ALBERT

Description: Introduction The acquisition of ABL1 Kinase Domain (KD) mutations represent the most frequent resistance mechanism in CP-CML patients (pts) treated with tyrosine kinase inhibitors (TKI). Currently, the standard assay relies on a poorly sensitive technique, Sanger Sequencing (SS). Thus, the detection of these mutations using SS might be too late to trigger a timely treatment change. In a national phase III academic trial (PETALs, EudraCT 2013-004974-82), we evaluated prospectively the value of a more sensitive technique, Next Generation Sequencing (NGS) to detect KD ABL1 mutations in newly diagnosed CP-CML patients randomized to get nilotinib 600 mg/d for 6 years ± Pegylated-IFN-α2a (Peg-IFN) 45 μg/wk for 2 years in combination. Methods Newly diagnosed CP CML pts ≤65 years were randomized 1:1 to get NIL 300 mg BID alone (M0 to M48, arm A) vs Peg-IFN alone for 30 days (M-1→M0) 30 mg/wk as priming, prior to NIL 300 mg BID + Peg-IFN 30 μg/wk 2 weeks, upgraded to 45 μg/wk thereafter, for up to 2 y (M0 to M24, arm B) followed by NIL alone for 4 more years unless pts enter a treatment-free remission phase. In addition to KD mutational analysis performed by SS as per protocol, patients also had KD mutational analysis performed by NGS at M3, M6, M12 and 6-monthly thereafter until achievement of a stable MMR, regardless of response. NGS assay was performed as previously described (Kizilors et al. Lancet Haematol 2019). Results Two hundred pts were randomized (99 in A, 101 in B), of which 96 patients (51/99 in A, 45/101 in B, p=0.399) underwent a KD mutational analysis performed by NGS as part of this study. The remaining 104 patients are currently being screened and the full dataset will be presented. Among the 96 patients tested, there was no difference in the distribution between the 2 arms with respect to gender, age [median 45 years (18-66)] or risk factors distribution (p=0.862 and 0.328 for Sokal and ELTS respectively in patients tested at 3 months). The median follow-up of this cohort is 45.0 (33.2-58.7) months. By 12 months, 11 patients [8/51 (11.8%) in A, 3/45 (6.6%) in B] had developed a KD mutation. After only 3 months of TKI therapy, 3 patients were found mutated (Y253H 2 pts, T315I 1 pt), of whom 2 pts were only detected using NGS. At M6, a KD mutation was found in 8 pts [A: 7 patients, B: 1 pt, (p= 0.055), of which 6/8 were not detected by SS, due to either low level Variant Allele frequency (VAF, n=5) or low level BCR-ABL transcript levels (n=1). Y253H mutations were found in 4 pts, T315I in 2 pts and E255K in 1 pt. Consecutively to KD mutation identification, 6/8 patients lost their response and were withdrawn from study (1 pt with a Y253H detected at M3 progressed to advanced phase), while 1 pt lost MMR at last follow-up and another pt with a mutation sensitive to nilotinib achieved MMR. KD mutations were detected while pts were in optimal response at M6 [BCR-ABL

Description: The combination of 2GTKI+pegylated IFN-α (Peg-IFN) is an attractive approach for first-line treatment of CP CML, inducing high rates of deep molecular responses in phase II trials. Thus, we evaluated nilotinib (NIL) alone versus NIL+Peg-IFN in newly diagnosed CP-CML patients (pts) in a randomised phase III trial (PETALs, EudraCT 2013-004974-82). Newly diagnosed CP CML pts ≤65 y, without prior history of arterial occlusion were randomized 1:1 to get NIL 300 mg BID alone (M0 to M48, arm A) vs Peg-IFN alone for 30 days (M-1→M0) 30 μg/wk as priming, prior to NIL 300 mg BID + Peg-IFN 30 μg/wk 2 wks, upgraded to 45 μg/wk thereafter, for up to 2 y (M0 to M24, arm B) followed by NIL alone for 4 more years unless pts enter treatment-free remission (TFR). The primary endpoint is the rate of MR4.5 by 1 y. As a secondary endpoint, pts reaching MR4.5 ≥2 y are allowed to stop NIL and enter a TFR phase in both arms. The trigger for treatment resumption is loss of MMR. All molecular assessments are centralised, quantifications are expressed as BCR-ABL/ABL1 (IS) in % with ≥32,000 copies of ABL1 as control. Two hundred pts were randomized (99 in A, 101 in B), 130 M and 35 F in each arm, median age of 46 (18-66) y. Median follow-up is 43.8 (34.3-55.9) Mo. Results are analysed in intention-to-treat. Sokal and EUTOS LTS scores were H in 25% and 2.5%, Int. in 33% and 16.5% and L in 42% and 81% pts respectively equally balanced. Median age is 46 (18-66) y, 18 pts (9%) had ACAs, all pts have a "Major" BCR transcript. CHR was obtained in 9.6% of pts at M0 (in B) and 88% of pts in A and 90.4% of pts in B at M1. CCyR rates at M3 were 63% vs 75% in A and B (p=ns), and BCR-ABL1 ≤1% at M6 were 87% in A vs 93% in B (p=ns). By M12, the rates of MMR were 68.1% vs 70.1% (p=0.44), MR4 were 34% vs 47.5% (p=0.041), MR4.5 were 15.9% vs 21.5% (p=0.049), MR5 11.7% vs 23.71% (p=0.023), in A vs B respectively. By M36 the rates of MMR were 83% vs 86.6% (p=0.31), MR4 were 70.2% vs 71.13% (p=0.50), MR4.5 were 37.2% vs 49.5% (p=0.05), MR5 33% vs 42.3% (p=0.12), in A vs B respectively The overall cumulative incidence of MR4.5 is superior in B (54.6 [43.7-65.5]%) vs A (44 [31.5-54]%) close to significance (unilateral Fisher test, p=0.05, see Figure). Seven patients were mutated by Sanger in A (5 Y253, 1 E255K, 1 T315I) vs 2 in B (2 T315I). One pt (A) progressed toward AP and then myeloid BC with a Y253H mutation, is still alive in CMR on Ponatinib. Twenty nine (29%) pts were withdrawn from study in A (toxicity 9, cancer 3, resistance 14, investigator decision 2, lost for FU 1) vs 26 (26%) pts for B (toxicity 13, resistance 8, investigator decision 5), 1 pt died from cervix cancer (A). Median overall doses of NIL delivered by M36 were 600 mg/d in both arms (p=ns). The median overall dose of Peg-IFN delivered in B by M24 was 37.5 mg/wk. The overall rate of grade 3-4 hematologic toxicities was 22%; with 2% and 7% thrombocytopenia, 4% and 6% neutropenia, and 1% and 1% pancytopenia in A vs B respectively. Major grade 3-4 non-hematologic toxicities consisted in 9% of cardiac disorders in A (2 coronaropathies, 1 myocardial infarction, 2 thoracic pains, 2 atrial fibrillation, 1 bradycardia, 1 palpitations, 1 pericarditis) vs 8% in B (2 coronaropathies, 1 myocardial infarction, 3 atrial fibrillation, 1 palpitations, 1 pericarditis), 4% vascular disorders in A (1 thrombophlebitis + PE, 1 transient ischemic attack, 1 PAOD, 1 carotid stenosis) vs 3% in B (1 thrombophlebitis, 1 PAOD, 1 transient ischemic attack). Three % of gastro-intestinal disorders were observed in A (2 pancreatitis, 1 anal fissure) vs 6% in B (2 pancreatitis, 1 anal fissure, 1 abdominal pain, 2 cholecystectomies); 5% auto-immune disorders in B (1 recurrent pericarditis, 2 hemolytic anemia, 1 ITP, 1 thyroiditis); 5 and 8 pregnancies (2 pts + 3 partner Arm 1, 3 pts + 5 partner Arm B), despite recommended contraceptive methods. Secondary tumours were diagnosed in 4% (1 breast, 1 cervix, 1 thyroid, 1 neuroendocrine) in A vs 2% of pts (1 neuroendocrine and 1 testis) in B. Of note 8% psychiatric episodes were reported in B pts (2 unsuccessful suicide attempts), vs 2% in A. We observed 9% lipase elevations in A, 6% in B, 2% cholestatic episodes in A, 6% in B; 3% of transaminase elevations in A vs 2% in B. Infections were detected in 3% A vs 7% in B. The combination of NIL + Peg-IFN seems to provide somewhat higher MR4.5 rates by M36 in newly diagnosed CP CML pts without inducing significant higher toxicities than NIL alone. Whether this will translate in higher TFR rates is under evaluation. Final updated results at M36 will be presented Disclosures Nicolini: Sun Pharma Ltd: Consultancy; Novartis: Research Funding, Speakers Bureau; Incyte Biosciences: Honoraria, Research Funding, Speakers Bureau. Etienne:Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Huguet:Servier: Honoraria; Amgen: Honoraria; Novartis: Honoraria; Incyte Biosciences: Honoraria; Jazz Pharmaceuticals: Honoraria; Pfizer: Honoraria; BMS: Honoraria. Guerci-Bresler:Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau. Charbonnier:Incyte Biosciences: Honoraria, Speakers Bureau; Novartis: Consultancy; Pfizer: Consultancy. Legros:Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Incyte Biosciences: Honoraria, Research Funding; BMS: Honoraria. Coiteux:Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Cony-Makhoul:BMS: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy; Incyte Biosciences: Honoraria, Speakers Bureau; Novartis: Consultancy. Roy:Incyte Biosciences: Consultancy. Rousselot:Pfizer: Research Funding; Incyte: Research Funding. Quittet:Novartis: Honoraria, Speakers Bureau. Ame:Incyte Biosciences: Honoraria, Speakers Bureau. Rea:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte Biosciences: Honoraria; BMS: Honoraria. Dulucq:Novartis: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Mahon:Novartis: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau. OffLabel Disclosure: Pegylated Interferon alpha 2 a is not licensed in this setting

Description: Introduction Deep molecular response (DMR) are now highly desirable goals in the treatment of CP-CML, especially in the front-line setting, because it can lead to a definitive treatment-free remission (TFR). However, such a goal is difficult to attain and does not concern the majority of patients (pts), but currently the precise number of pts able to access to TFR is unknown. Aims We aim to determine the number or newly diagnosed CP-CML pts reaching DMR, stable DMR, and access to TFR, on Imatinib (IM, Glivec®) first-line. Methods We retrospectively analyzed in an observational study, a cohort of newly diagnosed CP-CML pts treated with IM first-line 400 mg daily alone in our 3 reference centers between 2000→2018. All pts were followed according to the ELN recommendations 2006, 2010 and 2013. Clinical data were extracted from medical files, and responses (hematologic, cytogenetic, molecular) were analysed according to standard methods. Molecular results were standardised according to the ELN/Eutos programs since 2003, and were all expressed as BCR-ABLIS in %. DMR have been defined according to the ELN (NCP. Cross et al., Leukemia 2015). Stability of DMR has been defined as a stable if ≥2 years at least on 4 datapoints. TFR has been proposed to pts presenting the only current recommended criteria: MR4.5 ≥2 years at least on 4 datapoints [(Rea et al., Cancer 2018)], in the 3 centers involved, within clinical trials, pioneered in our country, or now as a clinical routine recommendation. Loss of MMR was the trigger for TKI resumption after IM cessation for TFR. Overall survival (OS), progression-free survival (PFS), failure-free survival (FFS, defined as progression to advanced phases death, loss of CHR, CCyR, or MMR, discontinuation of IM for toxicity, primary cytogenetic resistance) were analysed since IM initiation in intention-to-treat. Results Four hundred and eighteen pts have been included in this study, with a median age of 60.7 (48-70) years at diagnosis, with 57% males and 43% females. Sokal score (n=401) was low in 32%, intermediate in 51% and high in 17%. ACA were present at diagnosis in 5.5% of the pts (NA in 1.44%). Major BCR transcripts were found in 98% of pts, and atypical transcripts in 1.9%. CHR was reached in a median of 1 (0.85 to 1.64) month of IM,

Description: Abstract 2298 We previously reported that imatinib treatment could be safely discontinued in chronic myeloid leukemia (CML) patients who had achieved a sustained complete molecular remission (CMR) lasting for at least two years in succession. However, in the multicenter French STIM study, 60 % of patients loose CMR mainly within the first 6 months after discontinuation and the probability to remain in CMR 2 years after imatinib discontinuation is around 40 %. The molecular relapse originates in persisting leukemic cells that are undetectable by current RQ-PCR techniques. The sensitivity of these routinely used techniques is below a detection threshold corresponding to a 5-log reduction in the leukemic burden. We thus hypothesized that improving the sensitivity of leukemic cell detection would be of valuable help to better select those patients who can possibly be cured after imatinib treatment. In the present work, we have increased the sensitivity of the current RQ-PCR, based on repeated PCR and on an increased number of normal ABL copies that were analyzed, in order to augment the probability to amplify BCR-ABL. For each sample, 5 microg of RNA were used to synthesize 5 cDNA and for each cDNA, 2 PCR for BCR-ABL and one for ABL were carried out, i.e 10 BCR-ABL PCR points were performed per sample. For each microg of RNA the number of ABL copies was over 20 000, so the number of ABL copies analyzed was over 100 000. The detection threshold of BCR-ABL was calculated and considered at 40 cycles of PCR (theshold Ct). The control plasmid (pME-2) (Kindly given by Martin Müller and Andreas Hochhaus from European LeukemiaNet) used as standard curve dilution was also included to test the sensitivity and for instance 4 copies were detected in 19/20 cases, 2 copies 7/10 cases,1 copy in 2/10, 0.4 copy in 1/10. Thirty one samples from healthy donors or non CML patients (BCR-ABL negative) served as controls. Among the 310 (10 × 21) PCR for BCR-ABL we found only one positive well. 65 patients enrolled in the STIM study were analyzed at the time of imatinib discontinuation, using this new RQPCR technique. In the STIM study relapse was defined as the positivity of Bcr-Abl transcripts using classical QRT-PCR confirmed by a second analysis point indicating the increase in relation to the first analysis point performed on 2 successive assessments. Among 650 PCR for BCR-ABL, 46 wells were found positive. 9 patients were found at least 3 /10 positive PCR and among them 6 relapsed. 22 patients were found only at least 1 /10 positive and among them 15 relapsed. In 43 patients, BCR-ABL was never detected using the more sensitive RQ-PCR technique and 22 of them relapsed. To conclude, the use of a new RQPCR technique with a sensitivity of detection of BCR-ABL close to 6-log does not allow the prediction of molecular relapse following imatinib discontinuation in patients in CMR. Our results are in agreement with what was previously reported using PCR on genomic DNA. The persistence of leukemic cells in CMR patients does not automatically lead to CML relapse. Disclosures: Belanger: Novartis Pharma: Employment.

Description: Abstract 1684 The early assessment of molecular responses to tyrosine kinase inhibitors (TKI) used as a front line therapy for chronic phase chronic myeloid leukemia (CML) might represent an attractive surrogate marker for survival and help to discriminate poor prognosis patients (pts). So, we studied in a retrospective bicentric institutional analysis a series of 142 de novo CP CML pts diagnosed between 2000 and 2011, receiving imatinib (IM) 400 mg daily (n=85 pts), second generation TKI (TKI2) (n=57 pts) with 37 dasatinib (DAS) pts 100 mg daily and 20 nilotinib (NIL) pts 600–800 mg daily as front-line therapy, enrolled or not in clinical trials. All pts were assessed for their cytogenetic and molecular responses (local RQ-PCR for BCR-ABL, expressed as BCR-ABL/ABL ratios (IS) in %). Failure to TKI was defined as progression to accelerated phase or blast crisis, death, loss of complete hematologic response, loss of complete cytogenetic response, confirmed loss of major molecular response (MMR), discontinuation of TKI because unacceptable toxicity, primary cytogenetic resistance. The definition of progression included the same variables except the two last. Pts were considered in MMR with a BCR-ABL/ABL ratio ≤0.1% (IS). There were 87 males (61%) and 55 females with a median age of 55 years (21–83) at diagnosis. Six pts (4%) had an additional clonal abnormality, 4 a masked Philadelphia (Ph) chromosome, and 4 a variant Ph. Three pts had an atypical BCR-ABL transcript, all in IM400 group. Sokal scores were low for 37 (26%), intermediate for 61 (43%) and high for 43 (31%) (1 NA), and Euro scores were low for 49 (35%), intermediate for 78 (55%), and low for 14 (10%) (1 NA). These scores were similarly balanced in the IM400, NIL and DAS groups. The median time between diagnosis and TKI was 1 (0.9–1.28) month, not significantly different between TKI groups. The median follow-ups were 56, 37, and 19 months for IM400, NIL and DAS groups respectively. Fifteen percent, 6%, and 14% of pts achieved MMR at 3 months (M3) (p=ns); 31%, 37%, and 40% at M6 (p=ns); 55%, 70% and 71% at M12 (p=ns); 67%, 90% and 87% at M18 (p=ns) in IM400, NIL and DAS groups respectively, taken into account the limited numbers of pts at latest time-points in the 2 TKI2 arms. Twenty-three pts achieved 4-log molecular response MR4 in IM400, 10% in NIL and 11% in DAS at latest follow-ups. Four pts died, 3 in blast crisis in IM400 group and 1 for unrelated reason to disease or treatment in NIL group. A multivariate analysis adjusted on progression-free survival (PFS) performed, did not identify any significant parameter including age, gender, Sokal and Hasford scores, ACA, type of transcript, interval between diagnosis and TKI start. The progression-free survival (PFS) was 94 (83–100)% for pts achieving M3 MMR vs 86 (74–100)% if MMR occurred at later time points vs 86 (73–100)% for pts that never achieved MMR (p=0.02). There was no difference for PFS between the 3 TKI (log-rank p=0.9). None of the M3 MMR switched to another treatment, whereas 10 % of other pts did so for resistance (n= 10, 1 and 2 for IM400, NIL and DAS respectively), 9% for intolerance (n=10, 1 and 4), and 3% for other reasons (n=4, 0 and 0) with no difference between the 3 drugs (p=0.74). The failure-free survival (FFS) was 95% for M3 MMR pts vs 65% for pts with MMR occurring at later time points, versus 0% if MMR has not been achieved (p

Description: Despite the excellent efficacy of imatinib in chronic myeloid leukemia (CML), the response in patients is heterogeneous, which may in part be caused by pharmacogenetic variability. Imatinib has been reported to be a substrate of the P-glycoprotein pump. In the current study, we focused on the ABCB1 (MDR1) genotype. We analyzed the 3 most relevant single nucleotide polymorphisms of MDR1 in 90 CML patients treated with imatinib. Among the patients homozygous for allele 1236T, 85% achieved a major molecular response versus 47.7% for the other genotypes (P = .003). For the 2677G〉T/A polymorphism, the presence of G allele was associated with worse response (77.8%, TT/TA; vs 47.1%, GG/GA/GT; P = .018). Patients with 1236TT genotype had higher imatinib concentrations. One of the haplotypes (1236C-2677G-3435C) was statistically linked to less frequent major molecular response (70% vs 44.6%; P = .021). Hence, we demonstrated the usefulness of these single nucleotide polymorphisms in the identification of CML who may or may not respond optimally to imatinib.

Description: In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML.

Description: Introduction Imatinib has indeed revolutionized the treatment of chronic myelogenous leukemia (CML) since more than 15 years now, especially in CP. The first patients (pts) in this setting were treated with this compound within the IRIS phase III trial from Novartis, started in January 2000. Regular updates of the results of this study have been presented during various meetings until year 7, and academic studies have recently reported the outcomes of IM first-line CP CML pts after 66 months follow-up. However, little is known about the very long-term outcomes (〉8 years) of such first-line pts and these data might be of interest while generic forms of IM will be soon launched in this setting. In this study, we aimed to look at long-term outcomes in terms of efficacy and toxicities in first-line CP CML pts treated with branded form of IM (Glivec®). Methods This is a comprehensive retrospective analysis of first-line CP CML pts treated with IM first-line 400 mg daily since diagnosis and followed in 2 university reference centers for CML between 2000 and 2015, inside or outside academic or industrial clinical trials. All living pts have given their agreement for participation in this retrospective analysis. Pts have been analyzed in intention-to-treat, CML was defined according to ELN criteria [CP, accelerated phase (AP) and blast crises (BC)], Sokal, Euro and EUTOS scores have been calculated as published. Molecular biology tests have been performed according to ELN guidelines and BCR-ABL1/ABL1 were expressed as % on the international scale and 3 ELN conversion factors have been applied successively along time according to material exchanges performed with the central European laboratory in Mannheim. Cytogenetic and molecular responses have been defined according to the ELN criteria. Overall survival (OS) was calculated from the date of IM initiation until death at any time and for any reason; until progression to AP or BC at any time for progression-free survival (PFS); and until death, progression to AP or BC, failure on IM or IM treatment discontinuation for any cause including treatment-free remission (TFR), for event-free survival (EFS). The cut-off date for this analysis was the 20th of July 2,015. Results At time of analysis, 120 pts could be analyzed, with a median follow-up of 85.5 (1-194) months, 70 (58%) were males, with a median age of 55 (11-85) at IM initiation. Sokal score was high for 24(20%) pts, intermediate for 58 (49%), low for 34 (30.5%), unknown in 4 (0.5%) pts. Four (3.5%) pts had a variant Ph chromosome, 7 (6%) with additional chromosomal abnormalities, and 2 a masked Ph chromosome, 6 harbored atypical BCR-ABL1 transcripts excluded from analysis. Early molecular response (M3) was achieved in 86 (72%) pts, unreached in 20 (16%) pts, and unknown for 15 (12.5%) pts. It was predictive of Major Molecular Response (MMR) at 12 months (p=0.01, OR 5.35, 95%CI [1.3-31.94]), for MR4 rates at 24 months (p=0.03, OR 7.35 95%CI [1-328]) and for EFS (p=0.006) but not for OS and PFS in a multivariate Cox model analysis. MMR was achieved in 42% of evaluable pts at 12 months. Eutos, Euro and Sokal scores had no impact on OS, PFS and EFS. Five pts progressed to BC (1 myeloid, 4 lymphoid) within the 5 first years and died after allogeneic stem cell transplantation. The PFS rates were 97.5% at 2 years, 92% at 5 years, 88.6% at 10 and 14 years, EFS rates were 76% at 2 years, 60% at 5 years, 45% at 10 years and 21% at 14 years (figure 1), OS rates were 98% at 2 years, 95% at 5 years, 87% at 10 and 14 years. Figure 1: PFS and EFS in pts on IM first-line. (Dashed lines represent 95%CI). MR4.5 was achieved in 58 (48.5%) pts after a median of 46 (3-191) months and TFR strategy (or trial) was proposed in 28 pts (23.5%) and successful in 15 (12.5%) pts. At latest follow-up, after a median of 85.5 (4-180) months, 64 (53.5%) pts are still on IM, and 44 (37%) have switched to an alternative therapy for intolerance (17 pts, 14%) or resistance (16 pts, 13.5%, 7 with a BCR-ABL mutation) to IM and 11 for other causes (pregnancy, secondary tumors…). Overall, at latest follow-up, 10/120 pts died, 5 of CML progression and 5 from other causes. Conclusions After a very long median follow-up of more than 85 months, IM still consistently provides high rates of remission and survival, without disease progression and severe long-term toxicities. In addition, half of the pts reached the MR4.5 level, ≥2 years stable in 23.5% of the pts offering the possibility of a treatment-free strategy. Figure 1. Figure 1. Disclosures Nicolini: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ariad Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mahon:Novartis: Consultancy, Honoraria; ARIAD: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy, Honoraria. Etienne:Novartis: Consultancy, Honoraria; BMS: Honoraria.

Description: Abstract 2750 Poster Board II-726 Introduction Cellular uptake of imatinib is known to be mediated by Oct-1 but efflux by MDR1. In the cell, metabolism, particularly by CYP3A4, produces metabolites that could be carried by other transporters such as MRP2. Nilotinib seems to suffer less from the influence from such transporters. The aim of this study was to determine the impact of cellular exchange on the intracellular concentrations of imatinib, nilotinib or both in vitro but also among patients in order to improve the therapeutic drug monitoring of these medicines. Methods In vitro studies used the chronic myeloid leukemia cell line K562 overexpressing MDR1 (K562mdr) and wild-type K562 cells (K562wt). To investigate cellular uptake and the intra/extracellular concentration relationship, these were incubated with imatinib or nilotinib for 2h in the presence or absence of transporter inhibitors (verapamil, PSC833). Cells were precipitated by acetonitrile (with internal standards) and analysis was performed using HPLC-tandem mass spectrometry (Waters Acquity-TQD). The intracellular concentration of peripheral blood mononuclear cells (PBMC) of patients treated by these drugs was also investigated. Results In K562wt, the presence of verapamil, an inhibitor of both Oct-1 and MDR1, reduced imatinib intracellular concentration compared to the steady-state untreated K652wt, but the presence of PSC833, a specific inhibitor of MDR1, had little effect on the level of imatinib. K562mdr, that had lower steady-state imatinib cellular concentration than K562wt, recovered higher levels with verapamil but PSC833 allowed the greatest concentrations. Experiments investigating nilotinib found no difference between cell lines with or without inhibitors. Finally, the presence of both imatinib and nilotinib seemed to modify the steady-state concentration found individually. Intracellular imatinib concentration was correlated with that found in the culture medium (extracellular), but the gradient was different for each conditions for both K562wt and K562mdr, with or without inhibitors. At every concentration tested, we found the same differences in levels than in the kinetic study. Saturation was observed with imatinib, especially for untreated K562wt indicating probable involvement of active transport. Investigation of nilotinib under the same conditions did not show any difference in the gradient of each conditions and the relationship was more linear. The raw intracellular concentrations of PBMC obtained from patients (n=30) were very variable, especially for the imatinib. Correction by plasma concentration indicates individual patient's capacity for intracellular incorporation and a trend was found with response to the treatment. Conclusion Contrary to imatinib, nilotinib did not seem to be under influence of MDR1, nor of OCT1. The intracellular concentration appears as a good way to investigate pharmacokinetic resistance and could be an additional help for the management of the treatment, especially for imatinib. Disclosures: Mahon: Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria.

Description: Background: Transposable elements (TEs) are mobile pieces of DNA that can move ("jump") in our genome, thereby influencing a myriad of cellular processes. Long interspersed element-1 (LINE-1) retrotransposons comprise approximately 20 percent of the mammalian genome, and LINE-1 retrotransposition events can create genomic instability by unknown mechanism. In chronic myeloid leukemia (CML), BCR-ABL1 expression is associated with a high degree of chromosomal genetic instability. The aim of this study was to investigate LINE-1 retrotransposon ORF1 and ORF2 expression in CML patients and cell lines. Materials and methods: LINE-1 quantitative RT-PCR was performed on 48 CML patients and 35 control subjects before therapy and during imatinib therapy. In addition LINE-1 expression was carried out on BCR-ABL1 positive and negative cell lines treated by kinase inhibitors, depleted in BCR-ABL1 by RNA interference. The effect of gamma irradiation (50 Gy) on CML and non CML cell lines was investigated. Results: For CML patients at diagnosis, LINE-1 ORF1/ß-actin expression (median expression value 2.8 x 102) was significantly lower than for healthy donors (3.9 x 103) (P