Abstract: Background Tyrosine kinase inhibitors (TKIs) are the standard of care for patients (pts) with CML-CP. The current recommendation is to continue TKI therapy indefinitely.1 Results of several clinical trials indicate that pts achieving sustainable deep molecular response (DMR; defined as molecular response ≥ MR4) on imatinib (IM) may achieve long-lasting TFR. Nilotinib (NIL) at 300 mg bid induces higher rates of DMRs compared to IM.2 Also, DMRs can be achieved in pts with NIL (400 mg bid) who are switched after long-term IM.3 However, the optimal duration of consolidation treatment with NIL to increase the chances for successful and continuous TFR (≥ MR4) after stopping treatment is not yet known. Objective ENESTPath was designed to assess the proportion of pts (pretreated with IM and subsequently treated with NIL 300 mg bid) who can achieve a sustained DMR and maintain TFR without relapse for 12 months (mo) upon treatment discontinuation after different durations of treatment in consolidation phase. Methods ENESTPath is a randomized, phase 3 study enrolling pts with CML-CP who achieved a complete cytogenetic response (CCyR), but not MR4, after at least 24 mo of treatment with IM. After enrollment, pts were assigned to receive NIL at 300 mg bid for either 24 mo or 36 mo (arm 1 and arm 2, respectively). Pts with stable MR4 or better for at least 12 mo will enter the TFR phase. A stable MR4 was defined by 4 of the 5 preceding quarterly real-time quantitative RT-PCR (RQ-PCR) assessments ≥ MR4 and ≥ MR4 inthe last assessment performed by IS certified EUTOS laboratories. Results A total of 619 pts were enrolled in the study between May 2013 and June 2015. The present analysis reports the results of the first 300 pts (mean age, 50.8 years; 63.7% of males) enrolled and treated with NIL for 24 mo in induction and consolidation phase or who had discontinued earlier. Details of the baseline characteristics are given in Table 1. At data cutoff, 108 pts were in stable MR4; 101 (33.7%) were randomized and 7 (2.3%) were scheduled for randomization at that time point. 192 pts (64%) were not eligible for randomization, primarily due to lack of stable MR4 in 126 pts (42%), adverse events (AEs) or abnormal laboratory values in 44 pts (14.7%), and for other reasons in 22 pts (7.3%). The rates of MR4 at baseline*, 6 mo, 12 mo, 18 mo, and 24 mo were 14.3%, 43.3%, 45.7%, 43.7%, and 46%, respectively. By 24 mo of treatment with NIL, cumulative incidences of major molecular response (MMR), MR4, and MR4.5 of all treated pts not in respective MR at baseline were 93.2%, 69.3% and 42.1%, respectively (Figure 1). Further analysis showed that pts with MMR at baseline had a higher probability of achieving an MR4 than those lacking MMR at baseline, with a cumulative incidence of MR4 by 24 mo of 75.8% and 44.2%, respectively. No new safety signals were observed during the 24 mo consolidation with NIL. The majority of the AEs were low grade. Most common AEs irrespective of the relationship to the study drug were pruritus (19%), hypercholesterolemia (14.0%), rash (10.7%), asthenia (10%), and arthralgia (10%). The most common newly occurring or worsening all-grade biochemistry laboratory abnormalities included increase in total cholesterol (68.7%), increased ALT (54%), hyperglycemia (32.7%), and hyperbilirubinemia (37%); majority of them were grade 1 and 2. Newly occurring or worsening all-grade cytopenias include anemia (12.7%), thrombocytopenia (2.3%), leukopenia (2%), and neutropenia (1%). Grade 3 or 4 cardiovascular events (CVEs) were experienced by 6.7% of pts including ischemic heart disease (4.7%), peripheral artery occlusive disease (1.7%), and ischemic cerebrovascular events (0.7%) (Table 2). Conclusions This analysis of the first 300 pts after 24 mo of NIL treatment showed a cumulative incidence of MR4 in ~ 70% of pts who were not in MR4 at baseline with an advantage in favor of MMR at baseline. 108 pts (36%) were with stable MR4 at data cutoff and 192 pts (64%) discontinued the study due to very stringent protocol definitions of eligibility for randomization and AEs. Grade 3 or 4 AEs were consistent with the previous reports.4 References 1. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Chronic Myelogenous Leukemia V.1.2016 ©2016 National Comprehensive Cancer Network, Inc. 2. Hochhaus A, et al. Leukemia. 2016;30:1044-1054. 3. Hughes TP, et al. Blood. 2014;124:729-736. 4. Rea D, et al. Blood. 2015;125:[abstract 4040]. Disclosures Rea: Ariad: Honoraria; Pfizer: Honoraria; BMS: Honoraria; Novartis: Honoraria. Rosti:Roche: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Cross:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau. Niederwieser:Novartis: Research Funding, Speakers Bureau; Amgen: Research Funding, Speakers Bureau. Pregno:Novartis: Honoraria; BMS: Honoraria; ARIAD: Honoraria. Orlandi:Ariad: Honoraria; BMS: Honoraria; Novartis: Honoraria. Almeida:Celgene: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Shire: Speakers Bureau; Alexion: Speakers Bureau. Illes:University of Debrecen faculty of medicine department of hematology: Employment. Sagues:ICO-Girona/hospital Universtiari de Girona Dr. Josep Trueta: Employment. Haenig:Novartis: Employment. Supekar:Novartis: Employment. Shah:Cognizant: Employment; Novartis: Other: Vendor. Saglio:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Steegmann:Aria: Honoraria, Other: Research funding for Spanish CML Group; BMS: Honoraria, Other: Research funding for Spanish CML Group; Novartis: Honoraria, Other: Research funding for Spanish CML Group; Pfizer: Honoraria, Other: Research funding for Spanish CML Group. Baccarani:Pfizer: Consultancy, Honoraria, Speakers Bureau; Ariad: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau.

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

Abstract: Background Tyrosine kinase inhibitors (TKIs) are the standard of care for pts with CP-CML. Current recommendation is to continue TKI therapy indefinitely but previous studies indicate that pts with deep and sustained molecular responses (MRs) on imatinib (IM) may achieve long-lasting TFR. Nilotinib (NIL) at 300mg BID induces higher rates of deep MRs compared to IM and high dose NIL (400mg BID) enables a substantial proportion of pts who do not obtain MR4 (BCR-ABL1IS £ 0.01%) or MR4.5 (BCR-ABL1IS £ 0.0032%) with IM to reach such deep MRs levels, potentially compatible with TFR. However, optimal duration of treatment with NIL to ensure the highest rate of TFR after treatment discontinuation is unknown. Objective ENESTPath was designed to assess the optimal duration of NIL therapy that is necessary to achieve and maintain TFR upon treatment discontinuation in pts pretreated with IM. Methods ENESTPath is a randomized, phase III study enrolling CP-CML pts who after at least 2 years (yrs) of IM therapy achieved a complete cytogenetic response (CCyR), but not yet a MR4. After enrollment, pts were assigned to receive NIL at 300 mg BID for 2 yrs or 3 yrs (Arm 1 and Arm 2, respectively). Patients who will obtain a stable MR4 or better for at least 12 months (mo) will enter the TFR phase. Primary endpoint is to evaluate the proportion of pts in both arms who will remain in TFR for ≥1 yr after NIL discontinuation. Results 620 pts were enrolled in the study between May-2013 & Apr-2015. In this interim analysis, the first 300 pts (mean age 50.8 yrs; 63.7% male) enrolled and treated with NIL for ≥1 yr have been included. Baseline characteristics are detailed in the Table. By 12 mo of NIL treatment, cumulative incidences of newly acquired MR4 and MR4.5 were 57.4% and 30.5%, respectively. Further analysis of MR4 achievement showed that pts with a major molecular response (MMR: BCR-ABL1IS 〉 0.01% - ≤0.1%) at baseline had a higher probability to achieve a MR4 than those lacking MMR at baseline, with a cumulative incidence of MR4 by 12 months of 64.8% and 30.8%, respectively (Figure). Adverse events (AEs) were mostly of grade 1-2, manageable with supportive care or NIL dose interruption/reduction and included pruritus (19%), headache (9%), skin rash (9%), upper abdominal pain (8%) and constipation (7%). Grade 3-4 hematologic AEs were uncommon. The incidence of grade 3-4 laboratory abnormalities was low: lipase increase, hyperglycemia, ALT and AST increase, hyperbilirubinemia and hypercholesterolemia reported in 3.7%, 1.3%, 1%, 0.7%, 0.3%, 0.3% pts, respectively. Grade 3-4 ischemic cardiovascular events were experienced by 5% of pts including peripheral artery occlusive disease (1.7%) and coronary artery disease (3.7%) (1 pt experienced both AEs). Sub-analyses aiming to evaluate the impact of baseline SCOREa CV risk factor on the onset of arterial ischemic events are currently ongoing. Results on 168 pts showed grade 3-4 ischemic CV events in 19% of pts who were at very high or high risk (n = 47) compared to 1.7% of in pts with moderate or low risk (n = 121). During the first 12 mo, 48 (16%) pts discontinued NIL therapy: 32 discontinued due to AEs/laboratory abnormalities, 12 withdrew consent, 4 due to other reasons (protocol deviation, pregnancy and non-compliance). No patients left the study due to progression to AP/BP. Till date there were no on-treatment deaths. Conclusions This interim analysis shows that a switch to NIL at lower doses than in prior studies (300mg BID instead of 400mg BID) induces high rates of MR4 and MR4.5 in pts without such MR levels on IM. The safety profile of NIL at 300mg BID is consistent with that described in other prospective studies.Thus a switch to NIL for pts not achieving a deep MR during IM therapy is predicted to substantially increase the probability of achieving TFR requirements. A longer follow-up is necessary to assess what may be the best duration of NIL prior to treatment discontinuation. aRisk factors evaluated by applying the SCORE chart proposed by the European Society of Cardiology Table 1. Table 1. Figure 1. Figure 1. Disclosures Rea: Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Rosti:Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Cross:Qiagen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding. Hellman:Novartis: Research Funding; BMS: Research Funding. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Almeida:Shire: Speakers Bureau; Bristol Meyer Squibb: Speakers Bureau; Novartis: Consultancy; Celgene: Consultancy. Dezzani:Novartis: Employment. Pellegrino:Novartis: Employment. Costantini:Novartis: Employment. Walasek:Novartis: Employment. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Steegmann:Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding. Baccarani:NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract: This study aimed to determine the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells of CML patients at diagnosis and after nilotinib treatment.Microarray was performed on 30 CML patients at diagnosis as well as 8 patients after 12 months of nilotinib treatment using the latest generation Affymetrix GeneChip HTA 2.0 to further investigate genomic complexity. Bone marrow blood (in a range of 5-20 ml) samples were collected from patients at diagnosis and after 3, 6 and 12 months of nilotinib treatment. BM mononuclear cells were purified by density gradient centrifugation at 800 rpm for 20 min and soon after CD34+/lin- cells were isolated using Diamond CD34 Isolation Kit (Miltenyi Biotec). The purity of CD34+/lin-cells was about 97% as determined by flow citometry and the cells were counted at diagnosis and after the treatment with nilotinib (at 3, 6, and 12 months). BM CD34+/lin- cells of 30 patients at diagnosis and after 3 and 6 months of nilotinib were resuspended in RNAlater (Ambion, Life Technologies) and stored at -20°C until RNA extraction was performed. Total RNA was extracted from BM CD34+/lin- cells using MagMAX 96 Total RNA Isolation Kit (Ambion). BM CD34+/lin- cells were counted and a range of 100,000-800,000 has been noted at diagnosis. cRNA was prepared according to OvationPico WTA System V2 kit followed by Encore Biotin Module Kit (NuGEN). Ultimately, cRNA was hybridized to HTA 2.0 and signals were scanned by Affymetrix GeneChip Scanner 3000 following the manufacturer’s instructions. We observed that the number of CD34+/lin- cells dramatically decreased at 3 months (1,000-600,000), after 6 months (1,000-260,000) and after 12 months (100-130,000). In particular, CD34+/lin- cells were even less than 1000 after 12 months of nilotinib. If the number of CD34+/lin- cells after 12 months of nilotinib was less than 1000, we directly resuspended the cells in Prelude direct Lysis module (NuGEN) and microarray experiments were performed without RNA extraction. In this case, cRNA was prepared using Ovation One Direct System kit followed by Encore Biotin Module Kit (Nugen) and finally hybridized to the HTA 2.0. Microarray experiments were performed on CD34+/lin- cells of 30 CML patients at diagnosis with HTA 2.0. Moreover, we performed microarray experiments on CD34+/lin- cells of 8 CML patients at diagnosis vs. 8 CML patients after 12 months of nilotinib using HTA 2.0. Data was preprocessed and normalized using Robust Multi-array Average (RMA) algorithm. The Significant Analysis of Microarrays (SAM) was used to identify genes with statistically significant changes in expression in CML patients at diagnosis and at 12 months of treatment. P-values were corrected for multiple testing using false discovery rate. In conclusion, as far as we know, this is the first study which has isolated BM CD34+/lin-cells from CML patients at diagnosis and after nilotinib treatment. We observed a wide variety of the number of CD34+/lin- cells in 30 CML patients at diagnosis as well as after 3, 6 and 12 months of nilotinib. In particular, CD34+/lin- cells of CML patients at diagnosis were over 100.000. RNA was extracted and cRNA was prepared with OvationPico WTA System V2 kit followed by Encore Biotin Module Kit (NuGEN) and finally hybridized to HTA 2.0. On the other hand, the number of CD34+/lin- cells of CML patients after 12 months of nilotinib markedly decreased as low as 100 cells. In this case, we decided to avoid RNA extraction from the cells and we directly prepared cRNA using Ovation One Direct System kit and Encore Biotin Module Kit (Nugen) followed by the hybridization to the HTA 2.0 Array. To increase the statistical power in biomarker identification, we combined samples pre-processed with different experimental protocols (NuGEN) using ComBat, an empirical Bayes framework that adjusts data for batch effects and is robust to outliers in small datasets. Principal component analysis applied to the expression data before and after ComBat adjustment shows that the adopted normalization procedure effectively removes the systematic bias introduced by different protocols. Disclosures No relevant conflicts of interest to declare.

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

Abstract: Chronic myeloid leukemia (CML) is a disease of stemming from genetic damage to a hematopoietic stem cell. Despite nilotinib being a very effective drug for the treatment of CML, drug resistance can emerge. In the contest of the REL-PhilosoPhi34 study on behalf of the Rete Ematologica Lombarda, we performed an exploratory study aimed to identify the gene expression signature of bone marrow (BM) CD34+/lin- cells of CML patients at diagnosis as well as after 12 months of nilotinib to investigate the genes and pathways responsible for the response or resistance to nilotinib. Microarray experiments were performed using the latest generation Affymetrix GeneChip HTA 2.0 to further investigate genomic complexity. The study was developed in two steps as follows: In the first step we analyzed 30 CML patients and from the comparison between the BM CD34+/lin- cell counts from each patient at diagnosis and after 3 and 6 months of nilotinib, patients were divided into 2 classes: class 1 (n=24) showed highly reduced levels of CD34+/lin- cells while class 2 (n=6) demonstrated increasing levels of CD34+/lin- cells after 3 and 6 months of nilotinib, respectively (Fig.1). The 30 patients can be divided into 6 groups showing different patterns of CD34+/lin- cellularity (Figure 1). Data was preprocessed and normalized using Robust Multi-array Average (RMA) algorithm. The Significant Analysis of Microarrays (SAM) was used to identify genes with statistically significant changes in expression in CML patients. P-values were corrected for multiple testing using false discovery rate. No transcripts were selected as differentially expressed using this threshold. However, if a nominal significance level alpha equal to 0.05 is adopted together with a fold-change threshold equal to 2 (absolute value), 56 transcripts were selected in the comparison between the 2 groups of CML patients. Among them, we focused on NFKBIAgene which was over expressed in class 1 compared to class 2.NFKBIA is involved in 68 pathways regulating apoptosis (PI3K,NF-kB) and encodes IkBα protein belonging to the NF-kB pathway which is a potential downstream target of BCR-ABL1 due to its role in regulating cell survival and proliferation. Of note, 31/56 transcripts are located on chromosome 15 suggesting that this region could be crucial for transcriptional regulation of CML correlated to nilotinib response. The second step analyzed the GEP of CD34+/lin- cells of 8 patients at diagnosis (class 1) vs. the same 8 patients after 12 months of nilotinib (class 2) to investigate the gene expression changes and the pathways induced by nilotinib treatment. Data was preprocessed and analyzed as described above. 247 probes (corresponding to 51 genes and 180 non-annotated transcripts) were selected with a p-value lower than 0.05 after multiple testing correction and using a fold-change threshold equal to 3 (absolute value). Functional enrichment analysis was performed using DAVID and highlighted the following pathways and genes: ¯ Defense response and immune system: CAMP, CRISP3, CYBB, RGS1, CEACAM8, LTF (cell growth and differentiation), MNDA and HP (positive regulation of apoptosis) were over expressed in class 2 with high fold changes of 7.65, 9.95, 4.40, 3.47, 4.28, 3.61, 3.60, 3.95, respectively ¯ Transcriptional regulation and cell cycle: MMP8was under expressed in class 1 with the high fold change of 8,94 ¯ S100A12, S100A8, S100A9 (regulation of the cell cycle and differentiation) were over expressed in class 2 with the FC of 8.68, 3.96, 4.49, respectively ¯ RNA5S-ribosomal pseudogenes 310-311-312-313-314-315-316-317-320-363 were downregulated in class 2 ¯ 10 small-nuclear-RNAs and 11 small-nucleolar RNA were differently deregulated between the 2 classes ¯ APOC1, PLBD1 (lipid metabolism) were overexpressed and underexpressed in class 1, respectively In conclusion, this is the first study of GEP of CD34+/lin- cells with HTA 2.0 which demonstrated that 51 genes mostly involved in immune system, transcriptional regulation and cell cycle were significantly differently expressed from the comparison between CML patients at diagnosis and after 12 month of nilotinib. Ongoing studies are focused on examining the genetic and biologic mechanisms underlying nilotinib response or resistance to predict response or failure providing new insights into the molecular mechanisms in CML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.