Abstract: The TIDEL-II trial used imatinib (IM) upfront in patients (pts) newly diagnosed with chronic myeloid leukaemia in chronic phase (CML-CP), and switched selected pts to nilotinib (NIL) on the basis of IM intolerance or failure to achieve time-dependent molecular response. We previously reported major molecular response (MMR; BCR-ABL ≤0.1% IS) at 12 months (mths) and transformation-free survival (TFS) at 3 years. This abstract reports the final analysis with minimum follow-up of 60 months. Patients were enrolled across 27 Australasian sites in 2 equal and sequential cohorts. All started treatment with IM 600mg OD and dose escalated to IM 800mg OD if IM trough levels were 〈 1000ng/mL on day 22. A series of time-dependent molecular targets were set: BCR-ABL (IS) ≤10% at 3 mths (early molecular response: EMR), ≤1% at 6 mths and ≤0.1% at 12 mths. In cohort 1 (C1), pts failing to meet these targets dose escalated to IM 800 mg OD. Pts who failed to improve molecular response after another 3 mths, or were already on IM 800mg OD, switched to NIL 400mg BID. In cohort 2 (C2), pts who failed these targets switched to NIL directly. In addition, pts with grade III/IV or persistent grade II toxicity were also allowed to switch from IM to NIL. Data analysed were limited to 60 mths of follow-up. The study enrolled 210 pts with a median age of 49.7 years (range 16-81); 42% were female. Baseline demographics and outcomes were similar across 2 cohorts. Forty pts had day 22 IM trough 〈 1000ng/mL, and 31 had dose escalation. Switching to NIL occurred in 75 pts prior to 24 mths, 55 for failing TIDEL-II targets and 20 for IM intolerance. Table 1 summarises key results. In combination, 5 year overall survival (OS) and TFS and associated 95% confidence intervals, including withdrawn pts, were 95% (88-98%) and 92% (84-95%) respectively. Cumulative incidence by 60 mths of MMR was 86%, MR4 (BCR-ABL1 ≤ 0.01% IS) was 75% and MR4.5 (BCR-ABL1 ≤0.0032%) was 59%. Of the 181 pts achieving MMR on study, 44 (24%) did so after switching to NIL; of the 119 pts achieving MR4.5, 27 (23%) did so after switching to NIL. At 60 mths, 75 (36%) pts had withdrawn from study, and 14 were lost to follow up (including 12 with outstanding data queries). Of the 121 pts (58%) who remained on TIDEL-II until 60 mths, 33 were on NIL (30 in MMR), 77 on IM (76 in MMR); treatment was unknown for 11 (10 in MMR). The median dose of IM was 600mg OD. Of the 51 pts who dose escalated to IM 800mg OD (31 for low IM trough level and 20 for failing to achieve molecular targets), only 9 remained on this dose until mth 60 (5 and 4 pts form the respective groups). Eight pts transformed to accelerated or blastic phase; 5 within the 1st year, 2 in the 3rd and 1 in the 5th year; 2/8 occurred after study withdrawal. There were 14 deaths, mostly due to cardiac events (n=5) or progressive leukaemia (n=6). In all, 13/210 pts (6%) had cardiac, cerebral or peripheral vascular disease, 9 while on NIL and 4 having only had IM. Pts failing to meet molecular targets (analysed according to the 1st target failed) at 3, 6 and 12 mths numbered 25 (12%), 23 (11%) and 30 (14%) respectively with 19, 16 and 20 switching to NIL (subsequent molecular outcomes, Table 2). Pts failing to achieve EMR had poor achievement of MMR and MR4.5 (44% and 8% respectively by 60 mths). Of the 11 EMR failure pts who achieved MMR, only 4 remained in MMR at 60 mths. Pts failing to achieve BCR-ABL≤ 1% at 6 mths had similarly poor outcomes, with MMR and MR4.5 being 52% and 13% respectively. In pts failing to achieve MMR by 12 months, MMR and MR4.5 by 60 mths were 93% and 33% respectively. Twenty pts switched to NIL for IM intolerance prior to 24 mths: 9/20 already in MMR, and 3/20 already in MR4.5 at time of switching. For the remaining pts, MMR and MR4.5 were achieved by 100% and 88% after switching. The TIDEL-II strategy of combining IM and NIL compares favourably with other upfront treatment strategies, with MR4.5 of 59% by 60 mths. IM dose escalation to 800mg OD for target failure was not well tolerated: only 18% of pts who dose escalated maintained this dose at 60 mths. However, this did not appear to be detrimental to overall outcomes, which were similar in the 2 cohorts. Pts switching to NIL for IM intolerance, and for failing to achieve MMR by 12 months after meeting prior goals, had high rates of MMR, superior to those who failed their 3 and 6 mth targets. Early identification of pts at high risk of EMR failure who might benefit from more intensive or experimental therapies may be necessary to further improve outcomes. Disclosures Yeung: Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding. White:Novartis Pharmaceuticals: 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: Consultancy, Honoraria, Research Funding. Branford:Novartis: Honoraria, Research Funding, Speakers Bureau; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Otsuka: Research Funding; Ariad: Research Funding; Bristol Myers-Squibb: Honoraria; Cepheid: Consultancy. Butcher:Janssen: Consultancy; Roche: Consultancy; Novartis: Consultancy. Gottlieb:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Indee: Membership on an entity's Board of Directors or advisory committees. Arthur:Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Tam:Novartis: Honoraria. Mills:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Meeting attendance sponsorship. Hughes:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group; Ariad: 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.

Abstract: Abstract 2288 Previous studies have demonstrated that the achievement of an imatinib (IM) trough level of 1000ng/ml in CP-CML patients is associated with a better response. The aim of this sub-study was to perform a detailed assessment of IM plasma levels in the TIDEL II trial, in which patients are treated with IM 600mg/day upfront with subsequent dose escalation to 400mg BID, or switch to nilotinib if pre-determined molecular milestones are not met. Patients failing to achieve a trough level of 〉 1000ng/ml 22 days after the commencement of IM were scheduled to dose escalate to 400mg BID or maximum tolerated dose (MTD). 105 patients were enrolled in Cohort I of this trial. Plasma for trough IM PK testing was collected at day 8 and day 22, then at 3, 6, and 12 months, and IM levels were measured via HPLC. The median IM PK at day 22 was 1550ng/ml (R 0–4680) (Table 1). 21% (22/103 evaluable) of patients failed to achieve 1000g/ml at day 8, 20% (21/103) at day 22, 20% (18/91) at 3 mths, 29% (23/80) at 6 mths and 24% (13/55) at 12 mths. Overall 56/103 patients (54%) failed to achieve plasma IM levels of 〉 1000ng/ml at least once over the study period. In the majority of cases these reductions in IM level were transient. However, detection of an IM level 〈 1000ng/mL beyond day 22 was associated with a concomitant rise (median 1.9 fold; R: 0.6–122.5 fold) in BCR-ABL levels in 22/36 patients, and was sufficient in 40% of cases to trigger mutation analysis. The Y253H mutation was the only mutation found in one patient who had repeated low drug levels. In 3/9 patients the low drug level was associated with a loss of MMR, which was re-established when drug levels rose above 1000ng/ml again. In 3/8 patients in CCyR at the time of PK fall, CCyR was lost, and 2 of these 3 patients were removed from study for compliance. 16/21 patients who had an IM level 〈 1000ng/ml at day 22 dose escalated to 800mg (median 2.25 mths after commencement. R:1-3). 14/16 achieved IM levels 〉 1000ng/ml when reassessed 22 days later. There was a significant difference in BCR-ABL levels at 2 months between this cohort of patients (median 10.7% (IS) Range 0.5–79%) and those who achieved ≥1000ng/ml at day 22 (median 3.01% (IS) Range 0–52.5%. p=0.028). However by 6 months, when all 16 low PK patients had been dose escalated for at least 3 months there was no significant difference between the two cohorts ( 〈 1000ng/ml @ day 22 – median BCR-ABL – 0.149% Range 0–9.9% vs ≥1000ng/ml 0.153% Range 0–16.4%. p=0.944) supporting the efficacy of this approach. Furthermore at 3 and 6 mths post escalation there was no significant difference in PK between these 16 patients and those who achieved 1000ng/ml at day 22 (p=0.469 and 0.738 respectively) (Table 1). Twelve patients were dose reduced to 400mg for intolerance (median 3 mths after commencement; R:1-9). For these patients, IM levels at day 22 were significantly higher (median 2110ng/ml; R:920–3860ng/ml) than for all other patients (median 1500; R:0-4680ng/ml, p=0.036). All patients who dose reduced demonstrated a subsequent decrease in IM level (Table 1). assessed at 3, 6 and 12 months after therapy change In summary, there is wide interpatient variation in IM levels in patients on 600mg/day. Dose escalation generally resulted in increased IM levels, and improved response. Intolerance is associated with higher IM levels, however high IM levels are not exclusively observed in patients with intolerance. Plasma IM levels 〈 1000ng/ml occurred in approximately half of the patients at least once over the follow up period. Patients whose IM level fell below 1000ng/ml after day 22 often had an associated significant rise in BCR-ABL. Therefore, routine IM plasma testing provides an informative adjunct to BCR-ABL monitoring to guide subsequent treatment decisions. Table: Comparative trough IM levels in patients on 600mg/day, those dose escalated, and those dose reduced. Trough IM Plasma Level (ng/ml) median (Range) n= Day 8 Day 22 3mth 6 mth 12 mth All 1430(110–4070) 101 1550(0–4680) 103 1580(110–4940) 73 1505(0–4550) 70 1440(220–4870) 47 Dose increase D22* 1690(1210–3100) 16 1660(120–2800) 16 1550(0–3010) 9 Dose decrease* 1060(360–1850) 7 880(0–1900) 9 1535(860–2570) 6 Disclosures: White: Novartis: Honoraria, Research Funding; BMS: Research Funding. Slader: Novartis Pharmaceuticals: Employment, Equity Ownership. Osborn: Novartis Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding. Mills: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hughes: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria.

Abstract: Abstract 209 Background: Although the majority of chronic phase (CP) Philadelphia positive (Ph+) chronic myelogenous leukemia (CML) patients (pts) achieve good disease control with imatinib, some pts demonstrate suboptimal responses. Early dose escalation or switching to nilotinib, a more potent BCR-ABL kinase inhibitor, as soon as suboptimal molecular response is recognised may improve response and disease outcome. Aim: To optimise clinical and molecular outcomes in Ph+ CML using imatinib (IM) as frontline therapy with selective IM dose escalation based on pharmacokinetic (PK) results and switching to nilotinib (NIL) in case of suboptimal response, or IM-intolerance. Method: TIDEL-II is a multicentre, single arm prospective ALLG trial in de novo CP-CML pts with 2 separate sequential cohorts. In Cohort I, pts are treated with IM 600mg/d up-front, aiming for BCR-ABL RQ-PCR target values of ≤ 10%, 1%, and 0.1% IS (major molecular response, MMR) at 3, 6, and 12 months respectively. Pts who do not reach these treatment targets are classified as suboptimal responders. Dose escalation to 800mg/d or maximal tolerated dose occurs if trough IM level is 〈 1000ng/mL at day 22, or for suboptimal response. A switch to NIL (400mg bid) is triggered if molecular targets are still not met 3 months after IM escalation, or for loss or response, or for IM intolerance (Grade III/IV or persistent Grade II non-haematological toxicity). Results: 105 pts were assessed with median follow up of 18.9 months (range: 9–33) in cohort I. For pts with a minimum of 12 months follow up (n=80), complete cytogenetic response (CCR), MMR and complete molecular response (CMR)# rates at 12 months were 92%, 66% and 11% respectively. BCR-ABL levels at 3 months were predictive of MMR at 12 months, but not for CMR due to small pt numbers (Table 1). For pts who failed to achieve BCR-ABL of ≤10% at 3 months, the 12 month MMR rate was 25% (vs 5% in TIDEL-I where pts were also started on IM 600mg/d and suboptimal responders were dose escalated to IM 800mg/d). Of the 105 pts, 16 pts dose escalated IM due to a day 22 IM blood level 〈 1000ng/mL, after which 2/16 switched to NIL (1 suboptimal, 1 intolerant); all achieved CCR. Twelve pts dose escalated for suboptimal response, 7 subsequently switched to NIL for again failing treatment targets. In all, 21/105 pts (20%) switched to NIL: 7 for suboptimal response and 14 for intolerance. The median time to switching and the median pre-switch prescribed IM dose were 468 days & 800mg/d for the suboptimal group; and 183 days & 600mg/d for the intolerant group respectively. Of these, 20/21 achieved or remained in CCR. At the time of switching to NIL, 19/21 pts were not in MMR. With a median follow-up of 295 days post switch to NIL, 9/12 intolerant pts (75%) achieved MMR, whereas 1/7 suboptimal IM responders (14%) achieved MMR (median follow up after switching: 286 days). Only 7/105 pts (7%) discontinued treatment: 4 for non-compliance, 1 pt with a T315I mutation and 2 pts with blast crisis (BC). Progression to BC was associated with detectable mutations: 1 pt with 4 different mutations including T315I and 1 pt with H396P mutation. The progression rate to AP/BC was 2%. The overall mutation rate was 5/105 (5%). The 2 pts who progressed and the pt who discontinued when a T315I mutation was detected were among the 28 pts with BCR-ABL values 〉 1.0% at 3 months. In contrast, no resistant mutations were detected or transformations occurred in the 49 pts with BCR-ABL values ≤1.0% at 3 months. Conclusion: A strategy of selective intensification of BCR-ABL inhibitor therapy based on molecular response and PK values resulted in a 66% MMR rate by 12 months. Despite a minority of pts (20%) requiring a switch to NIL, this has enhanced the rate of MMR by 12 months when compared to IM intensification alone as seen in TIDEL-I where the rate of MMR and CMR by 12 months was 47% and 9% respectively. The IM intolerant pts demonstrated excellent response rates after switching to NIL. To date, the results from TIDEL-II compare favourably with other frontline strategies with regards to response and transformation rates. Disclosures: Yeung: Novartis Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding. Osborn:Novartis Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding. White:Novartis Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding. Branford:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding. Slader:Novartis Pharmaceuticals: Employment, Equity Ownership. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding. Mills:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hughes:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Meyers Squibb: Honoraria, Research Funding; Ariad: Honoraria.

Abstract: Regulatory T-cells (Tregs) play a critical role in preventing autoimmune and alloimmune reactions, including graft-versus-host disease (GVHD). Two recent clinical trials demonstrated that in patients undergoing hematopoietic stem cell transplantation, adoptive transfer of Tregs significantly reduced the incidence of grades II-IV GVHD. While Tregs significantly reduced GVHD severity, they did not eliminate GVHD. One potential way to augment Treg-mediated inhibition of GVHD is to increase Treg suppressive potency. We showed previously that Treg-specific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function (Science 328:372, 2010). However, it is unclear whether PKC-θ inhibition can boost Treg function in a systemic inflammatory condition like GVHD. Furthermore, the mechanism by which PKC-θ inhibition augments Treg function is unknown. In this study, we address these unanswered questions. Using a mouse MHC class I/II disparate acute GVHD model, we found that freshly isolated Tregs treated for 30 minutes with 10uM of the clinically available PKC-θ inhibitor AEB071 suppressed GVHD mortality (Fig 1A) and severity significantly better than DMSO treated Tregs. As Tregs exert much of their protective effect against GVHD early in the course of the disease, we analyzed proliferation of GVHD-causing conventional T-cells (Tcon) on D4 after transplant. We observed a significant reduction in Tcon proliferation in mice given AEB071 treated Tregs compared to DMSO treated Tregs. We then performed multi-photon microscopy on D4 after transplant using TEα-GFP Tcon, CD11c-eYFP antigen presenting cells (APCs) and wild-type Tregs. Compared to DMSO, AEB071 treated Tregs significantly increased Tcon velocity and displacement from APCs. Increased velocity and displacement are indicative of decreased Tcon-APC interactions, suggesting reduced priming when AEB071 Tregs are present. Mechanistically, AEB071 vs DMSO treatment of Tregs resulted in augmented expression of the suppressive molecules Neuropilin-1 (Nrp1) and Lymphocyte activation gene 3 (Lag3) after in vitro activation (Fig 1B, C) and in Tregs isolated from acute GVHD mice. Antibody blockade of Nrp1 and Lag3 in in vitro transwell suppression assays reduced the effect of AEB071 treatment, suggesting that these molecules may play a role in enhancing Treg function after PKC-θ inhibition. Flow cytometry analysis of phosphorylated proteins in activated Tregs revealed that PKC-θ inhibition resulted in reduced phosphorylation of the mTORC2 target FoxO3a, but not mTORC1 targets S6 and 4E-BP1. In addition, the mTORC2-specific phosphorylation site on Akt, serine 473, was reduced, whereas the mTORC1-specific site, threonine 308, was unaltered. Together, these data suggest reduced mTORC2 activity. Reduced phosphorylation increases Foxo3a nuclear translocation, which may result in increased Nrp1 and Lag3 expression, since Foxo3a has binding sites in both gene promoters. As both mTORC1 and 2 are involved in T-cell metabolism, we investigated the effect of AEB071 treatment on Treg oxygen consumption rate (OCR). Compared to DMSO, AEB071 treatment significantly increased Treg baseline and maximal OCRs after activation (Fig 1D). Increased OCR has been associated with increased Treg function. To identify additional alterations in phosphorylated proteins after PKC-θ inhibition, we performed a phosphoproteomic screen using in vitro expanded human Tregs treated with AEB701 or DMSO. We identified significant alterations in phosphorylation sites on 72 proteins, including reduced phosphorylation of an adaptor molecule that links PKC-θ to the intermediate filament vimentin. We found that vimentin is highly upregulated in Tregs compared to Tcon and that in Tregs, vimentin interacts with PKC-θ after activation. AEB071 treatment reduced the interaction between vimentin and PKC-θ. As with AEB071 treatment, Vimentin siRNA significantly increased Treg suppression in vitro compared to control transfected Tregs (Fig 1E), and augmented expression of Nrp1 and Lag3. AEB071 treatment of vimentin siRNA transfected Tregs did not further augment Treg function, suggesting an overlapping mechanism. In summary, our data demonstrate that PKC-θ interacts with mTORC2 and vimentin to modulate multiple aspects of Treg function, and that a brief incubation of Tregs with a PKC-θ inhibitor may be a viable method to enhance the efficacy of Treg therapeutics. Disclosures No relevant conflicts of interest to declare.

Abstract: Mucopolysaccharidosis type I (MPS IH; Hurler syndrome) is a congenital deficiency of α-L-iduronidase, leading to lysosomal storage of glycosaminoglycans that is ultimately fatal following an insidious onset after birth. Hematopoietic cell transplantation (HCT) is a life-saving measure in MPS IH. However, because a suitable hematopoietic donor is not found for everyone, because HCT is associated with significant morbidity and mortality, and because there is no known benefit of immune reaction between the host and the donor cells in MPS IH, gene-corrected autologous stem cells may be the ideal graft for HCT. Thus, we generated induced pluripotent stem cells from 2 patients with MPS IH (MPS-iPS cells). We found that α-L-iduronidase was not required for stem cell renewal, and that MPS-iPS cells showed lysosomal storage characteristic of MPS IH and could be differentiated to both hematopoietic and nonhematopoietic cells. The specific epigenetic profile associated with de-differentiation of MPS IH fibroblasts into MPS-iPS cells was maintained when MPS-iPS cells are gene-corrected with virally delivered α-L-iduronidase. These data underscore the potential of MPS-iPS cells to generate autologous hematopoietic grafts devoid of immunologic complications of allogeneic transplantation, as well as generating nonhematopoietic cells with the potential to treat anatomical sites not fully corrected with HCT.

Abstract: Abstract 451FN2 Background: While nilotinib and dasatinib produce faster responses than imatinib as first-line therapy in de novo Chronic Phase Chronic Myeloid Leukemia (CP-CML), an equally effective strategy may be to selectively use these more potent tyrosine kinase inhibitors (TKIs) only in patients who fail to achieve stringent early molecular targets or are intolerant. Aim: To update the molecular outcome and survival of patients in the TIDEL-II study. Method: TIDEL-II is a multicentre, single arm prospective ALLG trial for de novo CP-CML adult patients with two sequential cohorts each of 105 patients. All patients started on imatinib (IM) 600mg OD. Patients with IM trough levels 〈 1000ng/mL on day 22 were dose escalated to 800mg OD (IM800). All patients were monitored for achievement of time-dependent molecular targets - BCR-ABL RQ-PCR of 10%, 1% and 0.1% IS at 3, 6 and 12 months (mo) respectively. Patients in cohort I who failed to meet these targets had dose escalation to IM800. Those patients who again failed to achieve these targets after a further 3 mo were switched to nilotinib 400mg BID (NIL). Patients in cohort 2 who failed their time dependent targets switched to NIL directly without escalating to IM800. In both cohorts, switching to NIL was also permitted for grade III/IV or persistent grade II non-haematological toxicity or loss of response. Primary end point was MMR at 12 mo (BCR-ABL '0.1%IS), with CMR4.5 being a secondary end point (BCR-ABL ≤0.0032%IS). Results: At 12 mo 69% of patients achieved MMR. With median follow up (f/u) of 20mo, AP/BC progression occurred in 5 cases (2.4%) ( Table 1). The 3 mo molecular response was highly correlated with the MMR at 12mo and progression events (table 2). COHORT 1: Using intention to treat analysis (ITT) with median follow-up of 30 mo the rate of MMR at 12 and 24 mo is 66% and 81% respectively (n=105); CMR4.5 was 12% and 24%, respectively. In total, 34/105 (32%) patients switched to NIL, 12 for failure to achieve molecular targets, 19 for intolerance and 3 for loss of response. Only 2/12 patients who failed to meet targets on IM have subsequently achieved MMR on NIL (median f/u on NIL 14 mo). Fourteen patients switched for intolerance when not in MMR, and 9 subsequently gained MMR (64%) (median f/u on NIL 19 mo). Two patients progressed to AP/BC, both in the first 12 mo in patients taking IM. One progression related death and one fatal myocardial infarction (on NIL) have been reported. Fourteen (13%) of patients remain on IM800. COHORT 2: With a median f/u of 12 mo the rates of MMR and CMR4.5 at 12 mo (n=50) were 72% and 16%, respectively (ITT). To date, 35/105 patients, (33%) have switched to NIL, of which 23 switched for failure to meet molecular targets. Subsequently, 3/23 (13%) have achieved MMR (median 6 mo on NIL). Eleven patients have switched to NIL for intolerance, 7 of them not in MMR at time of switch; 6/7 reached MMR in the subsequent 6 mo (median 5 mo on NIL). Seven patients (7%) remain on IM800. Three patients progressed to AP/BC (3%), 2 on IM and 1 on NIL. Three deaths were reported (3%), 1 from cardiac causes and 1 from stroke, both patients on IM at the time; and 1 from CML progression. Relatively short f/u precludes a meaningful comparison of results between the 2 cohorts. Conclusion: The TIDEL-II strategy has achieved a higher rate of MMR at 12 mo of 69% compared to 47% achieved with the strategy of IM intensification previously utilised in the TIDEL-I study. The improvement in molecular response is mostly attributable to improved responses in patients intolerant of IM as deeper responses were uncommon with patients who failed their early molecular targets despite intensification of kinase inhibition. Molecular response at 3 mo is highly correlated with response and progression events, underscoring the importance of early molecular targets. Disclosures: Yeung: Novartis Pharmaceuticals: Research Funding; BMS Oncology: Research Funding. Osborn:Novartis Pharmaceuticals: Research Funding; BMS Oncology: Research Funding. White:Novartis Pharmaceuticals: Research Funding. Branford:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Research Funding; Ariad: Research Funding. Slader:Novartis Pharmaceuticals: Employment, Equity Ownership. Hiwase:CSL: Research Funding. Schwarer:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria; Hospira: Membership on an entity's Board of Directors or advisory committees. Arthur:Novartis Pharmaceuticals: Honoraria; BMS Oncology: Honoraria. Ross:Novartis: Honoraria, Research Funding. Mills:Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Sponsorship to professional meetings; BMS Oncology: Sponsorship to professional meetings. Hughes:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS Oncology: Research Funding.

Abstract: Mesenchymal stem cells (MSCs) can differentiate into non-hematopoietic cell types, including adipocytes, chondrocytes and osteocytes. MSCs have been isolated from multiple species, including humans, and multiple organs, including bone marrow, adipose tissue and umbilical cord blood. The beneficial effects of MSCs are being tested clinically in attempts to: improve hematopoietic engraftment, to treat osteogenesis imperfecta, graft-versus-host disease and autoimmune diseases, and as antitumor agents to deliver therapy for malignancies. Phase I clinical studies have not been associated with toxicities. We aimed to investigate the capacity of MSCs to aid in tissue healing after radiation induced injury in irradiated bone marrow transplant (BMT) recipients. To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using non-viral Sleeping Beauty transposons, and co-infused them with allogeneic bone marrow into irradiated reipients. Using in vivo whole body bioluminenscent imaging luciferase signals were shown to be increased between weeks 3 and 12 indicating expansion of MSCs. Unexpectedly, some mice (N=8/17) with the highest luciferase signals died and all surviving mice (N=9/17) developed foci of ectopic ossification in lungs. Two of mice also developed osteosarcomas in their extremities. This prompted us to characterize the transformed MSCs that originated from the donor MSCs. The transformed cells were aneuploid, lost their capacity to differentiate into mesenchyme-derived adipocytes and chondrocytes, and histologically identified as osteosarcomas. In addition, infusion of tumor cells resulted in malignant lesions in secondary recipients. Mapping of transposition sites in the genome and karyotype analysis indicated that the critical transformation event(s) occurred before infusion of the MSCs. Even though we have not encountered a transformation event in & gt;100 mice infused with MSC manipulated with transposons, we speculated that mutation by transposition was the inciting event. None of the identifiable transposition events occurred in a known proto-oncogene or tumor suppressor gene. This does not discount the possibility of insertional mutagenesis as the genomic lesion may have occurred on the chromosome which was subsequently disrupted or lost. Alternatively, genomic instability could have been a result of spontaneous unrepaired chromosomal lesion(s) that preceded the transposon insertion and resulted in osteosarcoma. These findings provide evidence of evolution of MSCs with osteogenic capacity into osteosarcoma in vivo and are clinically relevant as they document the potential of ex vivo manipulated MSCs for transformation into malignant disease.

Abstract: Vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1) are adhesive proteins induced on endothelium by cytokines. We examined the contribution of these adhesive proteins to human peripheral blood monocyte adherence to endothelium using transfected Chinese hamster ovary (CHO) cells stably expressing these proteins and monoclonal antibodies (MoAbs) to ELAM-1, VCAM-1, or CD49d/CD29 (VLA-4), the leukocyte receptor for VCAM-1. Monocytes bound to CHO cells transfected with cDNA of ELAM-1 or VCAM-1. Binding to ELAM-1 was inhibited by MoAb to ELAM-1 and binding to VCAM-1 was inhibited by MoAb to VCAM-1 or the alpha-chain of very late activation antigen-4 (VLA-4) (CD49d). Additive inhibition of adherence to unstimulated human umbilical vein endothelium (HUVE) was observed when monocytes were pretreated with both MoAb to CD49d and MoAb to CD18, the common beta-chain of the leukocyte beta 2 integrin receptors. Adherence of monocytes to HUVE stimulated by recombinant human tumor necrosis factor-alpha was not reduced by MoAbs to CD18, CD49d, or ELAM- 1 when used singly, but combinations of these MoAbs produced significant inhibition. We conclude that multiple receptor-ligand systems are involved in monocyte adherence to endothelium.

Abstract: Abstract 1690 Background: We have previously identified that low OCT-1 activity (OA) is a poor prognostic indicator in CP-CML patients treated with imatinib (IM). Importantly, a very low OA (OA≤4ng/200,000 cells) is associated with a significant risk of poor molecular response, kinase domain mutations and transformation. The TIDEL II strategy of early intervention, via dose escalation and/or switch to nilotinib (NIL) may reduce the incidence of poor response/therapeutic failure in CP-CML patients, particularly those with very low OA. Methods: Patients in Cohort I (n=105) of the TIDEL II trial were switched to NIL for either IM intolerance, or failure to demonstrate a clinical benefit from IM dose escalation which was triggered by failure to achieve time dependent molecular targets: ≤10% BCR-ABL by 3 m, ≤1% BCR-ABL by 6 m or ≤0.1% BCR-ABL by 12 m. In Cohort II (n=105) patients failing to achieve these molecular targets were switched directly to NIL without prior IM dose intensification. All patients, where possible, had OA measured at diagnosis. Only therapeutic changes prior to 24 months, and patients with a minimum of 6 months exposure to NIL are considered in this analysis. Results: (Table 1) Cohort I: Median follow-up 30 months. The overall rate of major molecular response (MMR) by 12 months was 66%. There was a significant difference in the rate of MMR between patients with low OA (n=49) compared to those with higher OA (n= 54): 49% vs 76%, p=0.007. The overall rate of MMR by 24 months was 81%. Again, patients with low OA (n=46) achieved MMR at a significantly lower rate compared to those with higher OA (n=54): 65% vs 91%, p= 0.003. Thirty patients have switched to NIL, 19/30 because of IM intolerance (av. time on IM 8.5m.) and 11/30 because of molecular target failure (av. time on IM 12.8m). 14/14 intolerant patients not in MMR at the time of switch have achieved MMR on NIL with an average log reduction of 2.8, and 9/18 have achieved CMR. In contrast, 1/9 patients switched for molecular target failure has achieved MMR on NIL, with an average log reduction of 0.65. Importantly, 3/19 withdrew from study due to CML related events. Cohort II: Median follow-up 12 months. To date, 22/105 patients have switched to NIL and have a minimum of 6 months follow-up: 11 for intolerance and 11 for molecular target failure. All patients switched for intolerance achieved and/or maintained MMR on nilotinib, with an average log reduction of 2.89. In contrast, 1/11 patients switched for molecular target failure achieved MMR, with an average log reduction of 0.95 and CCyR has been achieved in 5/10 patients not previously in CCyR. 2/11 of these patients have withdrawn from study. There was a significant difference in the time of switch to NIL, and the length of imatinib exposure between the 2 cohorts for intolerance, and a significant difference between the cohorts in the length of IM exposure for patients with target failure. However, this did not translate to a significant difference in molecular response between the 2 cohorts, suggesting the length of prior IM exposure is not a determinant of subsequent NIL response. Importantly in both cohorts, the OA of those patients switched to NIL based on molecular target failure was significantly lower than that of those who switched for intolerance (p=0.007 and p=0.003) and those patients remaining on IM (p=0.004). Conclusion: Switch to NIL significantly improves response in IM intolerant patients. The majority of patients who switch for molecular target failure on IM do not subsequently achieve MMR on NIL. This suggests that a low OA may delineate a group of CP-CML patients intrinsically insensitive to TKI therapy, for whom switch to NIL either following IM dose intensification (Cohort 1) or as a primary strategy (Cohort II) may not result in an improvement in response. A different first-line strategy may be more effective for this poor risk subgroup. Disclosures: White: Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Slader:Novartis Pharmaceuticals: Employment, Equity Ownership. Yeung:Novartis Pharmaceuticals: Research Funding; BMS Oncology: Research Funding. Osborn:Novartis Pharmaceuticals: Research Funding; BMS Oncology: Research Funding. Mills:Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Sponsorship to professional meetings; BMS Oncology:. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees.