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6589 Background: Despite impressive results achieved with IM 400 mg/day alone, only a minority of pts reached a complete molecular remission at 12-month. Higher dose of IM or its combination with other therapies might improve molecular remission. Design of the trial: the 3 experimental arms are IM 400mg daily in combination with Peg-IFN-α2a (Peg-IFNα2a, 90 μg weekly) or with Ara-C (20 mg/m2/day, days 15–28 of 28-day cycles) or IM 600mg daily. The reference arm is IM 400mg daily. All pts (over 18 years of age with Bcr-Abl positive CML) receive IM 400 mg/day as monotherapy days 1–14 and then start the assigned regimen for at least 12 months. The endpoints are overall survival (primary), rate and duration of hematologic, cytogenetic and molecular responses and tolerability. An interim analysis of the first 636 pts at 1 year from randomization will allow evaluation of molecular response rates, one of the experimental arm being selected for further comparison with IM 400. An experimental arm would be selected if it increased the 4 log reduction response rate at 12-month by at least 20 percentage points, (15% to 35%), with an acceptable tolerability. Results: This evaluation is based on a cohort of 370 pts with a median time of observation of 16 months, recruited between 9/2003 and 9/2005. [median age 53 yrs (18–81); Sokal distribution: 38% of pts low, 38% intermediate, and 24% high]. At 1 month 80% of pts achieved complete hematologic response. At 12 months, 138 pts (72%) achieved a major cytogenetic response, being complete in 120 pts (63%). Grade 3/4 hematologic toxicity occurred in 8% of IM400 pts, 9% of IM600 pts, 41% of IM+IFN pts and 33% of IM+Ara-c pts respectively. Dose of Peg IFN was reduced in 16% of pts, 45 μg per week being well tolerated. Grade 3/4 non hematological toxicity occurred in 11% of IM400 pts, 16% of IM600 pts, 10% of IM+IFN pts (maily skin rash) and 11% of IM+Ara-c pts. Discontinuation of experimental treatment occurred in 17% of IM600 pts, 36% of IM+IFN pts and 16% of IM+Ara-c pts. Conclusion: This first analysis confirmed both feasibility of IM combinations and high response rates. However a substantial hematological toxicity requires a careful assessment of pts. [Table: see text]

Predicting minimal residual disease (MRD) levels in tyrosine kinase inhibitor (TKI)-treated chronic myeloid leukemia (CML) patients is of major clinical relevance. The reason is that residual leukemic (stem) cells are the source for both, potential relapses of the leukemicclone but also for its clonal evolution and, therefore, for the occurrence of resistance. The state-of-the art method for monitoring MRD in TKI-treated CML is the quantification of BCR-ABL levels in the peripheral blood (PB) by PCR. However, the question is whether BCR-ABL levels in the PB can be used as a reliable estimate for residual leukemic cells at the level of hematopoietic stem cells in the bone marrow (BM). Moreover, once the BCR-ABL levels have been reduced to undetectable levels, information on treatment kinetics is censored by the PCR detection limit. Clearly, BCR-ABL negativity in the PB suggests very low levels of residual disease also in the BM, but whether the MRD level remains at a constant level or decreases further cannot be read from the BCR-ABL negativity itself. Thus, also the prediction of a suitable time point for treatment cessation based on residual disease levels cannot be obtained from PCR monitoring in the PB and currently remains a heuristic decision. To overcome the current lack of a suitable biomarker for residual disease levels in the BM, we propose the application of a computational approach to quantitatively describe and predict long-term BCR-ABL levels. The underlying mathematical model has previously been validated by the comparison to more than 500 long-term BCR-ABL kinetics in the PB from different clinical trials under continuous TKI-treatment [1,2,3]. Here, we present results that show how this computational approach can be used to estimate MRD levels in the BM based on the measurements in the PB. Our results demonstrate that the mathematical model can quantitatively reproduce the cumulative incidence of the loss of deep and major molecular response in a population of patients, as published by Mahon et al. [4] and Rousselot et al. [5]. Furthermore, to demonstrate how the model can be used to predict the BCR-ABL levels and to estimate the molecular relapse probability of individual patients, we compare simulation results with more than 70 individual BCR-ABL-kinetics. For this analysis we use patient data from different clinical studies (e.g. EURO-SKI: NCT01596114, STIM(s): NCT00478985, NCT01343173) where TKI-treatment had been stopped after prolonged deep molecular response periods. Specifically, we propose to combine statistical (non-linear regression) and mechanistic (agent-based) modelling techniques, which allows us to quantify the reliability of model predictions by confidence regions based on the quality (i.e. number and variance) of the clinical measurements and on the particular kinetic response characteristics of individual patients. The proposed approach has the potential to support clinical decision making because it provides quantitative, patient-specific predictions of the treatment response together with a confidence measure, which allows to judge the amount of information that is provided by the theoretical prediction. References [1] Roeder et al. (2006) Dynamic modeling of imatinib-treated chronic myeloid leukemia: functional insights and clinical implications, Nat Med 12(10):1181-4 [2] Horn et al. (2013) Model-based decision rules reduce the risk of molecular relapse after cessation of tyrosine kinase inhibitor therapy in chronic myeloid leukemia, Blood 121(2):378-84. [3] Glauche et al. (2014) Model-Based Characterization of the Molecular Response Dynamics of Tyrosine Kinase Inhibitor (TKI)-Treated CML Patients a Comparison of Imatinib and Dasatinib First-Line Therapy, Blood 124:4562 [4] Mahon et al. (2010) Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol 11(11):1029-35 [5] Rousselot et al. (2014) Loss of major molecular response as a trigger for restarting TKI therapy in patients with CP- CML who have stopped Imatinib after durable undetectable disease, JCO 32(5):424-431 Disclosures Glauche: Bristol Meyer Squib: Research Funding. von Bubnoff:Amgen: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria. Saussele:ARIAD: Honoraria; Novartis: Honoraria, Other: Travel grants, Research Funding; Pfizer: Honoraria, Other: Travel grants; BMS: Honoraria, Other: Travel grants, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding. Guilhot:CELEGENE: Consultancy. Mahon:NOVARTIS PHARMA: Honoraria, Research Funding; BMS: Honoraria; PFIZER: Honoraria; ARIAD: Honoraria. Roeder:Bristol-Myers Squibb: Honoraria, Research Funding.

Introduction. Chronic myeloid leukemia(CML) is a very rare disease in children and adolescents. The international registry of CML in children and adolescents gives us the opportunity to assess the prognostic relevance of variant t(9 ;22) and additional cytogenetic abnormalities at diagnosis on prognosis. Patients and Methods. We used clinical and biological characteristics and outcome of 239 children and adolescents less than 18 years to assess the impact of additional cytogenetic abnormalities and variant t(9 ;22) on the rate and the time to achieve a complete cytogenetic response (CCyR). Cytogenetic analysis was performed 24 or 48 hour culture on G-banded metaphases. Results. Overall, 17/239 children (7.1%) presented with additional cytogenetic finding at diagnosis: 5/239 children (2.1%) had a variant of the t(9;22), 10 children (4.2%) had other additional cytogenetic (numerical/structural) abnormalities (ACA), and 2/239 (0.8%) had both. Characteristics of the patients and phase of the disease are reported in Table 1. In the 7 patients with variant t(9;22), one (chromosome 1 in 3 patients, chromosome 8 in 2 patients and chromosome 14 in one patient) or two further chromosomes (chromosomes 8 and 17) were involved in 6 and one patient(s), respectively. In the 12 patients with ACA, 9 patients have one type of ACA, 2 patients have 2 type of ACA and one patient have 3 type of ACA. Regarding the patients in chronic phase at diagnosis (n=219), the cumulative incidence of complete cytogenetic response at 18 months was 88% (95% CI: 82 % - 93%) and 70% (95% CI: 42% - 93%) for patients without variant t(9;22) or ACA and for those with variant t(9;22) and/or ACA (p=0.151), respectively. Three deaths were recorded (among them one patient had ACA at initial diagnosis). With a median follow up of 43 months (range 1 – 161) the probability of 3 years overall survival was 99% (95% CI: 94 % - 100%) and 88% (95% CI: 39 % - 98%) for patients in chronic phase without variant t(9;22) or ACA and for those with variant t(9;22) and/or ACA (p=0.042), respectively. Conclusion. Additional chromosomal abnormalities at diagnosis in children with CML in chronic phase may have an impact on the outcome. A larger cohort of patient and a longer follow up is needed to confirm such findings. Table 1. Cytogenetic No variant t(9;22) or ACA Variant t(9;22) ACA Variant t(9;22) and ACA Number of patients 222 5 10 2 Sex (% males) 56% 60% 50% 100% Median age, yrs (range) 12 (1-18) 14 (9-17) 12 (5-16) 6 and 17 Phase of the disease (n) chronic accelerated blastic 208 12 2 3 1 1 7 1 2 1 0 1 Acknowledgment: The I-CML Ped study is supported by an unrestricted grant from Novartis Pharmaceutical Company Disclosures No relevant conflicts of interest to declare.

Abstract 2465 Background This study was designed to identify single nucleotide polymorphisms (SNPs) associated with probability of achieving a major molecular response (MMR) in chronic phase CML patients (pts) treated with imatinib. We used a non-commercial, pharmacogenetics-dedicated DNA chip containing 16561 SNPs covering 1916 candidate genes. We tested an exploratory cohort of pts treated with imatinib first line or after interferon therapy and then extended our finding in a validation set of pts included in the French SPIRIT trial (C Preudhomme, NEJM 2011) and randomized to the 400 mg or the 600 mg imatinib arm. Patients and methods Constitutive DNA samples from 312 pts were analyzed: samples from 91 pts in the exploratory set and 221 pts in the validation set (SPIRIT trial: 120 pts within the 400 mg imatinib arm and 101 pts within the 600 mg imatinib arm). We analyzed the expression of BCR-ABL/ABL standardized ratio as a function of the time elapsed since imatinib initiation for each patient using nonlinear (spline) interpolation and then derived the delay to achieve MMR or time of follow-up. With this method, we obtained a cumulative incidence curve of MMR in both groups. Relevant SNPs were identified using a COX model adjusted for covariates (sex, two first principal components and Sokal score when available). SNP with FDR (False Discovery Rate) below 30% in the exploratory cohort where subsequently investigate in the SPIRIT trial groups. Results Patient characteristics such as age, sex, Sokal score were homogeneous between exploratory and validations sets as well as within both arms of the SPIRIT trial. We confirmed the prognostic value of the Sokal score in term of cumulative incidence of MMR (CI-MMR) in both sets of pts (CI-MMR was 83%, 67% and 56% for low, intermediate and hight Sokal risk groups respectively, additive log rank, p < 0.001), as well as the significant improvement of CI-MMR within the SPIRIT trial for pts assigned to the 600 mg daily imatinib arm as compared to the 400 mg daily arm (CI-MMR was 80% in the 600 mg imatinib arm compared to 61% in 400 mg/day arm, log rank, p = 0.008). CI-MMR was similar between exploratory set and the 400 mg/day arm of the SPIRIT trial (63% vs 61%, log rank, p = 0.56) and higher in the 600 mg arm of the SPIRIT trial compared to the exploratory set: 80% vs 63% (log rank, p < 0.001), consistent with dose received in the exploratory set (imatinib 400 mg/d). Association study shows that 10 SNPs identified pts with significantly different probability to achieve MMR within the two first year of therapy (FDR less than 30% p < 0,01) in the exploratory cohort three of which belong to ABCG2 gene. In the SPIRIT trial (n=221), only two SNPs from ABCG2 locus were significantly associated with a higher probability of achieving MMR. We then analyzed separately both arms of the SPIRIT trial. All tree ABCG2-SNPs identified in the exploratory set of pts were significantly associated with CI-MMR in the 400 arm at the 5% level. In contrast, none of them were confirmed in the 600 mg arm. In order generalize our results and to get closer to the underling molecular structure of genotypes markers we performed haplotyping at locus of ABCG2 gene. Multivariate analysis distinguished two minor haplotypes (haplotype 1 and 3) linked to MMR achievement in pts receiving 400 mg/day of imatinib (from exploratory set and SPIRIT, n=211). Haplotypes 1 had G-C-G and haplotype 2 had G-T-G at rs12505410, rs13120400 and rs2725252 respectively and their frequencies were 26 and 6% respectively. Collapsing these haplotypes yielded a surrogate dominant marker highly associated to CI-MMR in this group (p < 0.001, figure 1A) whereas in the SPIRIT 600 mg arm, this phenomenon did not hold anymore (p=0.25, figure 1B). Interestingly, CI-MMR in the 400 mg/day harboring a copy of minor haplotypes (1 or 3) was comparable to the CI-MMR observed in pts receiving 600 mg/d imatinib lacking of at least one copy of the two minor haplotypes (75% vs 70% respectively, p=0.99) or whatever haplotype they had (75% vs 80%, p=0.37). Conclusion The ABCG2 gene product is a well-known protein involved in drug absorption in the bowel. Polymorphism of the ABCG2 gene has been implicated in drugs absorption including imatinib. We here confirm that ABCG2 haplotypes could distinguish patients at a lower probability to achieve MMR. We report for the first time that this unfavorable pharmacologic effect can be overcome in vivo by increasing imatinib daily dose from 400 mg to 600 mg. Disclosures: Rousselot: BMS, Novartis: Research Funding. Guilhot:ARIAD: Honoraria.

// Marc Delord 1 , Philippe Rousselot 2 , Jean Michel Cayuela 3,4 , Francois Sigaux 1,3 , Joelle Guilhot 5 , Claude Preudhomme 6 , Francois Guilhot 5 , Pascale Loiseau 7 , Emmanuel Raffoux 8 , Daniela Geromin 9 , Emmanuelle Genin 10 , Fabien Calvo 11,12 and Heriberto Bruzzoni-Giovanelli 11,12 1 Plateforme de Bioinformatique et Biostatistique, Institut Universitaire d’Hematologie, Universite Paris Diderot, Sorbonne Paris Cite 2 Service d’Hematologie et d’Oncologie, Hopital Mignot, Universite Versailles Saint-Quentin-en-Yvelines 3 Laboratoire Central d’Hematologie, Hopital Saint Louis 4 EA3518, Universite Paris Diderot, Sorbonne Paris Cite 5 Inserm CIC 0802, CHU de Poitiers, Poitiers 6 Laboratoire d’Hematologie, Inserm, U837, CHRU et Universite de Lille Nord, Institut de Recherche sur le Cancer de Lille 7 Service d’Immunologie et Histocompatibilite et INSERM U940, Hopital Saint Louis 8 Service des Maladies du Sang, Hopital Saint Louis 9 Plateforme de Ressources Biologiques, Hopital Saint Louis, Paris 10 Inserm U1078, CHU Brest, Universite Bretagne Occidentale, Brest 11 Pharmacologie, Universite Paris Diderot, Sorbonne Paris Cite, Paris 12 Centre d’Investigations Cliniques 9504 INSERM-AP-HP, Hopital Saint-Louis, Paris, France. Correspondence: Heriberto Bruzzoni-Giovanelli, email: // Keywords : CML, Imatinib, SNPs, ABCG2, Pharmacogenetic, molecular response, BCR-ABL Received : June 6, 2013 Accepted : July 16, 2013 Published : July 18, 2013 Abstract Pharmacogenetic studies in chronic myelogenous leukemia (CML) typically use a candidate gene approach. In an alternative strategy, we analyzed the impact of single nucleotide polymorphisms (SNPs) in drug transporter genes on the molecular response to imatinib, using a DNA chip containing 857 SNPs covering 94 drug transporter genes. Two cohorts of CML patients treated with imatinib were evaluated: an exploratory cohort including 105 patients treated at 400 mg/d and a validation cohort including patients sampled from the 400 mg/d and 600 mg/d arms of the prospective SPIRIT trial (n=239). Twelve SNPs discriminating patients according to cumulative incidence of major molecular response (CI-MMR) were identified within the exploratory cohort. Three of them, all located within the ABCG2 gene, were validated in patients included in the 400 mg/d arm of the SPIRIT trial. We identified an ABCG2 haplotype (define as G-G, rs12505410 and rs2725252) as associated with significantly higher CI-MMR in patients treated at 400 mg/d. Interestingly, we found that patients carrying this ABCG2 “favorable” haplotype in the 400 mg arm reached similar CI-MMR rates that patients randomized in the imatinib 600 mg/d arm. Our results suggest that response to imatinib may be influenced by constitutive haplotypes in drug transporter genes. Lower response rates associated with “non- favorable” ABCG2 haplotypes may be overcome by increasing the imatinib daily dose up to 600 mg/d.

Abstract 168 The SPIRIT phase III randomized multicenter open-label prospective trial was designed to compare 4-arms, IM-400 mg versus IM-600mg versus IM-400 mg + cytarabine at a dose of 20 mg/m2/day in cycles of 28 days, versus IM-400 mg + PegIFN at an initial dose of 90 μg per week. The planned molecular analysis after 1 year based on the outcome of 636 pts resulted in a highly significant superiority of superior molecular response (SMR) (0.01 % Bcr-Abl/Abl on IS) of the combination IM 400mg-PegIFN (N Engl J Med, 2010). As of December 31st 2010, date for closing accrual, 787 pts have been included. The current analysis provides update of the trial and additional information on relationship between molecular response and outcomes. Methods: Progression free survival (PFS) was defined by absence of accelerated phase (AP), blast crisis (BC), and death from any reasons, whichever came first. Rates were estimated by the Kaplan-Meier method and compared within groups by the log-rank test. In addition, time to progression (TTP) being defined as AP-BC only, was estimated by cumulative incidence function and compared within groups by the Gray test. Deaths unrelated with progression were then considered as competing events. Pts with available PCR samples at 3 months (N= 665 overall, 197 IM-400mg, 147 IM-600mg, 138 IM-AraC, 183 IM-PegIFN) were classified according to the BCR-ABL cut-off level of 10% IS at 3 and 6 months. Analyses of long term outcomes were then based on the kinetic of molecular response. Results. After a median follow-up of 68 months, out of the 787 pts, 59 PFS events (31 AP-BC; 28 deaths in CP) were recorded. Survival without progression at 60 months were for the IM-400mg, IM-600mg, IM-400mg + cytarabine and IM-400mg + PegIFN, 94%, 93%, 90% and 94% respectively (overall, p= 0.24). However we noticed that kinetic of molecular responses of pts who experienced AP-BC was very heterogeneous as showed in Fig 1. The accurate level of bcr-abl/abl transcript and the relevance of the IS conversion factor are questionable when values are above 10% or very low. Thus corresponding plots are shadowed in the Fig1. Then, when pts were stratified according to their molecular response at 3 months, 14 cases of AP-BC and 13 deaths without evidence of progression were recorded in the group of pts with a BCR-ABL ratio Conclusion: A 3-months BCR-ABL transcript below the level of 10% IS was associated with a PFS improvement. However, results which were observed with the addition of PegIFN or an increased dose of IM frontline do not confirm the relevance of the 10% BCR-ABL cut-off level as strong surrogate marker for progression to AP-BC. Pts assigned to the IM-PegIFN arm are at very low risk of progression to AP-BC even if their molecular response is delayed. Disclosures: Mahon: Novartis Pharma: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Rea:Bristol Myers-Squibb, Novartis, and Teva: Honoraria. Nicolini:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Ariad: Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria. Guerci-Bresler:Novartis, BMS: Speakers Bureau. Guilhot:ARIAD: Honoraria.