Personalized Approach To Treatment of Acute Myeloid Leukemia Using a High-Throughput Chemosensitivity Assay

Most patients with acute myeloid leukemia either relapse or fail to respond to initial therapy. Moreover, each patient’s AML contains multiple mutations that although varying from one population to another, are unique to that individual, prompting development of individualized approaches to AML therapy. We have devised a high-throughput sensitivity assay for 160 drugs; 45 are FDA approved and 115 are investigational, representing multiple mechanisms of action and signaling pathways. 30 primary patient blood marrow samples and 15 acute leukemia cell lines have been analyzed. Peripheral blood blasts from individual patients were thawed, viable cells isolated and purified to >80% using magnetic bead separations. In vitro chemotherapy cytotoxicity testing is performed in a 384 well high throughput format, with eight concentrations of each drug. The output is cell survival assessed via a luminescent detection method after a 4-day incubation with various drugs. Fitted curves (idbs XLfit) were derived from plots of survival versus drug concentrations used in the study. Leukemia cells are tested adherent to coated plates to mimic adhesion in the bone marrow microenvironment, a property that confers drug resistance. The assay exhibits excellent reproducibility from independently thawed samples from the same patients, with a Spearman correlation coefficient of 0.9 (p=1.6 X 10^-141). Gene expression microarrays were also performed for the same 30 patients.

Our assay of the 30 patients revealed that there were over 50 drugs that exhibited cytotoxicity in at least some patients. There was wide variation in the drug sensitivity patterns exhibited by the patient blasts samples, and each was unique. Notably, all patient samples were susceptible to several drugs with IC50s in the range that might predict clinical response. For patients who achieved complete remission, we showed statistically significant association with in vitro cytotoxicity in response to 7 drugs that are commonly employed to treat AML, including mitoxantrone (p=0.002 for 0.1 µM, p=0.01 for 0.3 µM), clofarabine (p=0.0009 for 0.1 µM, p=0.003 for 0.3 µM, p=0.007 for 1µM), daunorubicin (p=003 for 0.1 µM, p=0.005 for 0.3 µM), etoposide (p=0.02 at for 0.1 µM, p=0.01 for 0.3 µM, p=0.01 for 1 µM), and fludarabine (p=0.05 for 0.3 µM, p=0.002 for 1 µM). In addition, we used a multivariate statistical method to identify drug combinations that are effective in predicting the complete remission of AML patients. In particular, we found that the in vitro cytotoxicity data of a combination of three drugs (BAY 11-7085, TPCA-1, ON 01910.Na) are more predictive of the complete remission of patients compared to using each of these drugs individually (i.e. 91.2% accuracy compared to 84.8%, 84.2%, 79.1% respectively). We also performed linear regression analyses to study the relationship between in vitro cytotoxicity in the high throughput screen of each drug versus the patients’ clinical features (including complete remission, having received the tested drug, new diagnosis vs. relapse status, age, gender, bilirubin, albumin, lactate dehydrogenase, white blood count, platelet count, blast, absolute neutrophil count, % bone marrow blasts, hemoglobin, fibrinogen, cytogenetics risk category). The following patients’ clinical features showed significant correlations with the in vitro cytotoxicity of our assays in selected drugs: complete remission for mitoxantrone (p= 0.04 for 0.3 µM), flavoperidol (p= 0.02 0.1 µM), and fludarabine (p= 0.01 0.3 µM, p=0.02 for 1 µM); drug the patient received for clofarabine (p= 0.03 for 0.3 µM, p=0.007 for 1 µM); new diagnosis vs. relapse for clofarabine (p= p= 0.02 for 0.3 µM, p=0.006 for 1 µM).

This assay serves as the basis for a new clinical trial (ClinicalTrials.gov Identifier: NCT01872819) now open to enrollment for “personalized” leukemia therapy, for which patients with refractory AML are assigned drugs based on the results of this test. This new personalized approach to individualized therapy for refractory AML may provide novel drugs to patients and new insights into leukemia drug resistance.