BACKGROUND:Tyrosine kinase inhibitors (TKIs) have improved the survival of patients with chronic myeloid leukaemia. Many patients have deep molecular responses, a prerequisite for TKI therapy discontinuation. We aimed to define precise conditions for stopping treatment. METHODS:In this prospective, non-randomised trial, we enrolled patients with chronic myeloid leukaemia at 61 European centres in 11 countries. Eligible patients had chronic-phase chronic myeloid leukaemia, had received any TKI for at least 3 years (without treatment failure according to European LeukemiaNet [ELN] recommendations), and had a confirmed deep molecular response for at least 1 year. The primary endpoint was molecular relapse-free survival, defined by loss of major molecular response (MMR; >0·1% BCR-ABL1 on the International Scale) and assessed in all patients with at least one molecular result. Secondary endpoints were a prognostic analysis of factors affecting maintenance of MMR at 6 months in learning and validation samples and the cost impact of stopping TKI therapy. We considered loss of haematological response, progress to accelerated-phase chronic myeloid leukaemia, or blast crisis as serious adverse events. This study presents the results of the prespecified interim analysis, which was done after the 6-month molecular relapse-free survival status was known for 200 patients. The study is ongoing and is registered with ClinicalTrials.gov, number NCT01596114. FINDINGS:Between May 30, 2012, and Dec 3, 2014, we assessed 868 patients with chronic myeloid leukaemia for eligibility, of whom 758 were enrolled. Median follow-up of the 755 patients evaluable for molecular response was 27 months (IQR 21-34). Molecular relapse-free survival for these patients was 61% (95% CI 57-64) at 6 months and 50% (46-54) at 24 months. Of these 755 patients, 371 (49%) lost MMR after TKI discontinuation, four (1%) died while in MMR for reasons unrelated to chronic myeloid leukaemia (myocardial infarction, lung cancer, renal cancer, and heart failure), and 13 (2%) restarted TKI therapy while in MMR. A further six (1%) patients died in chronic-phase chronic myeloid leukaemia after loss of MMR and re-initiation of TKI therapy for reasons unrelated to chronic myeloid leukaemia, and two (<1%) patients lost MMR despite restarting TKI therapy. In the prognostic analysis in 405 patients who received imatinib as first-line treatment (learning sample), longer treatment duration (odds ratio [OR] per year 1·14 [95% CI 1·05-1·23]; p=0·0010) and longer deep molecular response durations (1·13 [1·04-1·23]; p=0·0032) were associated with increasing probability of MMR maintenance at 6 months. The OR for deep molecular response duration was replicated in the validation sample consisting of 171 patients treated with any TKI as first-line treatment, although the association was not significant (1·13 [0·98-1·29]; p=0·08). TKI discontinuation was associated with substantial cost savings (an estimated €22 million). No serious adverse events were reported. INTERPRETATION:Patients with chronic myeloid leukaemia who have achieved deep molecular responses have good molecular relapse-free survival. Such patients should be considered for TKI discontinuation, particularly those who have been in deep molecular response for a long time. Stopping treatment could spare patients from treatment-induced side-effects and reduce health expenditure. FUNDING:ELN Foundation and France National Cancer Institute.

Targeted inhibition of the oncogenic BCR-ABL1 fusion protein using the ABL1 tyrosine kinase inhibitor imatinib has become standard therapy for chronic myelogenous leukemia (CML), with most patients reaching total and durable remission. However, a significant fraction of patients develop resistance, commonly due to mutated ABL1 kinase domains. This motivated development of second-generation drugs with broadened or altered protein kinase selectivity profiles, including dasatinib and nilotinib. Imatinib-resistant patients undergoing treatment with second-line drugs typically develop resistance to them, but dynamic and clonal properties of this response differ. Shared, however, is the observation of clonal competition, reflected in patterns of successive dominance of individual clones. We present three deterministic mathematical models to study the origins of clinically observed dynamics. Each model is a system of coupled first-order differential equations, considering populations of three mutated active stem cell strains and three associated pools of differentiated cells; two models allow for activation of quiescent stem cells. Each approach is distinguished by the way proliferation rates of the primary stem cell reservoir are modulated. Previous studies have concentrated on simulating the response of wild-type leukemic cells to imatinib administration; our focus is on modelling the time dependence of imatinib-resistant clones upon subsequent exposure to dasatinib or nilotinib. Performance of the three computational schemes to reproduce selected CML patient profiles is assessed. While some simple cases can be approximated by a basic design that does not invoke quiescence, others are more complex and require involvement of non-cycling stem cells for reproduction. We implement a new feedback mechanism for regulation of coupling between cycling and non-cycling stem cell reservoirs that depends on total cell populations. A bifurcation landscape analysis is also performed for solutions to the basic ansatz. Computational models reproducing patient data illustrate potential dynamic mechanisms that may guide optimization of therapy of drug resistant CML.

Drug design of protein kinase inhibitors is now greatly enabled by thousands of publicly available X-ray structures, extensive ligand binding data, and optimized scaffolds coming off patent. The extensive data begin to enable design against a spectrum of targets (polypharmacology); however, the data also reveal heterogeneities of structure, subtleties of chemical interactions, and apparent inconsistencies between diverse data types. As a result, incorporation of all relevant data requires expert choices to combine computational and informatics methods, along with human insight. Here we consider polypharmacological targeting of protein kinases ALK, MET, and EGFR (and its drug resistant mutant T790M) in non small cell lung cancer as an example. Both EGFR and ALK represent sources of primary oncogenic lesions, while drug resistance arises from MET amplification and EGFR mutation. A drug which inhibits these targets will expand relevant patient populations and forestall drug resistance. Crizotinib co-targets ALK and MET. Analysis of the crystal structures reveals few shared interaction types, highlighting proton-arene and key CH-O hydrogen bonding interactions. These are not typically encoded into molecular mechanics force fields. Cheminformatics analyses of binding data show EGFR to be dissimilar to ALK and MET, but its structure shows how it may be co-targeted with the addition of a covalent trap. This suggests a strategy for the design of a focussed chemical library based on a pan-kinome scaffold. Tests of model compounds show these to be compatible with the goal of ALK, MET, and EGFR polypharmacology.

Dasatinib (DAS) and interferon-α have antileukemic and immunostimulatory effects and induce deep responses in chronic myeloid leukemia (CML). We assigned 40 newly diagnosed chronic-phase CML patients to receive DAS 100 mg o.d. followed by addition of pegylated interferon-α2b (PegIFN) after 3 months (M3). The starting dose of PegIFN was 15 μg/week and it increased to 25 μg/week at M6 until M15. The combination was well tolerated with manageable toxicity. Of the patients, 84% remained on PegIFN at M12 and 91% (DAS) and 73% (PegIFN) of assigned dose was given. Only one patient had a pleural effusion during first year, and three more during the second year. After introduction of PegIFN we observed a steep increase in response rates. Major molecular response was achieved in 10%, 57%, 84% and 89% of patients at M3, M6, M12 and M18, respectively. At M12, MR4 was achieved by 46% and MR4.5 by 27% of patients. No patients progressed to advanced phase. In conclusion, the combination treatment appeared safe with very promising efficacy. A randomized comparison of DAS±PegIFN is warranted.

Background: In chronic myeloid leukemia (CML) the combination treatment of tyrosine kinase inhibitors (TKIs) with interferon-α (IFN-α) has proved to be effective and well-tolerated. IFN-α has long-term immunomodulatory effects, and when combined to TKI therapy, it may increase the success rates for treatment free remission. In our recent clinical trial NordCML007, a low-dose pegylated IFN-α was combined with dasatinib therapy. As dasatinib is also known to have immunostimulatory effects (activation of T and NK cells and downregulation of regulatory T cells), we aimed to monitor the immune effects of dasatinib and IFN-α combination treatment.

Abstract Rationale: Dasatinib (DAS) and interferon have different modes of action and may have synergistic activity in CML, due to both antineoplastic and immunostimulatory mechanisms. Addition of pegylated interferon (PegIFN) to imatinib therapy in CP-CML has in previous clinical trials (French SPIRIT and NordCML002) resulted in deeper molecular responses. Thus, an optimal combination of DAS and PegIFN may increase the proportion of patients who reach deep molecular response with potential for treatment-free remission (TFR). Design: Newly diagnosed CP-CML patients were treated with DAS (Sprycel, BMS) 100 mg OD as single drug for three months. Thereafter weekly subcutaneous injections of Peg-IFN α2b (PegIntron, MSD) were added to DAS; from end of month 3 (M3) to M6, 15µg/week, thereafter 25µg/week until M15. Primary end points were safety and the rate of MMR at M12. The doses of PegIFN were lower than in the SPIRIT and NordCML002 studies to increase adherence. Population: Forty patients were included at 14 university centers. One patient was lost to follow-up after M6. All patients were included in analysis up to M12. Mean and median age was 48 years (range 19-71). The proportions of high risk patients were 25% (Sokal), 15% (Hasford), and 15% (EUTOS). Safety and dosing: Treatment was well tolerated with expected DAS and PegIFN related side effects. Six patients had seven serious adverse events (AEs), all hospitalizations. 1 episode each of bradycardia/atrial fibrillation (possibly PegIFN-related), headache (DAS), fever (PegIFN), anaphylaxis-like reaction (PegIFN), fever/malaise/headache (PegIFN), pneumonia and a knee effusion (both unrelated). One pleural effusion occurred (grade 2, 3%). Grade 3-4 neutropenia and thrombocytopenia occurred in 6 and 9 patients respectively. Prolonged hematological toxicity (>2 months) occurred in 8 patients, causing dosing problems in 5. One patient suffered grade 3 depression. Grade 3 flu-like symptoms occurred in 2 patients. One patient had lipase elevation grade 3 and one patient developed hypothyroidism attributed to PegIFN. Grade 2 dermal AEs like rash and acne occurred in about 20%, attributable to both drugs. 94% (DAS) and 76% (PegIFN) of assigned dose was given. Dose reductions occurred in 19 patients for DAS and 20 patients for PegIFN. Two patients discontinued DAS and switched to nilotinib, 1 for headache at M3 and 1 at M12 for lack of efficacy/hematological toxicity. Two patients could not start PegIFN for hematological toxicity (one lost to follow-up after M6). PegIFN was discontinued because of bradycardia/atrial fibrillation (1 patient), anaphylaxis (1 patient), flu-like syndrome (2 patients) and long-term hematological toxicity (2 patients). At 12 months 31/38 pats (82%) were still on PegIFN, a higher proportion than in the French Spirit or NordCML002 studies. Efficacy: We have used the DAS arm of the Dasision study (Kantarjian NEJM 2010) as a historical control. Early response at M3 was very similar between studies. In the present and the Dasision cohorts respectively, 18% vs 16% missed the 10% BCR-ABLIS landmark, 66% vs 56% achieved a CCyR and 8% vs 8% achieved MMR. At M6, three months after introduction of PegIFN, a steep increase in MMR rate was observed compared with Dasision. This was also reflected in deep responses, MR4.0 (see tables) and MR4.5 at M12, 18% vs 5%. The primary efficacy endpoint was MMR at M12, 82% vs 46%. Table 1.MMRDAS+PegIFN (%)DAS (Dasision)(%)Difference (%)M3880M6532726M9663927M12824636Table 2.MR4.0DAS+PegIFN (%)DAS (Dasision) (%)Difference (%)M3303M620614M938830M12481236 Progressions and treatment failure defined by ELN 2013: Failures: No progression was noted. At M3, 2 patients still had >95% Ph+ metaphases (MF). At M6, four patients (11%) had > 35% Ph+MF or >10% BCR-ABL levels. At M12, one patient failed CCgR and two more patients failed <1% BCR-ABL. No BCR-ABL mutations were detected in "failure" patients. Conclusion: The combination of DAS and low dose PegIFN could be safely administered in newly diagnosed CP CML. No unexpected autoimmune phenomena were observed, and pleural effusions were rare. Efficacy appears very promising with high early MMR rates and deep molecular responses. A randomized comparison DAS +/- PegIFN is warranted. Support: Study drug from BMS and MSD. Grant from BMS. Figure 1. Figure 1. Disclosures Hjorth-Hansen: Ariad: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Off Label Use: Dasatinib and Pegylated IFN combination in CML. Richter:Ariad: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding.

Abstract Background: The tyrosine kinase inhibitors (TKIs) have dramatically changed the natural history of chronic myeloid leukemia (CML) leading to significant improvement in clinical outcome and survival rates. The option of treatment cessation has recently become of utmost importance. Indeed, prospective trials suggest that imatinib therapy may be safely and successfully discontinued in CML pts with deep and sustained molecular responses (Mahon Lancet Oncol 2010, Ross Blood 2013). The major aim of the EURO-SKI study (European Leukemia Net Stop TKI study) was to define prognostic markers to increase the rate of patients in durable deep MR after stopping TKI. Further aims were the evaluation of harmonized methods of molecular monitoring, assessment of quality of life, and calculation of saved treatment costs per country. Methods: Adult CML patients in chronic phase CML on TKI treatment in confirmed deep molecular response (MR4, BCR-ABL <0.01%) for at least one year (>4 log reduction on TKI therapy for >12 months confirmed by three consecutive PCR tests) and under TKI treatment for at least 3 years were eligible. MR4confirmation was performed in a standardized laboratory (n=6). Primary endpoint was the assessment of the duration of MR (defined by continuous MMR) after stopping TKI. Patients (pts) after a prior TKI failure were excluded. According to protocol, an interim analysis was planned after 200 patients with eligible molecular results at month (mo) 6 were available to test the null hypothesis that relapse-free survival at 6 mo is less or equal 40%. Results: From June 2012 to June 2014, 498 CML pts in chronic phase from 10 countries were enrolled and included in the trial. From June 2012 to July 2013, 254 pts from 8 countries were registered; 54 were excluded (consent withdrawal n=1, protocol violation n=1, not eligible n=34, restart of TKI without relapse n=4, atypical or unknown transcript n=6, missing data n=8). Of the eligible 200 pts, 41.5% were female. Median age at diagnosis was 53.3 years (range, 13.8 to 85.5). In assessable pts 8.7% and 18.2 % were high-risk according to EUTOS and Sokal Scores. 103 pts were treated prior to the start TKI therapy, mostly with hydroxyurea or interferon. 1st-line TKI was imatinib in 97%, dasatinib in 1.5%, and nilotinib in 1.5% of pts. Twenty-four pts switched to second-line TKI therapy due to intolerance, 16 to dasatinib, 2 to imatinib, and 6 to nilotinib. The median time from diagnosis of CML to TKI cessation was 8 years (range, 3-19 years). TKI treatment duration was less than 5 years in 16%, 5-8 years in 36% and > 8 years in 48% of pts. Median duration of TKI treatment was 8 years (range, 3-12.6 years) and median duration of MR4 before TKI cessation was 5.4 years (range, 1-11.7 years). MR4duration was less than 2 years in 8%, 2-5 years in 37%, 5-8 years in 39% and >8 years in 16% of pts. For all eligible pts, a standardized European laboratory confirmed MR4 assessment. Since 123 of the 200 pts (61.5%, 95% CI: [54.4%; 68.3%]) remained without relapse the first 6 mo, the null hypothesis could be discarded (p<0.0001). Recurrence of CML, defined as loss of MMR, was observed in 43/92 pts (47%) treated <8 years, as compared to 23/87 pts (26%) treated for >8 years (p= 0.005). So far, there was a trend for prognostic significance of MR4 duration: 33/71 pts with MR4 <5 years (46%) lost MMR within 6 mo as compared to 28/87 pts (32%) with MR4duration >5 years (p=0.07). No significant difference was observed for relapse within 6 mo according to depth of molecular response at discontinuation (MR4 vs MR4.5 vs MR5). TKI cessation was a safe procedure but a substantial proportion of pts reported transitory musculoskeletal pain starting within weeks after imatinib discontinuation. The phenomenon was described in 30% of Swedish patients as a “TKI withdrawal syndrome” (Richter JCO 2014). Taking into account the cost of imatinib in Europe and time without treatment in the total study population at the most recent analysis, total savings for the community within the EURO-SKI trial were estimated at 7 million Euros. Conclusion: Employing a standardized molecular testing for patient selection within a TKI cessation trial in CML the chance to stay in treatment-free remission could be higher than previously reported. The EURO-SKI trial will further elucidate the prognostic factors but the preliminary results confirm (as reported in the STIM Study) the prognostic impact of the duration of TKI therapy before stopping. Disclosures Mahon: NOVARTIS PHARMA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BRISTOL MYERS SQUIBB: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria; PFIZER: Honoraria. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Hjorth-Hansen:Novartis: Honoraria; Bristol-myers Squibb: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Mayer:Janssen Research & Development: Research Funding; Roche: Research Funding; GlaxoSmithKline: Research Funding; Celgene: Research Funding. Almeida:Celgene: Consultancy; Novartis: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Bristol-Meyer Squibb: Membership on an entity's Board of Directors or advisory committees. Berger:Genzyme/Sanofi and Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Hochhaus:ARIAD Pharmaceuticals, Inc.: Research Funding. Saussele:Novartis: Honoraria, Research Funding, Travel Other; Bristol-Myers Squibb: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria, Travel, Travel Other.

Imatinib, a small molecule inhibitor of ABL, PDGFR and C-KIT, has revolutionized treatment of chronic myeloid leukaemia (CML). However, resistance to treatment is of increasing importance and often is due to point mutations in the Abl kinase domain (Abl KD). Here, we analysed clinical outcome and mutation status in two independent Nordic populations (n = 77) of imatinib-resistant CML patients. We detected BCR-ABL transcripts containing point mutations of residues in the P-loop, A-loop and other kinase domain residues in 32 patients (42%). In contrast to previous data, mutations in BCR-ABL were as frequently found in patients with primary resistance (56%) as with secondary resistance (53%). No T315I mutations were found in the study cohort. BCR-ABL splice variants were identified in a significant number of our cases (19%): BCR-ABL transcripts of variable length; a variant fusion transcript joining BCR exon 14 sequences to ABL exon 4; partial, in-frame-deletion of exon 4 due to induction of a cryptic splice site by the L248V and finally, alternative splicing of ABL exon 7 sequences. Though the majority of splice variants observed in this study do not encode functional proteins, alternative splicing appears to represent a common phenomenon in the biology of CML. We conclude that Abl KD point mutations represent a major mechanism of imatinib resistance. Other sequence irregularities were also detected, but their significance in conferring resistance is unclear. Diagnostic strategies looking for imatinib-resistant clones should be designed to detect a broader profile of BCR-ABL variants than just point mutations.

The BCR-ABL T315I mutation causes resistance to imatinib, nilotinib and dasatinib in chronic myeloid leukemia. Forty BCR-ABL positive patients with imatinib resistance were analyzed for T315I mutated clones after six months on nilotinib or dasatinib treatment by quantitative allele-specific ligation polymerase chain reaction with a sensitivity of 0.05%. Ligation polymerase chain reaction revealed 10 patients with more than 10−5 BCR-ABLT315I%/GUS (high levels), none of whom achieved major molecular response after 12 months, and a further 8 patients with 10−5 or below BCR-ABLT315I%/GUS (low levels) who all achieved major molecular response (P

Background: Thrombogenicity of atherosclerotic plaque largely depends on plaque morphology and their content of tissue factor (TF) and tissue factor pathway inhibitor (TFPI). The relationship between morphological composition of plaque (lipid-rich or calcified) and expression of TF and TFPI in circulating blood monocytes and within the plaques is not characterized.Objective: To investigate whether lipid-rich (echolucent) or calcified (echogenic) morphology of carotid atherosclerotic plaques is associated with differences in TF and TFPI expression in circulating blood monocytes and within carotid atherosclerotic plaques.Methods: We studied levels of monocyte TF and TFPI mRNA and protein expression and association with traditional risk factors for atherosclerosis in asymptomatic subjects with echolucent (n=20) or echogenic (n=20) carotid plaques, or controls without carotid atherosclerosis (n=20) determined by ultrasonography. Sections of calcified or lipid-rich carotid plaques obtained from symptomatic patients were assessed for TF and TFPI antigen expression.Results: TF and TFPI surface presentation, surface TF/TFPI ratio, and TF activity were higher in monocytes obtained from subjects with echolucent than with echogenic plaques or controls without carotid atherosclerosis. Multiple regression analyses revealed inverse association between serum apoA1 and monocyte surface TF antigen expression (p=0.007), and positive association between serum apoB and monocyte surface TFPI expression (p=0.028). Sections from lipid-rich carotid plaques contained 2.5-fold more TF and 1.5-fold more TFPI antigens relative to calcified lesions, also yielding a higher TF/TFPI ratio.Conclusions: Our findings indicate that circulating monocytes of asymptomatic individuals with echolucent lipid-rich carotid atherosclerosis express an imbalance between TF and TFPI expression cohering with changes found within advanced carotid atherosclerotic plaques obtained from symptomatic patients. (C) 2011 Elsevier Ltd. All rights reserved.

Biologic and clinical observations suggest that combining imatinib with IFN-α may improve treatment outcome in chronic myeloid leukemia (CML). We randomized newly diagnosed chronic-phase CML patients with a low or intermediate Sokal risk score and in imatinib-induced complete hematologic remission either to receive a combination of pegylated IFN-α2b (Peg-IFN-α2b) 50 μg weekly and imatinib 400 mg daily (n = 56) or to receive imatinib 400 mg daily monotherapy (n = 56). The primary endpoint was the major molecular response (MMR) rate at 12 months after randomization. In both arms, 4 patients (7%) discontinued imatinib treatment (1 because of blastic transformation in imatinib arm). In addition, in the combination arm, 34 patients (61%) discontinued Peg-IFN-α2b, most because of toxicity. The MMR rate at 12 months was significantly higher in the imatinib plus Peg-IFN-α2b arm (82%) compared with the imatinib monotherapy arm (54%; intention-to-treat, P = .002). The MMR rate increased with the duration of Peg-IFN-α2b treatment (< 12-week MMR rate 67%, > 12-week MMR rate 91%). Thus, the addition of even relatively short periods of Peg-IFN-α2b to imatinib markedly increased the MMR rate at 12 months of therapy. Lower doses of Peg-IFN-α2b may enhance tolerability while retaining efficacy and could be considered in future protocols with curative intent.

Mutations in the Bcr-Abl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia patients. Recommendations aimed to rationalize the use of BCR-ABL mutation testing in chronic myeloid leukemia have been compiled by a panel of experts appointed by the European LeukemiaNet (ELN) and European Treatment and Outcome Study and are here reported. Based on a critical review of the literature and, whenever necessary, on panelists' experience, key issues were identified and discussed concerning: (1) when to perform mutation analysis, (2) how to perform it, and (3) how to translate results into clinical practice. In chronic phase patients receiving imatinib first-line, mutation analysis is recommended only in case of failure or suboptimal response according to the ELN criteria. In imatinib-resistant patients receiving an alternative TKI, mutation analysis is recommended in case of hematologic or cytogenetic failure as provisionally defined by the ELN. The recommended methodology is direct sequencing, although it may be preceded by screening with other techniques, such as denaturing-high performance liquid chromatography. In all the cases outlined within this abstract, a positive result is an indication for therapeutic change. Some specific mutations weigh on TKI selection.

Abstract Abstract 1679 The BCR-ABL T315I mutation is associated with resistance against imatinib (IM), nilotinib (NI) and dasatinib (DA) in patients with chronic myeloid leukemia (CML). Although this mutation can be detected by different techniques and at different levels of the mutated clone, the prognostic significance of the absolute amount of the mutated allele is widely unknown. We therefore sought to determine absolute T315I allele levels after 6 months on second line NI or DA therapy to identify a cut off value for predicting the achievement of major molecular response after 12 months (MMR12; ≤0.1% BCR-ABL transcripts at international scale). Forty randomly selected BCR-ABL positive CML patients (n=24 male, median age 64, range 39–78 years) in chronic (n=31), accelerated (n=7), or blastic phase (n=2) with failure to IM were treated with DA 140 mg/day (n=20) or NI 800 mg/day (n=20) and analyzed by denaturing high-performance liquid chromatography (D-HPLC) and high-sensitive allele-specific ligation PCR (L-PCR) for quantification of mutated T315I BCR-ABL allele normalized by beta-glucuronidase (GUS) expression. This method was previously shown to obtain a dynamical detection range of 100% to <0.1% mutant T315I allele (3–3.5 log). The levels of the BCR-ABL T315I alleles at 6 months were correlated with the achievement of a MMR12 on second line treatment. The L-PCR investigator was blinded for the MMR12 results. On second line therapy, MMR12 was achieved by 12 (20%) patients. After 6 months, 37 (10 with MMR12) patients were available for D-HPLC assessment and showed a T315I mutation in 3 (8%) patients. None of these patients achieved MMR12. In comparison, 35 evaluable patients (8 with MMR12) showed >10−5 BCR-ABLT315I/GUS (n=10, 20%), <10−5 (n=8, 22%) or no (n=17, 48%) T315I mutation by L-PCR. All patients with >10−5 BCR-ABLT315I/GUS did not achieve MMR12, all patients with <10−5 developed MMR12 (p<0.0001). This pre-defined value of >10−5 BCR-ABLT315I/GUS as a negative predictor for MMR12 was applied to a new second independent group of 40 IM resistant patients (n=17 male, median age 60, range 32–79 years) all in first chronic phase, MMR12 n=9, 22.5%) equally distributed to NI and DA. In this group 12 (30%) patients showed >10−5 BCR-ABLT315I/GUS after 6 months and all but one did not achieve MMR12. In contrast, 5 patients with <10−5 showed MMR12, 3 patients did not (p=0.0181). In conclusion, absolute quantification of the mutant T315I allele is helpful to identify patients with a low chance of MMR12 on second line nilotinib or dasatinib treatment. Therefore, relevant mutations should be quantified into clinical trial protocols to identify certain amounts with prognostic significance of the mutated clones for a specific mutations on a given TKI treatment in CML. Disclosures: Lange: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hochhaus:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: Although the procoagulant reactivity of monocytes largely depends on expression and cell surface presentation of tissue factor (TF), little is known about the impact of tissue factor pathway inhibitor (TFPI) on regulation of TF function on the monocyte surface.Materials and methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy subjects and cryopreserved. We investigated TF and TFPI mRNA expression by reverse transcription-quantitative real-time PCR (RT-qPCR), surface presentation by flow cytometry and confocal microscopy, and TFPI-mediated regulation of TF functional activity on the surface of resting and LPS-stimulated PBMCs by TF activity assay and Calibrated Automated Thrombogram (CAT) assay.Results: Unstimulated PBMCs contained nearly no TF, but detectable TFPI protein levels. TFPI mRNA levels were 2-fold higher than TF, and the TFPI alpha mRNA isoform expression was higher than TFPI beta. LPS stimulation caused a parallel and sustained upregulation of both TFPI isoforms, concomitant with increased surface presentation of TFPI antigen. Stronger, but transient upregulation of TF mRNA and surface antigen was observed at 6 hrs of LPS stimulation. After LPS stimulation TF and TFPI were co-localized in the same areas of the monocyte membrane. Pre-incubation of PBMCs with anti-TFPI IgG significantly enhanced TF activity, shortened Lag-time, and increased thrombin generation. TFPI-dependent inhibition of TF was more prominent in resting than in LPS-stimulated cells.Conclusions: Our results support the concept that surface TFPI is an important regulator of procoagulant reactivity of human monocytes. (C) 2010 Elsevier Ltd. All rights reserved.

Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease that is often associated with several chromosomal and molecular abnormalities. Patients who have the Philadelphia (Ph) chromosome and associated BCR-ABL1 oncogene have a particularly poor prognosis. Currently, allogeneic haematopoietic stem cell transplantation (allo-HSCT) is the only known curative treatment for Ph+ ALL and facilitating allo-HSCT in eligible patients is a key treatment goal. However, many patients relapse after allo-HSCT, particularly those with measurable residual disease prior to transplantation, and a significant percentage of patients are ineligible for allo-HSCT, particularly older patients. Hence, many patients require additional/alternative therapies to prolong survival. Studies are ongoing to determine the most effective first-line drug regimens for patients who subsequently undergo allo-HSCT and ineligible patients. Tyrosine kinase inhibitors targeted to Bcr-Abl are important novel therapies for Ph+ ALL. Although imatinib administered in combination with chemotherapy is established as the current first-line strategy, relapse is common, even among allo-HSCT recipients. Emerging data indicate that more potent multi-targeted kinase inhibitors (including dasatinib, nilotinib, and bosutinib) have promising efficacy in the first- or second-line setting. Here, the evidence base for existing drug treatments for Ph+ ALL is discussed and emerging therapeutic strategies are explored.