Minimal residual disease (MRD) after allogeneic stem cell transplantation (SCT) for Ph+ acute lymphoblastic leukemia (ALL) is predictive of relapse. Imatinib administration subsequent to SCT may prevent relapse, but the role of scheduling and its impact on outcome are not known. In a prospective, randomized multicenter trial, we compared the tolerability and efficacy of post-transplant imatinib administered either prophylactically (arm A; n=26) or following detection of MRD (arm B; n=29). Prophylactic imatinib significantly reduced the incidence of molecular recurrence after SCT compared with MRD-triggered imatinib (40% vs 69%; P=0.046). Median duration of PCR negativity was 26.5 and 6.8 months, respectively (P=0.065). Five-year survival in both interventional groups was high (80 and 74.5%), despite premature discontinuation of imatinib in the majority of patients because of poor tolerability. Relapse probability was significantly higher in patients who became MRD positive (P=0.017). In conclusion, post-transplant imatinib results in a low relapse rate, durable remissions and excellent long-term outcome in patients with BCR-ABL1-positive ALL irrespective of whether it is given prophylactically or MRD-triggered. Reappearance of BCR-ABL1 transcripts early after SCT or at higher levels identifies a small subset of patients who do not benefit sufficiently from imatinib, and in whom alternative approaches should be explored.

Imatinib is highly effective in newly diagnosed, but not in relapsed, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). BCR-ABL tyrosine kinase domain (TKD) mutations are associated with acquired imatinib resistance, but their role in primary resistance is uncertain. Using highly sensitive ligation-PCR and denaturing high-performance liquid chromatography (DHPLC), we identified baseline TKD mutations in 21% and 42% of imatinib-naïve patients with newly diagnosed ( n =26) or recurrent ( n =65) Ph+ ALL, respectively ( P =ns). Within 4 weeks of starting the imatinib treatment, absolute levels of mutant bcr-abl transcripts increased significantly in patients with advanced, but not with de novo , Ph+ ALL. The net expansion of pre-existing mutant clones during imatinib treatment resulted in the rapid appearance of initially undetectable TKD mutations, which after 4 weeks were detectable in 70% of patients with advanced disease. There was a high degree of concordance between the type of mutations detected at relapse and during initial imatinib treatment. The profoundly different outgrowth dynamics of leukemic clones with bcr-abl mutations in imatinib-treated patients who differ in their disease history, provides clinical–translational evidence for a contributory role of non-mutational resistance mechanisms, possibly induced by prior chemotherapy. Moreover, the prevalence of pre-existing, clinically relevant TKD may have been underestimated in tyrosine kinase inhibitor-naïve patients with Ph+ ALL.

Abstract 247 Background: The presence of minimal residual disease (MRD) after allogeneic stem cell transplantation (SCT) for Ph+ ALL is highly predictive of eventual relapse. Imatinib (IM) has very limited efficacy in hematologic relapse of Ph+ALL, but may prevent leukemia recurrence if started when the leukemia burden is still very low and detectable only by molecular techniques. The optimal time for starting IM post transplant and the prognostic relevance of different bcr-abl transcript levels in relation to time after SCT have not been established. Aims: To determine the impact of post-transplant IM, given either prophylactically or after detection of bcr-abl transcripts (pre-emptively), on the overall incidence of MRD, remission duration, long-term treatment outcome and tolerability in pts. who underwent SCT for Ph+ALL in complete remission. Study Design: In a prospective, randomized multicenter trial, previously transplanted Ph+ ALL pts. (n=55) were assigned to receive imatinib prophylactically (n=26) or pre-emptively (n=29). SCT was performed in CR1 in 23 pts. and 27 pts. in the two groups, respectively. Five pts.were transplanted in CR2. Serial assessment of bcr-abl transcripts was performed by quantitative RT-PCR and additionally by nested-RT-PCR if the sensitivity of the qRT-PCR was below the quantitative range. Confirmatory testing of a second independent sample was not required, to reduce the risk of treatment delays. Samples were considered PCR negative only if the ABL copy number exceeded 10 4 . Imatinib administration was scheduled for one year of continuous PCR negativity. Results: IM was started in 24/26 pts. allocated to prophylactic IM and in 14/29 pts. in the pre-emptive arm. The majority of pts. received IM 400 mg/d (26/38 pts.), the other 12 pts. 600 mg IM daily. IM was started a median of 48 d after SCT in the prophylactic arm and 70 d after SCT with pre-emptive therapy. After a median follow-up of 30 mos. and 32 mos., respectively, 82% and 78% of pts. are alive in ongoing CR, 4 pts. died in CR. Five pts. transplanted in CR1 and 2/5 pts. transplanted in CR2 have relapsed (median follow-up 9 mos. and 10.5 mos., respectively). The frequency of MRD positivity was significantly lower in pts. assigned to prophylactic imatinib (10/26; 40%) than those in the pre-emptive treatment arm (20/29; 69%) (p=0.046 by chi 2 test). Only 9 of 29 pts. assigned to pre-emptive imatinib remained continuously PCR negative after SCT, with a median follow-up of 32 months (18–46 months) after SCT. The median duration of sustained, uninterrupted PCR negativity after SCT is 26.5 months with prophylactic and 6.8 months with pre-emptive administration of imatinib (p=0.065). The probability of remaing in CHR after SCT was significantly lower in partients who remained MRD negative after SCT (p=0.0002). Analysis of the kinetics of molecular relapse showed that detection of bcr-abl transcripts within 100 days of transplant, despite rapid initiation of IM, was associated with a significantly inferior EFS compared to first detection of MRD positivity more than 100 days after SCT. IM was discontinued prematurely in 54% pts. receiving imatinib prophylactically and in 64% of pts. receiving imatinib pre-emptively, mostly due to gastrointestinal toxicity. Accordingly, the time to IM discontinuation was 245 d and 191 d in the prophylactic and the pre-emptive treatment arms, respectively. Despite this early discontinuation rate, overall survival in the two treatment groups was 80% and 74.5% after 5 years, with no significant difference by log rank test (p=0.84). Conclusions: Prophylactic administration of imatinib significantly reduces the incidence of molecular relapse after SCT. Both interventional strategies are associated with a low rate of hematologic relapse, durable remissions and excellent long-term outcome in patients with Ph+ ALL. The presence of MRD both prior to and early after SCT identifies a small subset of patients with a poor prognosis despite post-transplant imatinib, and warrants testing of alternative approaches to prevent hematologic relapse. Disclosures: Schuld: Novartis: Employment. Goekbuget: Micromet: Consultancy. Ottmann: Novartis Corporation: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding.

Abstract 173 Imatinib (IM) in combination with either of a variety of chemotherapy regimens has become the mainstay of front-line treatment for younger patients with Ph+ ALL, followed by allogeneic SCT as a curative treatment option. Complete remission (CR) rates generally exceed 90% and overall treatment outcomes have improved as judged on the basis of phase II trials, but there is a paucity of long-term outcome data obtained from a large group of prospectively evaluated patients. Moreover, the impact of different front-line IM schedules on the outcome of patients undergoing allogeneic stem cell transplantation in first CR (CR1) as opposed to non-transplanted patients has not been thoroughly evaluated. We previously reported the results of a GMALL study comparing two alternating and concurrent schedules of IM and chemotherapy in successive patient cohorts (A1 and A2, respectively)(Wassmann et al, Blood 2006;108:1469). In a subsequent cohort (A3), IM was started simultaneously with induction chemotherapy according to the GMALL protocol 07/03, i.e. immediately after confirmation of the presence of a Philadelphia chromosome and/or bcr-abl translocation and completion of prephase therapy. We here report the long-term results of a prospective multicenter study of the GMALL study group including a total of 335 patients with newly diagnosed Ph+ ALL who received IM given at a single daily oral dose of 600 mg within 3 successive treatment cohorts. A1: IM administered between induction (IND) and first consolidation cycles (CONS1) and again after CONS1 (n=51); A2: IM given during the second half of IND and then continued throughout CONS1 until SCT (n=105); A3: IM initiated together with start of induction chemotherapy and continued throughout CONS1 until SCT (n=179). Minimal residual disease (MRD) was serially assessed by quantitative RT-PCR, mutational analyses was performed by D-HPLC and direct sequencing. The median age of all patients was 43 years (17-65 y), 57 (17%) patients were 55 years of age or older, 147 (44%) male and 182 (56%) female. CR rates in cohorts A2 and A3 were 89,4% and 85.7%, induction deaths occurred in 5.8% and 11.3% of patients, treatment failure was observed in 4.8% and 3% of pts., respectively. Initial responses are not reported for cohort A1 as only pts. already in CR or PR were eligible for study entry at this stage of the trial. The molecular response rate based on PCR negativity for bcr-abl transcripts after CONS1 was superior in cohort A3 with 33% (26/79) as compared to 12.5% (5/40) and 4.2% (2/47) in cohorts A2 and A1, respectively (p=0.01). Overall treatment outcome improved with earlier initiation and more prolonged administration of IM in the three successive patient cohorts: Overall survival (OS) at 4 years was 31%, 40% and 50% in cohorts A1, A2 and A3, respectively. There was a trend towards lower rates of pre-transplant relapse or treatment discontinuation of patients in CR in cohort A3 (4% and 1.1%, respectively) compared to cohorts A2 (8.7% and 5.8%) or A1 (11.8% and 5.9%). To date, 219 patients (66.4%) underwent SCT in CR1 (A1: n=39; A2: n=74; A3: n=106), with a median age of 39.5 years. The 3 year probability of DFS of pts. in cohort A3 who received myeloablative conditioning regimens combining TBI with cyclophosphamide or etoposide was 72%. DFS was not significantly different between matched sibling and matched unrelated donor SCT. The incidence of relapse after SCT was substantially lower among patients in cohort A3 (11.3%) than those in A2 (24.3%) or A1 (30.8%). Similarly, there was a trend to lower non-relapse mortality after SCT in A3 (20.8%) compared to A2 (25.7%) or A1 (33.3%). For all pts. transplanted in CR1 irrespective of treatment cohort, median OS was 57% after 3 yrs. and 52% after 7 yrs. Patients who did not undergo SCT in CR1 had a dismal outcome, with a median OS of 9.4 months and 14% alive after 3 years. In conclusion, earlier and more prolonged administration of IM in conjunction with chemotherapy is associated with superior treatment outcomes after SCT in newly diagnosed pts. with Ph+ ALL who are considered eligible for allogeneic SCT. SCT in CR1 remains the treatment of choice even in patients who achieve a good molecular response to initial therapy. Reducing induction as well as post-transplant mortality could have the greatest impact on further improving OS and DFS. Disclosures: Ottmann:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

In this review we describe general therapeutic options for adult patients with acute lymphoblastic leukaemia and focus on specific new treatment strategies. Current novel approaches include monoclonal antibodies against leukaemic-associated antigens, new formulations of existing chemotherapeutic agents, new antimetabolites and nucleoside analogons and targeted molecular therapy. Some of these agents have already been adopted into standard regimens, leading to dramatically improved outcomes for example in mature B-ALL and in the formerly most unfavourable subgroup Philadelphia chromosome (Ph)/BCR-ABL-positive ALL, while others still remain in early phases of development.

We report on the first successful allogeneic stem cell transplantation (SCT) in an HIV-infected patient with severe aplastic anemia (SAA) per- formed at a tertiary care institution. Highly active antiretroviral therapy (HAART) was administered until transplantation and restarted 34 days later with sustained virological response. The patient did however develop a rapid rise in HIV load during the interruption of HAART associated with an acute febrile illness. Due to the extended period between the onset of SAA until SCT, the posttransplant course was complicated by bacterial infections. Stage two skin GvHD, but no AIDS-defining opportunistic diseases were experienced. Neutrophils recovered to >0.5/nL on day +18 and the CD4 count reached 250/microL on day +71 and >500/microL on day +182. The patient is in good condition with an ECOG score of 0 twelve months after transplantation. This report demonstrates the feasibility of allogeneic stem cell transplantation in the HIV setting.

3015 Background: AMN107, a novel aminopyrimidine ATP-competitive inhibitor of Bcr-Abl. AMN107, is 10- to 50-fold more potent than imatinib against Bcr-Abl expressing cell lines, and is effective against most cell lines expressing imatinib-resistant Bcr-Abl mutants. In two p190-Bcr-Abl ALL cell lines, AMN107 was 30–40 times more potent than imatinib in inhibiting cellular proliferation and in inhibiting phosphorylation of p190 Bcr-Abl tyrosine kinase in cell lines and primary ALL cells. Methods: In an ongoing phase I study, AMN107 was given orally, once or twice daily dosing schedule. Results: Fifteen of 67 total pts entered had lymphoid disease: lymphoid blastic phase CML (CML LBP) 7 pts; Ph+ acute lymphocytic leukemia (Ph+ ALL) 8 patients. Median age was 53 years (range 28–80). AMN doses were: QD dose cohorts (mg/day); 50 (1 pt), 100 (2 pts), 200 (3 pts), 400 (1 pt), 600 (2 pts), 800 (4 pts), 1200 (1 pt); BID dose cohort; 400 mg BID (1 pt). Pts have been treated for 15 to 89 days. In the overall cohort, possible AMN-related Grade 3 or 4 hematologic adverse events were observed in 5/67 patients (3 responded) at doses ≥ 200 mg: Possible AMN-related non-hematologic Grade 3/4 events were: 600 mg, G3 skin rash (1 pt); ≥ 800 mg, G3 SGOT and/or SGPT elevations (2 pts), G3 indirect hyperbilirubinemia (2 pts), and at 1200 mg, G2 pancreatitis (1 pt). Clinical activity has been observed in 5 of the 14 with lymphoid disease treated with doses of ≥ 600 mg: hematological improvement (1 pt, Ph+ ALL), peripheral responses (2 pts, LBP), and bone marrow responses (marrow response/no evidence of leukemia, 2 pts, LBP) 1 pt with LBP had a cytogenetic CR. Mutational data are available for 3 of the responding patients: Y253H (hematologic improvement), T315I (peripheral response), and E355G (marrow response). Pharmacokinetic (PK) studies showed steady state was generally reached by day 8. Both Cmax and AUC were dose proportional from 50 to 400 mg with a median Tmax of 3 hours. The apparent half-life was 16 hours. Conclusions: AMN107 has significant activity in patients treated with ≥ 600 mg per day in imatinib-resistant CML LBP or Ph+ ALL Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis Novartis

WITHDRAWN. 31 Loss of terminal fucose residue expression associated with Bombay phenotype influences plasma von Willebrand factor and factor VIII antigen levels TA MCKINNON*, MA LAFFAN*, AF RIDDELL, A HANN* and JS O’DONNELL* *Haematology Department, Hammersmith Hospital and Imperial College, London, UK, Katherine Dormandy Haemophilia Centre, Royal Free Hospital, London, UK ABO blood group exerts a major quantitative effect on plasma VWF level. Group O individuals have significantly lower VWF:Ag levels. Furthermore, ABH antigenic determinants are carried on the N-linked glycans of circulating VWF according to the blood group of the individual. The mechanism through which these glycans determine plasma VWF levels remains unknown. To further investigate this mechanism, we have collected plasma samples from 47 individuals with the rare Bombay blood group phenotype. These individuals fail to express alpha-1,2 fucosyltransferase, and thus cannot express A, B or O(H) antigens regardless of their ABO genotype. Using a modified ELISA technique, we confirmed that Bombay plasma VWF in all cases did not express ABO(H) determinants. We found VWF : Ag levels in Bombay patients (median VWF : Ag = 0.69 IU/dl) to be significantly lower than those observed in groups AB, A or B (median VWF : Ag = 1.24, 1.01 and 0.94 respectively; p < 0.05). Moreover, Bombay VWF : Ag levels were also lower than those in group O individuals (median VWF : Ag = 0.78), although this difference failed to achieve statistical significance. VWF : CB was also reduced in the Bombay group (median VWF : CB = 28 Elucidating the role of MLL in adult murine haematopoiesis K MCMAHON*, S HADJUR*, U MENZEL, D KIOUSSIS and HJM BRADY* *Molecular Haematology and Cancer Biology Unit, Institute of Child Health, University College London, London, UK, Molecular Immunology, MRC NIMR, London, UK The gene MLL has a fundamental role in haematopoietic stem cell development. Research so far has concentrated on the role of MLL in embryonic haematopoiesis as MLL homozygous knockout embryos die in utero. Studies in foetal liver, yolk sac cells and chimaeras have shown that lack of MLL leads to a reduction of cells in the myeloid, B and Tcell lineages. However, the role of a given gene in haematopoiesis may differ in the context of the embryo and the adult mouse as shown by studies on other embryonic lethal genes such as SCL. To study the effect of MLL deletion on adult haematopoiesis we have generated conditional knockout mice, carrying an allele of MLL bearing Lox-P recombination sites (‘floxed’) that will be recombined in the presence of the Cre recombinase, truncating the gene. When bred to mice carrying the Cre recombinase, the resulting offspring will lack MLL only in specified haematopoietic lineages, bypassing the embryonic lethality caused by Hox deregulation in other parts of the embryo. We have successfully generated chimaeras bearing the ‘floxed’ alleles which have been bred to create conditional knockouts. These mice are being analysed to determine the impact of the absence of MLL on adult haematopoietic stem cell development and renewal. 0.71 IU/dl) compared to other blood groups, but the ratio of VWF : CB to VWF : Ag remained unchanged. Furthermore, VWF multimer pattern in the Bombay plasmas demonstrated no loss of HMW multimers. ABO blood group also influences plasma FVIII : Ag levels. In Bombay individuals, we found FVIII : Ag levels to be significantly higher (median FVIII : Ag = 0.91 IU/dl) than corresponding VWF : Ag levels (median VWF : Ag = 0.69 IU/dl), resulting in an elevation of the FVIII : VWF ratio compared to other ABO groups (medians 1.32 and 1.0 respectively) However in VWF-FVIII binding ELISA, the FVIII–binding capacity of Bombay VWF was not increased. Our findings support the hypothesis that alterations in terminal carbohydrate moiety expression on plasma VWF-FVIII constitute important determinants of both VWF : Ag and FVIII : Ag levels. 32 A randomised control trial of patient selfmanagement of oral anticoagulation compared with patient self-testing C GARDINER*, KE WILLIAMS*, IJ MACKIE, SJ MACHIN and H COHEN* *Department of Haematology, University College London Hospitals, London, UK, Department of Haematology, UCL, London, UK Several studies suggest that patient self-management (PSM) may improve the quality of oral anticoagulant therapy (OAT) as measured by time in INR target range. Whether this improvement is due to PSM itself, or more frequent testing is unclear. We performed a randomised control study to determine whether the quality of treatment afforded by PSM is superior to that achieved by patient self-testing (PST) alone. 13% of eligible patients from our hospital anticoagulant clinic (receiving long-term OAT for >8 months) agreed to participate. 104 patients aged 22 to 88 years (median = 59.8) were randomised to PSM (n = 55) or patient self-testing (PST) (n = 49). Following satisfactory completion of a nurse-led training course, patients in both groups measured their INR using the CoaguChek S (Roche Diagnostics) every two weeks, or more frequently if required, for a period of six months. The PST patients telephoned their INR result to a nurse specialist for interpretation and adjustment of warfarin dose, whereas those in the PSM group adjusted their own warfarin dose on the basis of their INR using an algorithm provided by the clinic. 77/104 (74%) patients completed the study (PSM = 41, PST = 36). The ‘drop out’ rates for both groups were similar, with difficulty in obtaining an adequate capillary sample the most common reason given. There was no significant difference in median time in target therapeutic range between the two groups; PSM 71.8% (95% CI 39.7%–92.4%) and PST 70.3% (95% CI 41.3%–92.6%). We conclude that, in the majority of suitably trained patients, the quality of OAT achieved through PSM is comparable to that obtained by self-testing patients managed by a specialised hospital anticoagulation clinic. PSM is therefore an effective model for selected patients. 33 A thromboelastograph study on the effects of tissuetype plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and thrombin activatable fibrinolysis inhibitor (TAFI) on whole blood fibrinolysis SL HARRIS*, MJ GALLIMORE*, DW JONES*, K TAPPENDEN* and M WINTER* *Kent Haemophilia Centre, Kent and Canterbury Hospital, Canterbury, Kent, UK, Department of Biosciences, University of Kent at Canterbury, Canterbury, Kent, UK Thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase which has been shown to reduce t-PA-induced fibrinolysis in plasma and whole blood in vivo. It has been shown, however, that there is a significantly lower risk of myocardial infarction (MI) in individuals with elevated TAFI levels. Urokinase-type plasminogen activator (u-PA) was thought to be involved in extracellular plasminogen activation and not fibrinolysis. However, it has recently been speculated that u-PA may have a role in plasminogen activation via single chain u-PA (scu-PA) association with platelets (which carry a novel receptor for u-PA). In the present study, we investigated whether fibrinolysis induced in whole blood by u-PA and t-PA was affected by increasing levels of TAFI and whether u-PA-induced fibrinolyis was affected by TAFI in whole blood in a different manner to t-PA. We used the thromboelastography analyser (TEG) to measure clot formation and fibrinolysis in blood containing u-PA and t-PA in the presence of various concentrations of TAFI. Following clot formation, fibrinolysis was monitored. Various concentrations of u-PA and t-PA were added to whole blood samples from five donors and the samples subjected to TEG. Concentrations of u-PA and t-PA were selected to give approximately 50% lysis 60 minutes after clot formation. The addition of increasing levels of TAFI in samples containing t-PA gave reduced fibrinolysis in a dose dependent manner. However, the addition of increasing levels of TAFI in blood samples containing u-PA did not show a dose dependent effect and in some cases actually increased fibrinolysis. In some donors, TAFI with u-PA gave reduced coagulation. Our results suggest that, the interaction of TAFI and u-PA may cause a different response in platelet rich clots compared to platelet poor clots and may be a significant factor in the lower risk of MI in individuals with higher TAFI levels. 34 Bleeding complications of oral anticoagulant therapy in a single centre ES GREEN*, S BOND*, S RHODES*, M TAYLOR* and C EMMAS *Swindon and Marlborough NHS Trust, Swindon, UK, Astrazeneca, UK Bleeding is the most serious complication of the use of oral anticoagulation in the prevention and treatment of thromboembolism. We prospectively audited the frequency and severity of over anticoagulation and bleeding in our Anticoagulant service at a District hospital serving a population of 330 000 patients (3900 anticoagulant patients) providing Anticoagulant Practitioner-led inpatient and outpatient anticoagulant dosing. Data was collected over 23 months including INR at time of event, whether complications necessitated hospital attendance and/or admission, investigations carried out and treatment given and an assessment of the associated costs made. There were 443 events. 146 were asymptomatic high INRs (>8). The majority of these were treated with oral vitamin K as outpatients. There were 297 bleeding episodes, 73 major and 224 minor. The most common sites of bleeding were epistaxis (87/297), gastrointestinal (66/297), haematuria (43/297), haematoma (22/297) and bruising (20/297). 152 patients required hospital admission. 23 patients died, 5 as a result of the bleeding episode. 48/443 patients had received anticoagulant therapy for less than 4 weeks at the time of the event, 145/443 were unstable, 224/443 were stable and 26/443 were GP monitored patients with no INR data available. At the time of the bleeding event 48% of patients had an INR abov