Purpose:The organic cation transporter OCT-1mediates active transport of imatinib.We recently showed that low OCT-1activity is a major contributor to suboptimal response in chronic myeloid leukemia (CML) patients treated with imatinib. The relevance of OCT-1activity and efflux pumps in determining intracellular uptake and retention (IUR) of dasatinib was assessed. Experimental Design: The effect of OCT inhibitors on [ 14 C]dasatinib and [ 14 C]imatinib IUR was compared using peripheral blood mononuclear cells from newly diagnosed CML patients. The role of efflux transporters was studied using ABCB1-and ABCG2-overexpressing cell lines and relevant inhibitors. Results: Unlike imatinib, there was no significant difference in the dasatinib IUR at 37jC and 4jC (P = 0.8), and OCT-1 inhibitors including prazosin did not reduce dasatinib IUR significantly. In CML mononuclear cells, prazosin inhibitable IUR was significantly higher for imatinib than dasatinib (6.38 versus 1.48 ng/200,000 cells; P = 0.002; n = 11). Patients with high OCT-1 activity based on their imatinib uptake had IC 50 dasatinib values equivalent to patients with low OCT-1acti-vity. Dasatinib IUR was significantly lower in ABCB1-overexpressing cell lines compared with parental cell lines (P < 0.05). PSC833 (ABCB1inhibitor) significantly increased the dasatinib IUR (P < 0.05) and reduced IC 50 dasatinib (from 100 to 8 nmol/L) in K562-DOX cell line. The ABCG2 inhibitor Ko143 significantly increased dasatinib IUR in ABCG2-overexpressing cell lines and reduced IC 50 dasatinib . Conclusion: Unlike imatinib, dasatinib cellular uptake is not significantly affected by OCT-1 activity, so that expression and function of OCT-1 is unlikely to affect response to dasatinib. Dasatinib is a substrate of both efflux proteins, ABCB1and ABCG2.

Measurement of OA pretherapy is a predictor for the long-term risk of resistance and transformation in patients with imatinib-treated CML. Early dose-intensity may reduce the negative prognostic impact of low OA. We propose that OA could be used to individualize dosage strategies for patients with CML to maximize molecular response and optimize long-term outcome.

No abstract

In chronic myeloid leukemia (CML) cell lines, brief exposure to pharmacologically relevant dasatinib concentrations results in apoptosis. In this study, we assess the impact of intensity and duration of Bcr-Abl kinase inhibition on primary CD34(+) progenitors of chronic phase CML patients. As CML cells exposed to dasatinib in vivo are in a cytokine-rich environment, we also assessed the effect of cytokines (six growth factors cocktail or granulocyte-macrophage colony-stimulating factor (CSF) or granulocyte-CSF) in combination with dasatinib. In the presence of cytokines, short-term intense Bcr-Abl kinase inhibition (>or=90% p-Crkl inhibition) with 100 nM dasatinib did not reduce CD34(+) colony-forming cells (CFCs). In contrast, without cytokines, short-term exposure to dasatinib reduced CML-CD34(+) CFCs by 70-80%. When cytokines were added immediately after short-term exposure to dasatinib, CML-CD34(+) cells remained viable, suggesting that oncogene dependence of these cells can be overcome by concomitant or subsequent exposure to cytokines. Additional inhibition of Janus tyrosine kinase (Jak) activity re-established the sensitivity of CML progenitors to intense Bcr-Abl kinase inhibition despite the presence of cytokines. These findings support the contention that therapeutic strategies combining intense Bcr-Abl kinase inhibition and blockade of cytokine signaling pathways can be effective for eradication of CML progenitors.

No abstract

We have previously demonstrated in CML patients enrolled to the Australian TIDEL trial, (600mg imatinib upfront in newly diagnosed patients) that patients with high OCT-1 activity, measured in patient blood mononuclear cells prior to imatinib start, achieve a superior molecular response, compared to those with low OCT-1 activity 1. Furthermore, the impact of low OCT-1 activity could be partially overcome with increased imatinib dose. We now prospectively test the predictive value of OCT-1 activity on the achievement of a major molecular response (&lt;0.1 BCR-ABL IS) by 12 months, in CML patients enrolled to the TOPS trial (randomised 400 vs 800 mg imatinib). A subset of 131 TOPS2 patients had OCT-1 activity measured prior to the start of therapy, as part of the Global Novartis Correlative Science Studies. 41 had high OCT-1 activity (&gt;7.2ng/200,000 cells) as defined in our original study. Patients with high OCT-1 activity had a markedly superior rate of MMR, on either standard or high dose imatinib (table 1). Significantly, a greater proportion of patients with low OCT-1 activity achieved MMR on the high dose arm compared to those on standard dose. This finding was not evident in the high OCT-1 activity group. The % of patients achieving MMR by 12 months (n) Total Low OCT-1 Activity High OCT-1 Activity p- value Total 48% (90) 90% (41) &lt;0.001 400mg 59% (34) 24% (17) 94% (17) &lt;0.001 800mg 62% (97) 53% (73) 87% (24) 0.044 p-value 0.27 0.012 0.64 Table 1: The % of patients achieving MMR based on OCT-1 activity and Randomised dose. The median OCT-1 activity for those patients achieving a MMR (n=80) was 6.05ng/200,000 cells compared to 3.9 for those patients failing to achieve MMR (n=51:p=0.003). Of the 131 patients, trough imatinib levels were available on 61. A greater proportion of patients with a trough imatinib plasma level of &gt;1000ng/ml 3 (n=50) at 1 month achieved MMR (88%) compared to those with plasma levels of &lt;1000ng/ml (n=11:45%: p=0.032). Importantly, OCT-1 activity is not significantly different comparing those patients with trough levels &gt;1000ng/ml (5.6ng/200,000 cells) at 1 month to those with lower trough levels (7.3ng/200,000 cells: p=0.117). This indicates that OCT-1 activity is not providing a surrogate marker of imatinib PK. Dividing the imatinib PK data into quartiles, there is no significant difference in the % of patients achieving MMR based on trough imatinib levels, in patients with high OCT-1 activity. In contrast significantly fewer patients with low OCT-1 activity and low trough levels achieve MMR by 12 months. (Table 2) The % of patients achieving MMR by 12 months (n) Imatinib PK (ng/ml) Total Low OCT-1 Activity High OCT-1 Activity P value Quartile 1 &lt;1600 47% (15) 12% (8) 86% (7) 0.013 Quartile 2 &gt;1600 &lt; 2500 80% (15) 67% (6) 89% (9) 0.469 Quartile 3 &gt;2500 &lt; 3500 80% (15) 77% (13) 100% (2) 0.654 Quartile 4 &gt;3500 75% (16) 60% (10) 91% (6) 0.559 Table 2: The percentage of patients achieving MMR based on quartile analysis of imatinib PK at day 29 In the Australian cohort of 60 patients where detailed molecular response data is available the median molecular response in the 4 subgroups at 12 months shows a significant difference between the 400 mg group with low and high OCT-1 activity (Median BCR-ABL 0.2% IS v 0.02% IS p=0.03) but no difference in the 800 mg groups (low OCT-1 activity v high Median BCR-ABL 0.05% IS v 0.03% IS p=0.139). These analyses support our original proposal that OCT-1 activity defined at diagnosis has a major impact on molecular response and raises the possibility of patient-specific dosing. Patients with low OCT-1 activity are likely to achieve superior molecular responses if they receive imatinib at doses greater than 400 mg, whereas we could not find evidence of a molecular benefit to high dose imatinib for patients with high OCT-1 activity. The clinical value of monitoring trough imatinib drug levels remains to be clearly defined but it is likely to be greatly enhanced if it assessed in the context of the patient’s OCT-1 activity.

No abstract

507 The major active influx protein for imatinib into target BCR-ABL positive cells is the organic cation transporter OCT-1. We have previously demonstrated that the functional activity of the OCT-1 protein (OCT-1 activity) is predictive of molecular response in TIDEL (trial of imatinib 600 mg/day with selective dose intensification in untreated CP-CML) The OCT-1 activity (OA) is measured in mononuclear cells from untreated CML patients by calculating the intracellular concentration of 14-C imatinib less the intracellular concentration in the presence of OCT-1 inhibition. To address the question of whether OA is predicting only the rate of response, we now investigate the impact of OA on response and progression at 5 years. There is a significant difference in the achievement of MMR (p=0.007) and CMR by 60 months (p=0.032) (Table 1). Six patients developed kinase domain mutations over the course of this study. 5/6 had low OA. Significantly, for the first time addressing Event Free Survival (events defined as loss of CHR, MCR or CCR, progression to AP or BC or change of therapy due to unsatisfactory efficacy), we demonstrate that more patients with high OA are event free at 5 years when compared to patients with low OA (Table 1). To determine whether the detrimental effect of low OA on survival was more significant in those patients with OA in the lowest quartile (Q1) we compared the response of Q1 patients to all other patients (Table 2). These data demonstrate importantly, that patients in Q1 have significantly poorer outcomes, than the remainder of the patient cohort. In previous analyses we have shown that the effects of a low OA can be partially overcome by higher imatinib doses. Limiting the analyses to those patients receiving <600mg average daily dose over the first 12 months there was a significant difference in the achievement of MMR (low OA (n=11) 27%: high OA (n=12) 92% p=0.021) and EFS (36% vs 75% p=0.03). In patients receiving ≥600 mg there was no significant difference between the groups, reinforcing the importance of dose. In 45 patients we examined the expression of OCT-1 mRNA for prediction of MMR, CMR, EFS and mutation development. Dividing the patients into low and high OCT-1 expression about the median we found that the level of mRNA is not predictive of MMR (low–60% vs high 78 p=0.241) CMR (low–45% vs high 55 p=0.456) EFS (low–55% vs high 70 p=0.315) or mutation development (low–18% vs high 14% p=0.666). These data indicate that the level of OCT-1 mRNA is not sufficiently discriminating to predict response and progression. While our previous studies demonstrated that OA could predict the rate of decline in BCR-ABL over the first 12-24 months, this update demonstrates for the first time, that this assay can identify nearly all patients (>80%) who fail to achieve MMR in the long term. Most importantly OA is also strongly predictive of resistance and progression events. Functional assessment of OCT-1 Activity provides prognostic information that is more discriminating than assaying the level of OCT-1 mRNA. This long term study reinforces the notion that OA is an important predictive variable in CP-CML patients treated with IM. It provides further evidence that OA is a critical variable to consider in future trials of imatinib and a key factor to enable individualization of imatinib dose to optimize the long term outcome for CML patients. Disclosures: White: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding. Manley:Novartis: Employment. 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.