further studies. Disclosures: No relevant conflicts of interest to declare.

Abstract 3409 A score aiming at early identification of CML patients showing sensitivity to second generation TKIs was proposed by the Hammersmith group. The score was created by analizing 80 patients and was based on 3 prognostic factors: previous cytogenetic response to imatinib, Sokal risk and recurrent neutropenia during imatinib. Subsequently, the score was validated in a small series of 28 patients. Aim of our study was to confirm the validity of this score and to establish its strength on a large group of CML patients resistant to imatinib and treated with second generation TKIs. One hundred twenty-seven patients were collected from 6 different Italian hematologic centers. There were 66 males and 61 females, median age 54 years (range 25–80). Twenty-seven patients received interferon before imatinib. Thirty patients had primary resistance, whereas 97 patients received second-generation TKI after acquired resistance to imatinib. The application of Hammersmith score was possible in 118 patients with available data: 52 patients were identified as good risk, 27 patients as intermediate risk and 38 patients as poor risk. The 1-year cumulative incidence of complete cytogenetic response (CCR) was 73% in good risk patients, 40% in intermediate risk patients and 23% in poor risk patients (p=0.0001). Similarly, the cumulative incidence of major molecular response (MMR) was 52% in good risk, 28% in intermediate risk and 13% in poor risk category (p=0.001). In the evaluation of event-free survival (EFS), events were considered loss of hematologic or cytogenetic response, disease progression, death for any cause, toxicity: the estimated 2-year event-free survival (EFS) was 89% in good risk, 70% in intermediate risk and 55% in poor risk group (p=0.0001). Progression-free survival (PFS) was defined as survival without evidence of accelerated or blastic phase: the estimated 2-year PFS was 97% in good risk, 93% in intermediate risk and 87% in poor risk category (p=0.05). Kaplan Meier estimated 2-year overall survival (OS) was 100% in the good risk, 93% in the intermediate risk and 82% in the poor risk category (p=0.001). In conclusion, as suggested by Milojkovic et al, some prognostic factors before starting second generation TKIs might predict cytogenetic response and outcome. As far as we known, the so-called Hammersmith score was not yet validated in large series of patients: we demonstrated that this score was able to discriminate patients at high risk of failure and consequent progression before treatment with second generation TKIs. Disclosures: No relevant conflicts of interest to declare.

Abstract 1752 Poster Board I-778 Iron overload (IO) occurs in a large proportion of patients affected by myelodysplastic syndromes; however data on the mechanisms responsible for IO and on the consequences on organ damages are scanty. IO in MDS may result both from ineffective erythropoiesis and from reiterative transfusions. The role of iron chelation therapy in MDS is deduced from thalassemia experience. We evaluated parameters of iron metabolism and erythropoiesis in 171 consecutive chelation naïve MDS patients. Patients were classified according WHO criteria, and prognostic scores evaluated according IPSS and WPSS criteria. CMML patients (n= 7) were also included in the analysis. Serum ferritin, iron, transferrin, transferrin saturation and non-transferrin-bound iron (NTBI) were measured. Growth-differentiation factor-15 (GDF-15) and hepcidin-25 were also determined, parallel to hemoglobin, endogenous erythropoietin (EPO) and LDH. All the parameters studied were highly variable within the analyzed cohort, as expected due to the great heterogeneity of MDS. As already observed, ferritin levels were higher than normal range in MDS patients (1091 ± 1135 ng/ml) and significantly increased in transfused ones (1890,55 ± 1833 ng/ml). EPO production was stimulated in all MDS patients (215, 40; range 85-1011 U/L), respect to normal range, and physiologically inversely correlated with hemoglobin levels (p Disclosures No relevant conflicts of interest to declare.

Abstract 2211 Poster Board II-188 Dasatinib is a 2nd generation tyrosine-kinase inhibitor active in CML patients resistant or intolerant to Imatinib; at present there is no data on its toxicity and efficacy in unselected elderly patients. To highlight this issue, 97 patients treated with Dasatinib when aged > 60 years were retrospectively evaluated from 16 Italian Centers on a “real-life” basis, including all patients treated at each Center independently from enrolment or not in controlled clinical trials.There were 52 males and 45 females, median age at Dasatinib start was 69.5 years (IR 65.0 – 73.3), Sokal Risk at diagnosis was low in 26 patients, intermediate in 37, high in 15 and not valuable in 19. Forthy-five patients (46.4%) were primarily resistant, 11 (11.4%) were intolerant and 41 (42.2%) had secondary resistance to Imatinib; all patients were in CP when Dasatinib was started. Median time from diagnosis to Dasatinib treatment was 85.0 months (IR 44.8 – 120.0); 53/97 patients (54.6%) had been pretreated with IFN ± Ara-C before Imatinib, all patients received Imatinib at standard dose (400 mg/day) followed in 50/97 (51.5%) by increased dose (600 – 800 mg/day) with an overall median period of Imatinib treatment of 48.6 months (IR 26.9 – 67.0). In addition, 28/97 patients (28.8%) received other 2nd line treatment (10 Nilotinib, 14 HU +/- other drugs, 3 Imatinib + HU or IFN and 1 allogeneic transplant) before Dasatinib. Starting dose of Dasatinib was 140 mg/day in 47 patients, 100 mg/day in 44 patients and ≥ 50 mg/day in 6 patients, respectively. After a median period of treatment of 15.6 months (IR 7.6 – 23.0) all patients were evaluable for toxicity; on the whole, grade 3 – 4 hematological and extra-hematological toxicities were reported in 36/97 (37.1%) and 27/97 (27.8%) patients, respectively. A grade 3 – 4 hematological toxicity occurred in 25/47 (53.1%) patients receiving 140 mg as compared to 10/44 (22.7%) patients receiving 100 mg (p 60 years resistant/intolerant to Imatinib; in particular, when employed at the current recommended dose of 100 mg/day, it is very effective and has a favourable safety profile also in heavily pretreated elderly subjects. Disclosures: Vitolo: Roche: . Pane:Novartis: Research Funding; Ministero dell'Università/PRIN: Research Funding; Regione Campania: Research Funding; Ministero della Salute/Progetto integrato Oncologia: Research Funding.

Dasatinib(SPRYCEL®) is a kinase inhibitor active against BCR-ABL and SRC family kinases. A phase I trial of dasatinib has shown CHR and MCyR at total daily doses of 100 mg and 140 mg daily in both BID and once daily (QD) schedule in CML-CP patients (pts). Phase II studies of dasatinib have demonstrated the efficacy and safety of a 70 mg twice daily (BID) dose in CML-CP pts with resistance or intolerance to imatinib. We analyzed safety and adverse events (AE) data in 27 pts with CML (CP), imatinib resistant (85%) or intolerant (15%), who have been treated with dasatinib from May 2006 to July 2007 in a compassionate use in Italy. A possible relationship of AE observed during dasatinib with imatinib toxicity and previous types of treatment was analyzed. Median age was 52 years at diagnosis and 58,6 years at start of dasatinib. Sokal was low in 22%, intermediate in 33%, high in 15%, not available in 30%. The median time from CML diagnosis to enrollment in the compassionate use was 78 months. Imatinib dosage, administered during the previous 3 months, was 400 mg/d in 40% of pts, 400–600 mg/d in 50% and >600 mg/d in 10% . Prior treatment for CML included interferon alpha in 48%, chemotherapy in 18%, nilotinib in 18% of the cases and stem cell transplant (SCT) in 1 case. Additional chromosome anomalies and mutations were observed in 18% and 55% of cases, respectively. Dasatinib was administered 70 mg BID, and dose escalation to 100 mg BID and reduction to 50 mg BID or 40 mg BID were allowed for inadequate response or AE. Hematological toxicity during imatinib treatment was reported in 55% of the cases as first episode, and 30% as second episode; extrahematological toxicity was also 55%. Following dasatinib treatment, 70% of the patients had some degree of hematologic toxicity with 21 events. 95% of these AE occurred in the first 2 months of treatment, with 80% in the first month. A second episode of hematolological toxicity was observed in 26% of the cases between months 2 and 15 (median mo. 5). Overall, neutropenia was observed in 11 patients (grade 4 in 5), thromobocytopenia in 11(grade 3 or less) and anemia in 4(grade 4 in one).13 patients have complained of non. hematological toxicity, with 15 AE with grade >2 occurring in the first 40 days. Further AEs were rare and non clinically relevant. The only grade 4 AE was pulmonary oedema; grade 3 AE were fever, arthralgia, liver toxicity, fluid retention; one grade 2 pleural effusion was reported. 48% of cases reduced dosage, whereas only one increased due to lack of response: this patient is now undergoing an allogeneic SCT One patient died, after stop for fluid retention, because of sepsis. All the other patients are still on therapy with a follow-up ranging from 2 to 15 months (median 6 mo). Hematological toxicity during dasatinib was not related to the number of previous treatments and to the time on imatinib. In conclusion, therapy with dasatinib is generally well tolerated, and side effects are reduced by a correct use and clinical surveillance of the patients; in addition, we found that both hematological and extrahematological toxicities decline with time and are not related to the previous treatment.

Multiple myeloma (MM) is an incurable disease due to the neoplastic proliferation of plasma cells. Histone deacetylase (HDAC) inhibitors are a novel class of agents that can induce tumour cell growth arrest, differentiation and/or apoptosis in vitro and inhibit tumour growth in animals. ITF2357, an orally effective member of the family of HDAC inhibitors, is a potent inducer of apoptosis and death of MM cells (Golay et al. Leukemia, 2007). It has also a potent inhibitory activity on secretion of pro-inflammatory cytokines by stromal and mesenchimal stem cells. For these properties, ITF2357 has been given safely to patients with Crohn’s disease and plaque psoriasis as an anti-inflammatory drug. We report the preliminary results of a phase II multiple dose clinical trial of oral ITF2357 in patients with relapsed or progressive MM, who had received at least two previous lines of treatment. Primary endpoint was to determine the maximum tolerated dose of ITF2357 administered every 12 hours for 4 consecutive days followed by 3 days of rest every week during the first cycle (i.e., first 4 weeks). Up to 12 weeks of treatment with ITF2357 were scheduled. Concomitant oral dexamethasone was given at a maximum dose of 20 mg every week. Fifteen patients (aged 52–77 years) have been enrolled so far. The first 6 patients received 150 mg ITF2357 every 12 hours for 4 consecutive days every week. Because 2 of them experienced a grade 3 gastro-intestinal toxicity (diarrhea) during the first cycle, the subsequent 9 patients received 100 mg ITF2357 every 12 hours with the same weekly schedule. None of them experienced a dose-limiting toxicity during the first cycle. The median duration of treatment was 7 weeks, range from 2 (1 patient) to 12 weeks (5 patients). Thrombocytopenia(≥ grade 2) was the most common side effect, which we observed in 11 cases: grade 4 thrombocytopenia occurred in 4 patients. Grade 3–4 gastro-intestinal toxicity was observed in 4 patients. No grade 4 neutropenia was registered. Three patients experienced 4 serious adverse events: pneumonia in one, severe deterioration of general conditions requiring hospitalization in another and both events in the last one. Two months after completing the 12 weeks of treatment one patient developed atrial fibrillation which required therapy. Two other patients had transient ECG abnormalities without need of hospitalization or therapy. We observed 1 partial remission, 4 stable diseases (SD) and 10 disease progressions. At last follow-up, 6 patients remain alive (3 in SD and 3 with disease progression) and 9 are dead (all for progressive MM). Although ITF2357 is tolerable when administered orally at the dose of 100 mg every 12 hours for 4 consecutive days every week, it is unlikely that it may play a significant therapeutic activity when used alone in advanced MM. However, we noted some evidence of anti-myeloma activity in a group of patients with deteriorated clinical conditions and advanced disease. Therefore, further clinical investigation is warranted to clarify how best to exploit this drug when given in combination with other active drugs to MM patients.

Imatinib is an effective therapy for chronic phase CML, but patients may became irresponsive due to the development of resistance, caused by amplification of the BCR-ABL genomic locus or by point mutations within the kinase domain of BCR-ABL, which prevents drug binding. Novel dual SRC/ABL kinase inhibitors with higher potency against native and imatinib-resistant mutants of BCR-ABL have substantial clinical utility, but at least one mutation remains resistant to any kinase inhibitor (T315I). Thus, the search for alternative drugs effective in CML is still cogent. Treatment of CML cells with histone deacetylase inhibitors (HDIs) of the class of hydroxamic acid analogues promotes proteasomal degradation of Bcr-Abl, associated with apoptosis, in synergy with imatinib. We evaluated whether HDIs of other classes, namely short chain fatty acids like butyrates and valproic acid, could exert the same effects and we intended to dissect the determining molecular mechanisms. The human CML cell lines K562, KBM, LAMA-84 S and LAMA-84 R and primary imatinib-resistant CML-BC cells were grown in the presence of valproic acid (VPA) at the escalating doses 0.2 mM to 2mM or the mannose ester of butyric acid D1 (0.2–1mM) for 24 and 48 hrs. Apoptosis was induced in a time and dose dependent manner by VPA and D1 (annexin V test and flow cytometric analysis after propidium iodide uptake). Imatinib was synergistic with both HDIs in inducing apoptosis and cell proliferation arrest (MTT-assay). VPA and D1 were able to induce after 48 hrs of incubation a significant decrease in the number of copies of BCR-ABL determined by real time-PCR, paralleled by a substantial decrease in Bcr-Abl protein expression, shown in western blots of total cell lysates from CML cells. This decrease in the expression of protein kinase could account for the synergy with imatinib, but also for the reversal of resistance in mutated Bcr-Abl CML cells and is consistent with what previously observed. We also analysed the expression of Hsp-90 (protein chaperone of Bcr-Abl) and found it quantitatively unmodified but hyperacetylated by the treatment with both HDIs. As little is known of the ability of short chain fatty acids to induce acetylation of non-histone proteins, we compared the acetylated proteome of CML cells treated and not treated with HDIs, alone and in combination with imatinib, by 2D western blot versus a pan-acetylated antibody, followed by MALDI-TOF mass spectrometry for protein identification. 22 proteins were positively identified with a high degree of confidence, with the majority of these being cytoplasmic. At least two chaperone proteins were identified as target of acetylation after VPA and D1 treatment of CML cells, other targets were proteins involved in the synthesis and stability of RNA. Phosphorylation of proteins, evaluated by 2D western blot, was not significantly affected by HDIs. Short chain fatty acids are indeed not the most potent HDIs, but have been used successfully in clinical trials. Our observations contribute to the dissection of proteome modifications by HDIs and may help extrapolate the molecular effects of different HDIs on CML cells so to improve their use as single drugs or in combination with imatinib or new SRC/ABL inhibitors.

Acute myeloid leukemia (AML) is a disease characterized by a block of maturation. Genes coding for core binding factors are rearranged in a considerable subset of AML cases and result in an altered interaction of core binding factor (CBF) subunits with transcriptional coregulators (NCoR/SMRT). Recruitment of histone deacetylase is also altered in AML, and a subsequent transcriptional repression of target genes involved in myeloid maturation is determined. We determined here the effects of two histone deacetylase inhibitors, sodium butyrate and the stable prodrug xylitol butyrate derivative (D1), on a t(8;21)-positive cell line (Kasumi-1) as well as primary AML blasts. Exposure (24-96 h) to butyrates (1 mM) of Kasumi-1 cells induced histone H4 acetylation, whereas H3 acetylation was unchanged. Induction of morphological and immunophenotypic granulocytic maturation (96 h), also confirmed by an increased expression of CAAT/enhancer binding protein alpha, was observed. Inhibition of proliferation and apoptosis via activation of caspase-9 was also observed. In primary AML blasts, butyrates (0.5 mM) increased histone H4 acetylation of 18 of 19 cases tested. Terminal granulocytic maturation was observed in all cases (5 of 5) characterized by chromosomal translocations involving CBF, whereas in non-CBF cases, maturation was incomplete (4 of 8) or absent (4 of 8). Our data indicate the possibility to effectively remove, in CBF AML cases, the maturation block generated by histone deacetylase stable recruitment, contributing to a possible development of molecularly targeted therapies of AML.

Abstract 1118 Poster Board I-140 Background The median age of an unselected population of Ph+ CML patients is close to 60 years. In the prognostic classifications (Sokal, Blood 1984; Hasford, JNCI 1998) that were elaborated before the introduction of IM, age was a significant and important prognostic factor. The most recent IM studies have not clarified the prognostic importance of age and IM therapy is still denied to several elderly patients. Aim to asses the relationship between age (less and more than 65 years) and outcome, in CML patients treated front-line in early chronic phase (ECP). Methods We analyzed the data of 559 previously untreated ECP patients who were assigned to receive IM 400 mg daily (76%) or 800 mg daily (24%) in three controlled, prospective studies of GIMEMA (Clin Trials Gov. NCT00514488 and NCT00510926; and an observational study of IM 400 mg). The median follow-up is currently 42 (extremes 1 – 64) months. There were 115 patients more than 65 years old (median age 71 years), while 444 (79%) were less than 65 years (median age 46 years). The proportion of patients who were treated with IM 800 mg daily was the same in both age groups. Results The cumulative complete cytogenetic and major molecular response rates were identical in the two age groups (88% vs 88% and 82% vs 83%, respectively). However, overall survival (86% vs 93%, p = 0.01), failure-free survival (72% vs 81%, p=0.03) and particularly event-free survival (calculated based on the intention-to-treat principle, where events were any failure [according to the European LeukemiaNet criteria – Baccarani, Blood 2006] and treatment discontinuation for any cause) (60% vs 71%, p=0.006) were significantly inferior in the older age group. All these difference were mainly due to comorbidities leading to more deaths in CP (table). Conclusions/Methods These data show that response to IM was not affected by old age. Survival curves were affected because of age-related complications and comorbidities. Age should never be a contraindication to IM treatment. Acknowledgments: European LeukemiaNet, COFIN, University of Bologna and BolognAIL. Disclosures Saglio: Novartis: Honoraria. Baccarani:Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Mayer Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau.