ALBERT

Description: Background and rationale In chronic myeloid leukemia (CML) about half of patients (pts) achieving a deep and stable molecular response (MR) with tyrosine kinase inhibitors (TKIs) may discontinue TKI treatment without disease recurrence. As such, treatment free remission (TFR) has become an ambitious goal of treatment. Given the evidence that deepness and duration of molecular response are necessary but not sufficient requisites for a successful TFR, additional biological criteria to possibly identify more and better CML patients suitable for an efficacious discontinuation are today focus of research in CML. Leukemia stem cells (LSCs) are supposed to be the reservoir of disease. We first showed in a cross-sectional study including 112 pts in TFR for a median of 31 months (mos) that residual circulating CD34+/CD38-/CD26+ CML-specific LSCs were still detectable in the majority of CML pts despite stable and deep molecular response. This evidence suggested that the level of BCR-ABL transcript only may not reflect the actual residual CML LSCs burden and that there could be a "threshold" of LSCs predicting a successful TFR. Aims To further study the behavior of residual LSCs during TKI discontinuation we designed a prospective multicentered study (AIRC IG 20133 study) in which we monitored circulating CD26+ LSCs in CML pts from the time of TKI discontinuation until molecular relapse. Methods CML pts meeting the current molecular criteria for TKI withdrawal entered this multicenter study. At TKI stop (baseline) and at +1, +2, +3, +6, + 12 mos after discontinuation and at any time if molecular relapse, CML pts were evaluated for peripheral blood number of CD34+/CD38-/CD26+ LSCs by centralized flow-cytometry analysis and for BCR-ABL transcript level by standard (IS) quantitative RT-PCR assay. Results 49 consecutive CML pts were enrolled in the study so far. Pts characteristics at diagnosis, type of TKI, disease response and treatment duration before discontinuation are shown in Tab. 1. After a median time of 7 mos since TKI stop (range 1-24), 13/49 (26.5%) pts lost their molecular response and restarted TKI treatment. Median time to relapse after discontinuation was 4 mos (range 2-7). 36/49 (73.4%) pts are still in TFR after a median time of 7.5 mos (range 1-24). If considering a cut-off of 6 mos from discontinuation as the period with higher risk of relapse, 14/36 pts actually in TFR have discontinued treatment for ≤ 6 mos (range 1-6) while 22/36 pts are in TFR for a median of 10 mos (range 7-24). Regarding residual CML LSCs evaluation, at baseline 23/49 (46%) pts had still measurable circulating CD26+LSCs with a median number of 0.0204µ/L (range 0.0077-0.1197), while 26/49 (54%) had no detectable CD26+ LCSs. Considering the small number of molecular relapses no statistical difference in number of residual CD26+ LSCs at time of discontinuation was shown between pts losing vs maintaining TFR (13 pts median CD26+ LSC 0.0237/µ/L, range 0-0.1197 and 36 pts median CD26+ LSCs 0.0204/µ/L, range 0-0.1039, respectively). However, the number of pts with undetectable CD26+ LSCs at baseline was 6/13 (45%) and 20/36 (55%) in the two subgroups, respectively. Considering subsequent time points, the 13 relapsed pts showed a small yet progressive increase of residual CD26+ LSCs number until molecular relapse, while the 36 pts in TFR showed a fluctuation of CD26+ cells number. However, Kendall rank correlation coefficient, Mood test and bi-linear relation model of the whole cohort showed no correlation between BCR-ABL/ABLIS ratio and number of residual CD26+ LSCs either at baseline or at each time points after discontinuation, thus confirming our previous observations. Conclusions Yet very preliminary our results showed that CD26+ LSCs are detectable at time of TKI discontinuation and during TFR. Moreover, at least for the observation median time of the study (7.5 mos) the persistence of "fluctuating" values of residual CD26+ LSCs do not hamper the possibility to maintain a stable TFR. Due to the short follow up and the small number of molecular relapsed pts we could not find a threshold of CD26+ LSCs predictive of TFR loss. Our data may suggest other factors then LSCs "burden" to play an active role in controlling disease recurrence. Additional studies evaluating CD26+ LSCs ability to modulate the immune system through a variable expression of immune response inhibitory molecules and through their interactions with effectors cells are ongoing. Table Disclosures Bocchia: Novartis: Honoraria; Incyte: Honoraria; BMS: Honoraria. Pregno:Bristol Myers Squibb: Honoraria; Incyte: Consultancy, Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Abruzzese:Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy. Crugnola:Novartis: Honoraria; Incyte: Honoraria. Iurlo:Pfizer: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Galimberti:Roche: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau. Liberati:Bristol & Mayer: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria.

Description: Although the success of imatinib mesylate therapy represents an exciting advance in targeted cancer therapy, it has still to be determined whether responses to this p210 inhibitor in chronic myeloid leukemia (CML) patients will be durable. In fact most of clinical studies agree on the evidence of a persistent molecular disease in the majority of treated patients and altough the absolute level of bcr-abl transcript may vary over the treatment, yet a molecular complete response is of rare observation. In addition, discontinuation of imatinib exerts always in rapid loss of response. In accordance to this the persistence of malignant progenitors in patients in complete cytogenetic response (CCR) after short term imatinib treatment, has been recently demonstrated. In particular, Bathia et al. showed in 12/15 patients studied after a median time of 10 months of imatinib treatment a median of 11% of residual CML CD34+ progenitors in the bone marrow (by FISH Dual Fusion bcr/abl analysis)while only 3/15 patients had no detectable residual CD34+ cells. Less is known about residual Ph+/CD34+ cells surviving after a prolonged therapy with this targeting drug. Thus, we evaluated the amount of bone marrow residual CD34+ cells in 17 CML patients in stable CCR after a long lasting treatment with imatinib. At the time of evaluation, the patients were on conventional dose (400mg) Imatinib for a median time of 48 months (range 36–58 months) having achieved a CCR status (conventionally defined as the complete absence of t(9;22) on caryotypic analysis) within 3 to 6 months of treatment. However all of them still showed molecular disease as detected by nested RT-PCR. Bone marrow CD34+ cell-enriched populations were selected from mononuclear cells using immunomagnetic column separation and were evaluated after cytospin by FISH using a bcr-abl Dual Color Extra Signal Probe(LSI bcr-abl ES, Vysis), that is able to detect bcr-abl fusion in interphase nuclei with a false positive signal rate close to 0. A minimum of 100 CD34+ nuclei per each sample were evaluated. Interestingly, in 8/17 patients no Ph+/CD34+ cells were detected, while in the remaining 9/17 patients a median of 2% (range 0.5–11%) of bcr-abl positive progenitors were still observed. In this small selected serie of patients prolonged treatment with imatinib appears to be correlated with a lower, yet detectable, amount of residual bone marrow Ph+/CD34+ cells when compared to previously published data. This result could be partly explained with the different specificity and sensitivity of the probe used (bcr/abl ES

Description: We have shown that vaccinations with a p210-derived peptide vaccine (CMLVAX100) induced a strong peptide-specific CD4+T cell proliferation in the majority of vaccinated chronic myeloid leukemia (CML) patients. CD4+ T cell response was strictly mediated by the longest peptide included in the vaccine (b3a2-25) and correlated with disease response as about 65% of vaccinated patients showed reduction of minimal residual disease still persisting during imatinib treatment. The role of b3a2-25-peptide specific CD4+ T cells as potential mediator of antitumor response has not yet been fully elucidated. The majority of vaccine-induced T cells resulted to be CD4+/CD25+/Foxp3+, but despite this phenotype they showed no regulatory/inhibitory activity on naïve T cells. A smaller proportion resulted instead to be perforin+, thus potentially cytotoxic. To evaluate if b3a2-25-peptide specific CD4+T cells could exert a direct cytotoxic effect, we used CML-derived JURL-MK2 cell line expressing b3a2-p210 as target. Effector cells were b3a2-25-specific CD4+ T cells freshly isolated from 3 vaccinated patients and further in vitro expanded in the presence of IL-2 and b3a2-25 peptide. After in vitro expansion, peptide-specific CD4+/CD25+/Foxp3+ population represented about 60% of total CD4+ isolated cells, while peptide-specific CD4+/perforin+ cells counted for about 9%. Cellular cytotoxicity measurement was then carried out by flow cytometry using a fluorescent dye to label target cells and a fluorescent-DNA dye to determine the dead cells. Briefly, 1×104 JURL target cells labelled with DIOC18 green fluorescent dye were mixed with b3a2-25-specific CD4+ T (effector cells) at various E:T ratio (5:1, 10:1, 20:1, 35:1) and incubated 4 hours at 37°C. After co-incubation, Propidium Iodide (PI) red fluorescent was applied and the samples were run on flow cytometer for the determination of DIOC+/PI+ dead cells. Spontaneous cell death was determined by incubation at 37°C of target cells alone and maximum cell death was determined by incubation of target cells with 2% paraphormaldeide. Control experiments included different effector cells: freshly isolated CD4+ T cells from the same 3 vaccinated patients not further in vitro expanded (in this experimental condition the percentage of b3a2-25-specific CD4+/CD25+/Foxp3+ is about 10% of total CD4+ cells while b3a2-specific CD4+/perforin+ is about 2%); CD4+ T cells in vitro stimulated with IL-2 (without b3a2-25 peptide) from healthy subjects or from not previously vaccinated CML patients. Our results showed a specific killing of JURL-MK2 cells only in the presence of expanded peptide-specific CD4+ T cells from all 3 vaccinated patients with a linear increase of DIOC+/PI+ target cells from 5.3% (E:T 5:1) to 33% (E:T 35:1). No cytotoxicity was observed when CD4+ cells were expanded from healthy donors of from not vaccinated patients ruling out the possibility of killing mediated by a “non specific” activation of CD4+ cells due to IL-2 exposure. On the contrary, specific cytotoxicity appeared to correlate to the increased percentage of peptidespecific CD4+ cells obtained after in vitro re-stimulation with b3a2-25 peptide, as only background killing was observed when freshly isolated CD4+ T cells from all 3 vaccinated patients were cultured with JURL-MK2 cells. In conclusion, CMLVAX100 induced b3a2- 25-peptide specific CD4+ T cells appear to exert direct cytotoxity toward b3a2-CML JURL-MK2 cells. To our knowledge this is the first time that CML-peptide specific CD4+/cytotoxic T cells are induced in vivo and they could mediate the minimal disease reduction observed after CMLVAX100 vaccinations in CML patients. Experiments focused on determining which subtype, CD4+/CD25/Foxp3 or CD4+/perforin+, is the main effector of cytotoxicity as well as experiments clarifying if CD4+ cytotoxicity is mediated by HLA-DR molecules presentation of b3a2-p210 derived peptides are ongoing.

Description: Rituximab 375 mg/sqm weekly for 4 weeks has significant activity in patients with idiopathic thrombocytopenic purpura (ITP). In this setting, different biological and clinical evidence suggests the possible use of lower doses of Rituximab. Twenty-eight adult patients, median age 43 years (range 16–71 years), with previously treated, active and symptomatic ITP were treated prospectively with Rituximab at the fixed dose of 100 mg iv weekly for 4 weeks. Exclusion criteria were positive HIV, HBsAg and pregnancy test, any B-cell lymphoprolipherative disease or other malignancies. Response assessment was evaluated considering the rate of overall and complete responses (OR = platelet count ≥ 50 x 109/L; CR= platelet count ≥ 100 x 109/L and discontinuation of steroid therapy, if present), the time to response (TTR; time necessary to reach a platelet count ≥ 50 x 109/L), the time to complete response (TCR; time necessary to reach a platelet count ≥ 100 x 109/L) and the duration of response. B-cell count and pharmacokinetics (PK) were monitored and related with clinical outcome. Stepwise logistic regression was used to assess whether response was associated with age, gender, diagnosis-Rituximab interval and basal CD20+ve lymphocytes. Results were considered statistical significant when P ≤ 0.05. All patients completed the therapeutic program receiving the four infusions of Rituximab as scheduled, none experiencing short term toxicity. All patients achieved B-cell depletion. OR and CR were achieved in 21/28 (75%) and 12/28 (43%) patients, respectively. CR rate was associated with younger age (OR=0.92 CI95%[0.85;0.99]). The median TTR and TCR were 31 and 44 days, significantly longer then those observed with standard dose in patients with similar characteristics (Haematologica2003;88:538). After a median follow-up of 11 months (range 3–18), 7/21 (33%) patients relapsed, and 3 needed further treatments. PK data showed a concentration time-course profile of Rituximab that was super-imposable, once corrected for the difference in the dose, to that observed previously in patients treated with standard dose, diseases with a median value of 4.1 micrograms/milliliter (range 0–11.9), 12 weeks after the start of treatment. In patients with ITP, low dose Rituximab led to short and mid-term response rates similar to standard dose but with slower timing of response.

Description: In the past five years, the body of data concerning imatinib mesylate had certainly defined the role of this drug as very effective first line debulking therapy for CML patients. However, both the persistence of molecular disease in most of patients together with the evidence that discontinuation of imatinib inevitably exerts on a rapid loss of response, suggest that with imatinib alone the cure of CML is unlikely. CMLVAX100 is a peptide-based vaccine that specifically targets p210-b3a2 in CML cells through the induction of a peptide-specific T cell response in b3a2-CML vaccinated patients. We recently published the preliminary results of a pilot study testing the immunological response and antitumor activity of vaccinations with CMLVAX100 in 10 patients showing persistent residual disease during treatment with imatinib. We here report the results on a larger cohort of patients and with a longer follow up. Up to date 21 patients were enrolled in this study. After a median time of 24 months (range 12–50) of imatinib treatment, patients started vaccinations with CMLVAX100 showing different degrees of persistent residual disease: 8/21 patients presented with molecular disease, 10/21 showed residual cytogenetic disease (range 2–45% of Ph+ metaphases), while 3/21 presented only an hematological response (100% of Ph+ metaphases). No improvement of their residual disease was documented for a median of 12 months (range 6–24) before starting vaccinations. Vaccine treatment plan included 6 vaccinations at 2 weeks interval (immunization) and for responder patients additional boosts of vaccine every 4–6 months. So far 18/21 patients completed the immunization and are evaluable for response; in addition 8/18 received 4 further boosts of vaccine. After immunization, CMLVAX100 induced a prompt immunological response in most of the patients as 17/18 patients showed peptide specific T cell response in vitro and 12/18 developed a positive delayed type hypersensitivity response in vivo. After 6 vaccinations 6/10 patients with persisting cytogenetic disease reached a complete cytogenetic response (CCR), with 3 of them achieving an undetectable level of bcr-abl transcript. In addition, 3/5 evaluable patients starting vaccinations with persistent molecular disease, further reduced their bcr-abl level, wth one reaching molecular negativity. None of the 3 patients vaccinated in hematological remission had cytogenetic response. Of the 8 patients who underwent 4 additional boosts of vaccine, one reached a complete molecular response, 5 maintained the response obtained after immunization, while 2 patients who previously achieved an undetectable level of bcr-abl transcript, lost the complete molecular response, while maintaining CCR, thus suggesting that a 6 months interval between boosts could be too long to maintain an efficient immune control on residual leukemic cells. In conclusion, we confirm that CMLVAX100 has a synergistic antitumor effect with imatinib in CML patients with persistent minimal residual disease. However, follow-up data recommend that closer boosts of vaccine are necessary in order to maintain an optimal level of response.

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

Description: Introduction. The application of Pediatric-Type Therapy (PTT) programs to adults with ALL can improve outcome significantly despite higher age-related toxicity. Recent series reported survival rates ≥ 50%, but only few combined PTT with Minimal Residual Disease (MRD) study for risk-oriented Hematopoietic Cell Transplantation (HCT) and/or explored the value of specific PTT element such as higher dose, lineage-targeted MTX up to 5 g/m2. Methods. To improve over prior data, NILG protocol 10/07 (Clinical.Trials.gov NCT-00795756) for unselected adult patients aged 18-65 years combined PTT together with MRD study for risk/MRD-based HCT. The 8-course program consisted of a 5-drug complete remission (CR) induction (cycle no. 1; imatinib added if Ph+) followed by 3 modified BFM blocks (no. 2, 4 and 6), 3 lineage-targeted MTX blocks (no. 3, 5 and 7; MTX 5 g/m2 for T-ALL and 2.5 g/m2 for B-ALL [1.5 g/m2 if age 〉 55 years or Ph+]; no. 3 and 7 with high-dose Ara-C 2 g/m2 x4, no. 5 with L-Asp 10,000 IU/m2 x2) and reinduction (no. 8). CNS prophylaxis was with triple intrathecals or liposomal cytarabine (Haematologica 2015;100:786). MRD was studied molecularly with sensitive probe(s) (sensitivity 10-4 or greater) on marrow samples obtained at end of induction (week 4, w4) and after cycles 3 (w10), 5 (w16), 7 (w22) i.e. after 1st, 2nd and 3rd lineage-targeted MTX block. Patients were risk-stratified at diagnosis and after MRD analysis for the purpose of allocation to HCT or conventional maintenance. The HCT allocation cohort consisted of predefined very high-risk patients (vHR: WBC 〉100, highly adverse cytogenetics, pre-T/mature T-ALL) regardless of MRD, of HR patients without MRD study (HR: late CR; B-ALL with WBC 〉30 or pro-B phenotype), and of HR or standard-risk (SR) patients with MRD ≥ 10-4 at w10/16 or positive at w22. Conversely, the maintenance allocation cohort consisted of SR and HR patients with MRD 〈 10-4 at w10/16 and negative at w22 and of SR patients without MRD study. A family related/unrelated donor search was activated at diagnosis in order to proceed to HCT soon after cycle no. 3 when needed. Results. 205 patients were enrolled, with a median age of 41 years (range 17-67 years, 11% 〉 60 years). 55% were male, 42 had Ph+ ALL, 119 Ph- B-ALL and 44 T-ALL. Of 163 patients with Ph- ALL, 45% were SR, 13% HR and 42% vHR. CR rate was 98% in Ph+ ALL and T-ALL, and 83% in Ph- B-ALL (88% vs 58% in patients ≤ vs 〉 60 years, P .0013). The MRD study was successful in 109/142 CR patients with Ph- ALL (77%), contributing to the final risk classification in 63 patients, of whom 41 were MRD responsive (65%) and 22 MRD resistant (35%). Altogether, 55 CR patients constituted the maintenance allocation group (39%) and 87 the HCT allocation group (61%), which included mainly vHR patients (n=61, 43%) selected for HCT independently of MRD study results. According to intention-to-treat, median OS is not reached (53% at 5 years, figure) and median DFS is 4.8 years (48% at 5 years). In Ph- ALL, 5-year OS/DFS are 74%/61% in T-ALL (medians not reached) and 48% each in B-ALL (medians 3.9 and 4.7 years). Median OS is not reached in both HCT and maintenance allocation groups (58% and 73% at 5 years, respectively, P .078), with a median DFS of 4.7 years (48% at 5 years) versus not reached (59% at 5 years) (P .19). Treatment adherence was good with some exceptions in maintenance allocation group (6 HCT, 11%) and a transplant realization of 68% (53 allogeneic; 6 autologous) in HCT allocation group. With HCT, 5-year incidence of nonrelapse mortality was 17%. The MRD analysis proved that DFS of patients achieving an MRD response 60 years. 5-year OS and DFS of 55% and 52% respectively in Ph- patients aged up to 65 years represent an improvement over prior NILG study (5-year OS and DFS of 36% and 35% respectively). MRD was essential in orientating the HCT choice in SR and HR patients and retained a major prognostic role in all patients. Optimizing the early MRD response with new immunotherapeutics and clarifying the role of HCT in MRD responsive vHR patients are some relevant topics of future research. Figure Figure. Disclosures Ciceri: MolMed SpA: Consultancy. Vitolo:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gallamini:Millenium Takeda: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 3413 Introduction Imatinib mesylate is highly effective in inducing rapid hematologic and cytogenetic responses in the vast majority of chronic myeloid leukemia (CML) patients. Yet, discontinuation of treatment is associated with disease relapse probably due to the persistence of resistant leukemic stem cells representing a reservoir of the disease. On this regard it has been reported that BCR-ABL positive progenitor cells can still be detected in patients in complete cytogenetic response (CCyR) after short term of imatinib treatment (Bhatia R, et al. Blood. 2003;101:4701-4707) but also after a stable long lasting CCyR (Bocchia M, et al. Leukemia. 2008;22:426-428). Compared to imatinib, the second generation Tyrosin Kinase Inhibitor (TKI) nilotinib appears to eradicate more rapidly the bulk of CML cells, inducing high rate of CCyR and major molecular response (MMolR) after a very short period of treatment (78% CCyR and 52% MMolR at 3 months) (Rosti G, et al. Blood. 2009;114(24):4933-8). Despite nilotinib is a more potent TKI, it didn't appear to be more effective in eliminating in vitro CML progenitors than imatinib (Konig H, et al Leukemia. 2008;22:748-755). Up to date no data evaluating the persistence of Ph+ stem cells in early chronic phase CML patients during first line treatment with nilotinib have been reported. Patients, materials and methods We investigated the presence of residual CD34+/Ph+ cells in 24 CML patients in CCyR during first line nilotinib treatment. Patients were enrolled in 2 clinical trials (GIMEMA CML0307 and CAMN107A2303) and evaluation of residual leukemic stem cells was performed during a routine bone marrow aspirate after receiving specific patients informed consent. Bone marrow purified CD34+ cells were evaluated for BCR-ABL fusion gene by fluorescent in situ hybridization (FISH) analysis. A minimum of 100 interphase nuclei of purified CD34+ cells was considered optimal for FISH analysis. Results All 24 patients have been treated exclusively with nilotinib since diagnosis (17/24 at 400mg bid; 5/24 at 300mg bid; 2/24 at 400mg/day). At the time of analysis all 24 patients were in CCyR for a median time of 27 months (range 6–29) after being treated for a median time of 30 months (range 9–30) with this second generation TKI. Regarding molecular response 20/24 (83%) were in MMolR while only 1/24 (4%) was in CMolR. Harvest, purification and subsequent FISH analysis of bone marrow CD34+ cells was optimal in 15/24 (63%) patients, suboptimal in 5/24 (21%) patients (less than 100 interphase nuclei analyzed) and not adequate in 4/24 (16%) patients (less than 50 interphase nuclei). With respect to leukemic stem cells, residual CD34+/Ph+ cells were found only in 1/20 (5%) evaluable patients. Of note, in this patient 140 CD34+ interphase nuclei were analyzed and only 1 was found bcr-abl positive (0.7%). Conclusion Our study shows for the first time that in patients in CCyR during front line Nilotinib treatment residual CD34+/Ph+ stem cells are very rarely detected. These results quite differ from what was previously found in imatinib treated patients (Bocchia M, et al. Leukemia. 2008;22:426-428). In fact in the present series, only 1/20 (5%) patients treated with nilotinib in CCyR for a median time of 27 months showed residual CD34+/Ph+ cells, while in our prior study residual leukemic CD34+ cells were still detectable in 14/31 (45%) imatinib treated patients in stable CCyR (median of 39 months). Despite the limited number of patients studied, this novel evidence may support the better short term clinical results observed with nilotinib as first line treatment in CML. Disclosures: Rosti: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Roche: Speakers Bureau.

Description: Background: The European LeukemiaNet (ELN) response criteria are widely used to decide, at given time points, when the treatment with tyrosine-kinase inhibitors (TKIs) of CML patients should be continued (optimal response, OR), when a careful monitoring is required (warning, W) or when the therapy should be changed (failure, F). The 2013 ELN response criteria are the same for all chronic phase CML patients, irrespective of the prescribed TKI, but the time to response is influenced by the first-line TKI. Despite faster responses, a clear survival advantage of 2nd generation TKIs over imatinib (IM) has not been demonstrated yet. A validation of the 2013 ELN response definitions and an analysis of their prognostic impact in IM-treated patients may provide important information. Aims: The aim of our study was to assess the significance of 2013 ELN response criteria in CML patients treated frontline with IM, investigating whether or not optimal responders, warnings or failures at 3, at 6 and at 12 months have a different long-term outcome. Methods: 559 patients enrolled within 3 prospective clinical trials (NCT00514488, NCT00510926, observational trial CML/023) were analyzed (ITT population of each study). The 3-month response according to 2013 ELN criteria was not fully evaluable due to missing cytogenetic analysis in 452/559 patients, so we focused on the early molecular response (EMR, BCR-ABL 〈 10% at 3 months), corresponding to OR. The responses at 6 and 12 months were retrospectively defined according to 2013 ELN criteria: F, BCR-ABL 〉 10% and/or Ph+ 〉 35% at 6 months, BCR-ABL 〉 1% and/or Ph+ 〉 0 at 12 months; OR, BCR-ABL 〈 1% and/or Ph+ 0 at 6 months, BCR-ABL 〈 0.1% at 12 months; W: intermediate conditions. As the ELN criteria changed over time, not all the failures switched to alternative treatment. Progression: transformation to advanced phases (2013 ELN definitions) at any time, including after treatment discontinuation. Overall survival (OS): all the deaths at any time (in-study or off-study) were included. Leukemia-unrelated death: known cause of death, no progression, CCyR and/or MMR 〈 6 months prior to death; all other deaths were classified as leukemia-related (LRD). The cumulative incidence of LRD was estimated considering the competing risk of leukemia-unrelated death. Results: The median follow-up was 76 months (66-99 months). The patients with OR at 3 months were 82%; the patients with OR-W-F at 6 months were 76%, 14% and 10%, respectively; the patients with OR-W-F at 12 months were 65%, 20% and 14%, respectively. The OS, the progression-free survival (PFS) and the cumulative incidence of LRD according to the presence-absence of EMR were 87-81% (p=0.015), 85-81% (p=0.035) and 11-5% (p=0.019), respectively. Combining the Sokal score and the EMR, the patients were divided into 4 groups, low and intermediate risk/responders, low and intermediate risk/not responders, high risk/responders, high risk/not responders: the OS and the cumulative incidence of LRD across the 4 groups were 88%, 84%, 86% and 70% (p=0.005, Figure 1) and 3%, 9%, 10% and 20% (p